Prof Christian Eggeling

Research Area: Immunology
Technology Exchange: Cellular immunology, In vivo imaging, Microscopy (Confocal) and Microscopy (Video)
Scientific Themes: Immunology and Molecular, Cell & Systems Biology
Web Links:
Far-field fluorescence Nanoscopy: (a) Confocal (upper) and STED (lower) images of Abberior Star 635P-phalloidin labeled microvilli at the cell surface of fixed HeLa cells, using an Abberior-Resolft setup equipped with a pulsed diode laser for excitation and a pulsed Ti:Sa for STED. Scale bar: 5 μm. Insets: close-ups of the lower left edge of the cell, scale bar: 1 μm. (b) Two-color confocal (upper) and STED (lower) images of Abberior STAR 488-immunolabeled nuclear pore complexes (green) and Abberior STAR 440Sx-immunolabeled alpha-tubulin in microtubules (red) in fixed HeLa cells (sample kind gift of Leica Microsystems). Scale bar: 5 μm.

Far-field fluorescence Nanoscopy: (a) Confocal (upper) and STED (lower) images of Abberior Star ...

STED microscopy of membrane interactions. (a) The “raft” principle: Lipids and proteins may interact to transiently coalesce into signaling platforms. (b) The movement of phospholipids in live PtK2 cells is free (I) while that of sphingolipids is hindered due to transient interactions with proteins (II). These differences in diffusion cannot be observed by the diffraction-limited ~ 250-nm large confocal detection area, which averages over any nanoscopic details (left), but by the < 50-nm large observation area of the STED microscope, which reaches the size of complexes (right). (c) Detected fluorescence count-rate over time of single diffusing dye-labeled phospholipids (upper panels) and sphingolipids (lower panels): The STED recordings (right panels) but not the confocal recordings (left panels) observe differences between molecular transits through the observation area of non-interacting (I) and interacting (II) lipids.

STED microscopy of membrane interactions. (a) The “raft” principle: Lipids and proteins may ...

The main research interests of my laboratory are focused on the application and development of ultra-sensitive, live-cell fluorescence microscopy techniques with a spatial resolution down to the molecular level (super-resolution microscopy or nanoscopy), superior to conventional optical microscopes. These super-resolution microscopes will be used to unravel nanoscopic changes at the molecular level in living cells following cellular immune responses. We are planning to visualize previously un-detectable molecular interactions (such as protein-protein and protein-lipid interactions), which will shed new light on different molecular pathways triggered at the cell surface and intracellularly during antigen presentation by dendritic cells and T cell activation. A list of ongoing and future projects is summarized below:

Breaking the resolution limit of conventional microscopy

Studies of complex biological systems such as of living cells demand the use of non-invasive and very sensitive analysis technique such as far-field fluorescence microscopy. Its only drawback is the limited spatial resolution: the diffraction of light prevents that objects closer than about 200 nm can be discerned. As a consequence, important details of for example the cellular immune response cannot be disclosed. A remedy to this physical limit is the on-off switching of fluorescence, ensuring that the measured signal stems from a region of the sample that is much smaller than these 200 nm. Examples of such super-resolving microscopes or nanoscopes are based on Stimulated Emission Depletion (STED) far-field microscopy, on the use of photoswitchable fluorescent markers (RESOLFT or (f)PALM/(d)STORM/… microscopy), or on the optical shelving into the fluorescence marker’s dark state (GSD(IM) microscopy). These techniques deliver a spatial resolution of down to below 40 nm in the living cell and, as a consequence, details of cellular structures and protein aggregations can be imaged and analyzed with much larger details. We apply and develop these techniques further to get new insights into different immunological processes.

Single-molecule super-resolution microscopy of membrane dynamics

Many cellular responses lead to subtle changes on the molecular level, demanding not only for a superior spatial resolution of the analyzing method but also for the sensitivity to monitor single molecules over time and space. The combination of STED microscopy with single-molecule sensitive fluorescence-detection tools such as Fluorescence Correlation Spectroscopy (FCS) as well as the fast spatio-temporal tracking of single labeled molecules (single-particle tracking, SPT) allows for the disclosure of complex dynamical processes otherwise impeded by the limited spatial resolution of conventional far-field microscopy. For example, STED-FCS or SPT offered us to gain novel insights into important cellular processes, such as lipid-lipid, lipid-protein, and protein-protein interactions and the formation of so-called “lipid-rafts” in the cellular plasma membrane. These molecular interactions play an important role in the cellular immune response. We will therefore apply and further develop the STED-FCS and SPT nanoscopy techniques to highlight important molecular processes on the plasma membrane as well as inside the cell during immunological reactions. 

Super-resolution analysis of molecular organization and dynamics at the surface of T cells and antigen presenting cells

The organization and interaction of different molecules at the surface of immune cells such as T cells or antigen-presenting cells (APC) are a key mechanism to the cellular response of the human immune system. We will use aforementioned super-resolution techniques such as STED(-FCS), RESOLFT or (f)PALM/(d)STORM or single-molecule tracking to disentangle these molecular mechanisms at the spatial scale of interest (< 200nm). Examples include the interaction of the T cell receptor (TCR), the Src-type tyrosine kinase (Lck) and lipids during T cell activation, as well as diffusion dynamics of antigen presenting proteins such as Major histocompatibilitycomplex (MHC) or CD1 molecules (in their loaded and unloaded state). We also use our nanoscopes to reveal novel details of the (nanoscale) organization and dynamics of the cellular cytoskeleton during T cell - APC interaction and the formation of the immunological synapse.

Nanoscopic cellular changes during virus cycling

Infectious agents such as influenza virus, human immunodeficiency virus (HIV), Dengue fever virus (DENV) or Hepatitis C virus (HCV) usually cause severe disease symptoms with world-wide thousands of death cases each year. A successful treatment and prevention of these viral infections require the understanding of the full pathway of infection by the virus down to the cellular and molecular level. Furthermore, it is desirable to investigate subtle changes of the organization and dynamics of protein or lipid molecules during the virus-host cell interactions, e.g. during the virus replication cycle starting from virus entry/fusion, over viral replication to the release of the replicated virus. Optical (super-resolution) fluorescence microscopy is a perfect tool to observe these events in the living cell since it allows the investigation of specific molecules without disturbing the sample under study. For this reason, we set up a super-resolving STED microscope in category 3 to investigate subtle changes in the (living) cell during virus infection and cycling. Examples include HIV, DENV and flu.

Wolfson Imaging Centre Oxford and Nanoscopy Oxford

The WIMM has (with the help of funding from the Wolfson foundation and the MRC) established a new optical microscope facility for live-cell studies (the Wolfson Imaging Centre Oxford, http://www.imm.ox.ac.uk/wolfson-imaging-centre-oxford) and hired new researchers with an intense scientific record in this field; Prof. Christian Eggeling (the scientific director of the facility), Dr Christoffer Lagerholm (the facility manager) and Dr Dominic Waithe (the image analyst). They bring along strong expertise on super-resolution microscopy. Integrated into the WIMM and closely collaborating with other optical microscopy experts throughout Oxford (Micron Bioimaging Unit in the Department of Biochemistry headed by Prof Ilan Davis, http://www2.bioch.ox.ac.uk/microngroup/micron_home.php; and the Nanoscopy Oxford initiative, NanO) the whole range of classical and super-resolution optical microscopes for live-cell studies are now open for Oxford-wide researchers, bringing up the possibilities for novel cutting-edge research in all scientific fields.

Name Department Institution Country
Prof Simon J Davis Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Vincenzo Cerundolo Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Veronica J Buckle Nuffield Division of Clinical Laboratory Sciences Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Graham Ogg Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof David G Jackson Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Dr Kerstin Luhn Investigative Medicine Division Oxford University,
Dr Geraldine Gillespie (NDM) NDM Research Building Oxford University, NDM Research Building United Kingdom
Dr Sergi Padilla-Parra (NDM) Structural Biology University of Oxford United Kingdom
Prof Jan Rehwinkel Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Galiani S, Waithe D, Reglinski K, Cruz-Zaragoza LD, Garcia E, Clausen MP, Schliebs W, Erdmann R, Eggeling C. 2016. Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins. J Biol Chem, 291 (33), pp. 16948-16962. | Show Abstract | Read more

Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future.

Eggeling C, Honigmann A. 2016. Closing the gap: The approach of optical and computational microscopy to uncover biomembrane organization. Biochim Biophys Acta, 1858 (10), pp. 2558-2568. | Show Abstract | Read more

Biological membranes are complex composites of lipids, proteins and sugars, which catalyze a myriad of vital cellular reactions in a spatiotemporal tightly controlled manner. Our understanding of the organization principles of biomembranes is limited mainly by the challenge to measure distributions and interactions of lipids and proteins within the complex environment of living cells. With the recent advent of super-resolution optical microscopy (or nanoscopy) one now has approached the molecular scale regime with non-invasive live cell fluorescence observation techniques. Since in silico molecular dynamics (MD) simulation techniques are also improving to study larger and more complex systems we can now start to integrate live-cell and in silico experiments to develop a deeper understanding of biomembranes. In this review we summarize recent progress to measure lipid-protein interactions in living cells and give examples how MD simulations can complement and upgrade the experimental data. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Colin-York H, Shrestha D, Felce JH, Waithe D, Moeendarbary E, Davis SJ, Eggeling C, Fritzsche M. 2016. Super-Resolved Traction Force Microscopy (STFM). Nano Lett, 16 (4), pp. 2633-2638. | Show Abstract | Read more

Measuring small forces is a major challenge in cell biology. Here we improve the spatial resolution and accuracy of force reconstruction of the well-established technique of traction force microscopy (TFM) using STED microscopy. The increased spatial resolution of STED-TFM (STFM) allows a greater than 5-fold higher sampling of the forces generated by the cell than conventional TFM, accessing the nano instead of the micron scale. This improvement is highlighted by computer simulations and an activating RBL cell model system.

van der Velde JH, Oelerich J, Huang J, Smit JH, Aminian Jazi A, Galiani S, Kolmakov K, Guoridis G et al. 2016. A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization. Nat Commun, 7 pp. 10144. | Show Abstract | Read more

Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with 'self-healing' properties. To date, utilization of such fluorophore derivatives is rare due to their elaborate multi-step synthesis. Here we present a general strategy to covalently link a synthetic organic fluorophore simultaneously to a photostabilizer and biomolecular target via unnatural amino acids. The modular approach uses commercially available starting materials and simple chemical transformations. The resulting photostabilizer-dye conjugates are based on rhodamines, carbopyronines and cyanines with excellent photophysical properties, that is, high photostability and minimal signal fluctuations. Their versatile use is demonstrated by single-step labelling of DNA, antibodies and proteins, as well as applications in single-molecule and super-resolution fluorescence microscopy. We are convinced that the presented scaffolding strategy and the improved characteristics of the conjugates in applications will trigger the broader use of intramolecular photostabilization and help to emerge this approach as a new gold standard.

Sezgin E, Can FB, Schneider F, Clausen MP, Galiani S, Stanly TA, Waithe D, Colaco A et al. 2016. A comparative study on fluorescent cholesterol analogs as versatile cellular reporters. J Lipid Res, 57 (2), pp. 299-309. | Show Abstract | Read more

Cholesterol (Chol) is a crucial component of cellular membranes, but knowledge of its intracellular dynamics is scarce. Thus, it is of utmost interest to develop tools for visualization of Chol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently labeled Chol analogs. Unfortunately, the introduction of the label may influence the characteristics of the analog, such as its localization, interaction, and trafficking in cells; hence, it is important to get knowledge of such bias. In this report, we compared different fluorescent lipid analogs for their performance in cellular assays: 1) plasma membrane incorporation, specifically the preference for more ordered membrane environments in phase-separated giant unilamellar vesicles and giant plasma membrane vesicles; 2) cellular trafficking, specifically subcellular localization in Niemann-Pick type C disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS)-based and super-resolution stimulated emission depletion-FCS-based measurements of membrane diffusion dynamics. The analogs exhibited strong differences, with some indicating positive performance in the membrane-based experiments and others in the intracellular trafficking assay. However, none showed positive performance in all assays. Our results constitute a concise guide for the careful use of fluorescent Chol analogs in visualizing cellular Chol dynamics.

Waithe D, Clausen MP, Sezgin E, Eggeling C. 2016. FoCuS-point: software for STED fluorescence correlation and time-gated single photon counting. Bioinformatics, 32 (6), pp. 958-960. | Show Abstract | Read more

MOTIVATION: Fluorescence Correlation Spectroscopy (FCS) is a popular tool for measuring molecular mobility and how mobility relates to molecular interaction dynamics and bioactivity in living cells. The FCS technique has been significantly advanced by its combination with super-resolution STED microscopy (STED-FCS). Specifically, the use of gated detection has shown great potential for enhancing STED-FCS, but has also created a demand for software which is efficient and also implements the latest algorithms. Prior to this study, no open software has been available which would allow practical time-gating and correlation of point data derived from STED-FCS experiments. RESULTS: The product of this study is a piece of stand-alone software called FoCuS-point. FoCuS-point utilizes advanced time-correlated single-photon counting (TCSPC) correlation algorithms along with time-gated filtering and innovative data visualization. The software has been designed to be highly user-friendly and is tailored to handle batches of data with tools designed to process files in bulk. FoCuS-point also includes advanced fitting algorithms which allow the parameters of the correlation curves and thus the kinetics of diffusion to be established quickly and efficiently. AVAILABILITY AND IMPLEMENTATION: FoCuS-point is written in python and is available through the github repository: https://github.com/dwaithe/FCS_point_correlator Furthermore, compiled versions of the code are available as executables which can be run directly in Linux, Windows and Mac OSX operating systems. CONTACT: dominic.waithe@imm.ox.ac.uk.

Fritzsche M, Fernandes RA, Colin-York H, Santos AM, Lee SF, Lagerholm BC, Davis SJ, Eggeling C. 2015. CalQuo: automated, simultaneous single-cell and population-level quantification of global intracellular Ca2+ responses. Sci Rep, 5 pp. 16487. | Show Abstract | Read more

Detecting intracellular calcium signaling with fluorescent calcium indicator dyes is often coupled with microscopy techniques to follow the activation state of non-excitable cells, including lymphocytes. However, the analysis of global intracellular calcium responses both at the single-cell level and in large ensembles simultaneously has yet to be automated. Here, we present a new software package, CalQuo (Calcium Quantification), which allows the automated analysis and simultaneous monitoring of global fluorescent calcium reporter-based signaling responses in up to 1000 single cells per experiment, at temporal resolutions of sub-seconds to seconds. CalQuo quantifies the number and fraction of responding cells, the temporal dependence of calcium signaling and provides global and individual calcium-reporter fluorescence intensity profiles. We demonstrate the utility of the new method by comparing the calcium-based signaling responses of genetically manipulated human lymphocytic cell lines.

Reglinski K, Keil M, Altendorf S, Waithe D, Eggeling C, Schliebs W, Erdmann R. 2015. Peroxisomal Import Reduces the Proapoptotic Activity of Deubiquitinating Enzyme USP2. PLoS One, 10 (10), pp. e0140685. | Show Abstract | Read more

The human deubiquitinating enzyme ubiquitin-specific protease 2 (USP2) regulates multiple cellular pathways, including cell proliferation and apoptosis. As a result of alternative splicing four USP2 isoenzymes are expressed in human cells of which all contain a weak peroxisome targeting signal of type 1 (PTS1) at their C-termini. Here, we systematically analyzed apoptotic effects induced by overexpression and intracellular localization for each isoform. All isoforms exhibit proapoptotic activity and are post-translationally imported into the matrix of peroxisomes in a PEX5-dependent manner. However, a significant fraction of the USP2 pool resides in the cytosol due to a weaker PTS1 and thus low affinity to the PTS receptor PEX5. Blocking of peroxisomal import did not interfere with the proapoptotic activity of USP2, suggesting that the enzyme performs its critical function outside of this compartment. Instead, increase of the efficiency of USP2 import into peroxisomes either by optimization of its peroxisomal targeting signal or by overexpression of the PTS1 receptor did result in a reduction of the apoptotic rate of transfected cells. Our studies suggest that peroxisomal import of USP2 provides additional control over the proapoptotic activity of cytosolic USP2 by spatial separation of the deubiquitinating enzymes from their interaction partners in the cytosol and nucleus.

Cited:

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Scopus

Hell SW, Sahl SJ, Bates M, Zhuang X, Heintzmann R, Booth MJ, Bewersdorf J, Shtengel G et al. 2015. The 2015 super-resolution microscopy roadmap JOURNAL OF PHYSICS D-APPLIED PHYSICS, 48 (44), pp. 443001-443001. | Show Abstract | Read more

© 2015 IOP Publishing Ltd.Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio)physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of 'super-resolution' far-field optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physics addresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough discussion on the concepts underlying super-resolution optical microscopy, the potential of different approaches, the importance of label optimization (such as reversible photoswitchable proteins) and applications in which these methods will have a significant impact. Mark Bates, Christian Eggeling

Neuhaus A, Eggeling C, Erdmann R, Schliebs W. 2016. Why do peroxisomes associate with the cytoskeleton? Biochim Biophys Acta, 1863 (5), pp. 1019-1026. | Show Abstract | Read more

Attachment of peroxisomes to cytoskeleton and movement along microtubular filaments and actin cables are essential and highly regulated processes enabling metabolic efficiency, biogenesis, maintenance and inheritance of this dynamic cellular compartment. Several peroxisome-associated proteins have been identified, which mediate interaction with motor proteins, adaptor proteins or other constituents of the cytoskeleton. It appears that there is a species-specific complexity of protein-protein interactions required to control directional movement and arresting. An open question is why some proteins with a specific role in peroxisomal protein import have an additional function in the regulation of cytoskeleton binding and motility of peroxisomes.

Platta HW, Brinkmeier R, Reidick C, Galiani S, Clausen MP, Eggeling C. 2016. Regulation of peroxisomal matrix protein import by ubiquitination. Biochim Biophys Acta, 1863 (5), pp. 838-849. | Show Abstract | Read more

Peroxisomes are organelles that play an important role in many cellular tasks. The functionality of peroxisomes depends on the proper import of their matrix proteins. Peroxisomal matrix proteins are imported posttranslationally in a folded, sometimes even oligomeric state. They harbor a peroxisomal targeting sequence (PTS), which is recognized by dynamic PTS-receptors in the cytosol. The PTS-receptors ferry the cargo to the peroxisomal membrane, where they become part of a transient import pore and then release the cargo into the peroxisomal lumen. Subsequentially, the PTS-receptors are ubiquitinated in order to mark them for the export-machinery, which releases them back to the cytosol. Upon deubiquitination, the PTS-receptors can facilitate further rounds of cargo import. Because the ubiquitination of the receptors is an essential step in the import cycle, it also represents a central regulatory element that governs peroxisomal dynamics. In this review we want to give an introduction to the functional role played by ubiquitination during peroxisomal protein import and highlight the mechanistic concepts that have emerged based on data derived from different species since the discovery of the first ubiquitinated peroxin 15years ago. Moreover, we discuss future tasks and the potential of using advanced technologies for investigating further details of peroxisomal protein transport.

Vicidomini G, Ta H, Honigmann A, Mueller V, Clausen MP, Waithe D, Galiani S, Sezgin E, Diaspro A, Hell SW, Eggeling C. 2015. STED-FLCS: An Advanced Tool to Reveal Spatiotemporal Heterogeneity of Molecular Membrane Dynamics. Nano Lett, 15 (9), pp. 5912-5918. | Show Abstract | Read more

Heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events, such as of lipids in plasma membrane bioactivity. However, these dynamics can often only be visualized by single-molecule and super-resolution optical microscopy techniques. Using fluorescence lifetime correlation spectroscopy (FLCS, an extension of fluorescence correlation spectroscopy, FCS) on a super-resolution stimulated emission depletion (STED) microscope, we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells. STED-FLCS allows an improved determination of spatiotemporal heterogeneity in molecular diffusion and interaction dynamics via a novel gated detection scheme, as demonstrated by a comparison between STED-FLCS and previous conventional STED-FCS recordings on fluorescent phosphoglycerolipid and sphingolipid analogues in the plasma membrane of live mammalian cells. The STED-FLCS data indicate that biophysical and biochemical parameters such as the affinity for molecular complexes strongly change over space and time within a few seconds. Drug treatment for cholesterol depletion or actin cytoskeleton depolymerization not only results in the already previously observed decreased affinity for molecular interactions but also in a slight reduction of the spatiotemporal heterogeneity. STED-FLCS specifically demonstrates a significant improvement over previous gated STED-FCS experiments and with its improved spatial and temporal resolution is a novel tool for investigating how heterogeneities of the cellular plasma membrane may regulate biofunctionality.

Clausen MP, Sezgin E, Bernardino de la Serna J, Waithe D, Lagerholm BC, Eggeling C. 2015. A straightforward approach for gated STED-FCS to investigate lipid membrane dynamics. Methods, 88 pp. 67-75. | Show Abstract | Read more

Recent years have seen the development of multiple technologies to investigate, with great spatial and temporal resolution, the dynamics of lipids in cellular and model membranes. One of these approaches is the combination of far-field super-resolution stimulated-emission-depletion (STED) microscopy with fluorescence correlation spectroscopy (FCS). STED-FCS combines the diffraction-unlimited spatial resolution of STED microscopy with the statistical accuracy of FCS to determine sub-millisecond-fast molecular dynamics with single-molecule sensitivity. A unique advantage of STED-FCS is that the observation spot for the FCS data recordings can be tuned to sub-diffraction scales, i.e. <200 nm in diameter, in a gradual manner to investigate fast diffusion of membrane-incorporated labelled entities. Unfortunately, so far the STED-FCS technology has mostly been applied on a few custom-built setups optimised for far-red fluorescent emitters. Here, we summarise the basics of the STED-FCS technology and highlight how it can give novel details into molecular diffusion modes. Most importantly, we present a straightforward way for performing STED-FCS measurements on an unmodified turnkey commercial system using a time-gated detection scheme. Further, we have evaluated the STED-FCS performance of different commonly used green emitting fluorescent dyes applying freely available, custom-written analysis software.

Sezgin E, Waithe D, Bernardino De La Serna J, Eggeling C. 2015. Spectral imaging to measure heterogeneity in membrane lipid packing ChemPhysChem, 16 (7), pp. 1387-1394. | Show Abstract | Read more

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Physicochemical properties of the plasma membrane have been shown to play an important role in cellular functionality. Among those properties, the molecular order of the lipids, or the lipid packing, is of high importance. Changes in lipid packing are believed to compartmentalize cellular signaling by initiating coalescence and conformational changes of proteins. A common way to infer membrane lipid packing is by using membrane-embedded polarity-sensitive dyes, whose emission spectrum is dependent on the molecular order of the immediate membrane environment. Here, we report on an improved determination of such spectral shifts in the emission spectrum of the polarity-sensitive dyes. This improvement is based on the use of spectral imaging on a scanning confocal fluorescence microscope in combination with an improved analysis, which considers the whole emission spectrum instead of just single wavelength ranges. Using this approach and the polarity-sensitive dyes C-Laurdan or Di-4-ANEPPDHQ, we were able to image - with high accuracy - minute differences in the lipid packing of model and cellular membranes.

Sezgin E, Davis SJ, Eggeling C. 2015. Membrane nanoclusters-tails of the unexpected. Cell, 161 (3), pp. 433-434. | Show Abstract | Read more

The existence, nature, and role of highly ordered membrane domains, often referred to as lipid rafts, have been highly debated by cell biologists for many years. In this issue, Raghupathy et al. describe molecular mechanisms leading to the formation of ordered lipid-protein clusters.

Lehnert S, Hessler S, Galiani S, Clausen M, Tabor A, Eggeling C, Groemer T, Alzheimer C, Huth T. 2015. Illuminating the interaction between beta-secretase BACE1 and KCNQ potassium channels ACTA PHYSIOLOGICA, 213 pp. 134-134.

Eggeling C. 2015. Super-resolution optical microscopy of lipid plasma membrane dynamics. Essays Biochem, 57 pp. 69-80. | Show Abstract | Read more

Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS, and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS, the STED-FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid-protein interactions and the traditional lipid 'raft' theory.

Sezgin E, Davis SJ, Eggeling C. 2015. Membrane Nanoclusters—Tails of the Unexpected Cell, 161 (6), pp. 1472-1472. | Read more

Andrade DM, Clausen MP, Keller J, Mueller V, Wu C, Bear JE, Hell SW, Lagerholm BC, Eggeling C. 2015. Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane--a minimally invasive investigation by STED-FCS. Sci Rep, 5 pp. 11454. | Show Abstract | Read more

Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes.

Eggeling C, Willig KI, Sahl SJ, Hell SW. 2015. Lens-based fluorescence nanoscopy. Q Rev Biophys, 48 (2), pp. 178-243. | Show Abstract | Read more

The majority of studies of the living cell rely on capturing images using fluorescence microscopy. Unfortunately, for centuries, diffraction of light was limiting the spatial resolution in the optical microscope: structural and molecular details much finer than about half the wavelength of visible light (~200 nm) could not be visualized, imposing significant limitations on this otherwise so promising method. The surpassing of this resolution limit in far-field microscopy is currently one of the most momentous developments for studying the living cell, as the move from microscopy to super-resolution microscopy or 'nanoscopy' offers opportunities to study problems in biophysical and biomedical research at a new level of detail. This review describes the principles and modalities of present fluorescence nanoscopes, as well as their potential for biophysical and cellular experiments. All the existing nanoscopy variants separate neighboring features by transiently preparing their fluorescent molecules in states of different emission characteristics in order to make the features discernible. Usually these are fluorescent 'on' and 'off' states causing the adjacent molecules to emit sequentially in time. Each of the variants can in principle reach molecular spatial resolution and has its own advantages and disadvantages. Some require specific transitions and states that can be found only in certain fluorophore subfamilies, such as photoswitchable fluorophores, while other variants can be realized with standard fluorescent labels. Similar to conventional far-field microscopy, nanoscopy can be utilized for dynamical, multi-color and three-dimensional imaging of fixed and live cells, tissues or organisms. Lens-based fluorescence nanoscopy is poised for a high impact on future developments in the life sciences, with the potential to help solve long-standing quests in different areas of scientific research.

Sezgin E, Waithe D, Bernardino de la Serna J, Eggeling C. 2015. Spectral imaging to measure heterogeneity in membrane lipid packing. Chemphyschem, 16 (7), pp. 1387-1394. | Show Abstract | Read more

Physicochemical properties of the plasma membrane have been shown to play an important role in cellular functionality. Among those properties, the molecular order of the lipids, or the lipid packing, is of high importance. Changes in lipid packing are believed to compartmentalize cellular signaling by initiating coalescence and conformational changes of proteins. A common way to infer membrane lipid packing is by using membrane-embedded polarity-sensitive dyes, whose emission spectrum is dependent on the molecular order of the immediate membrane environment. Here, we report on an improved determination of such spectral shifts in the emission spectrum of the polarity-sensitive dyes. This improvement is based on the use of spectral imaging on a scanning confocal fluorescence microscope in combination with an improved analysis, which considers the whole emission spectrum instead of just single wavelength ranges. Using this approach and the polarity-sensitive dyes C-Laurdan or Di-4-ANEPPDHQ, we were able to image-with high accuracy-minute differences in the lipid packing of model and cellular membranes.

Milovanovic D, Honigmann A, Koike S, Göttfert F, Pähler G, Junius M, Müllar S, Diederichsen U et al. 2015. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains. Nat Commun, 6 pp. 5984. | Show Abstract | Read more

The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein-protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes.

Betzig E, Hell SW, Moerner WE, Eggeling C, Kador L, Sauer M, Welter K. 2014. Nobel Prize in Chemistry: Fascinating glimpses into the nanoworld Chemie in Unserer Zeit, 48 (6), pp. 425.

Spillane KM, Ortega-Arroyo J, de Wit G, Eggeling C, Ewers H, Wallace MI, Kukura P. 2014. High-speed single-particle tracking of GM1 in model membranes reveals anomalous diffusion due to interleaflet coupling and molecular pinning. Nano Lett, 14 (9), pp. 5390-5397. | Show Abstract | Read more

The biological functions of the cell membrane are influenced by the mobility of its constituents, which are thought to be strongly affected by nanoscale structure and organization. Interactions with the actin cytoskeleton have been proposed as a potential mechanism with the control of mobility imparted through transmembrane "pickets" or GPI-anchored lipid nanodomains. This hypothesis is based on observations of molecular mobility using various methods, although many of these lack the spatiotemporal resolution required to fully capture all the details of the interaction dynamics. In addition, the validity of certain experimental approaches, particularly single-particle tracking, has been questioned due to a number of potential experimental artifacts. Here, we use interferometric scattering microscopy to track molecules labeled with 20-40 nm scattering gold beads with simultaneous <2 nm spatial and 20 μs temporal precision to investigate the existence and mechanistic origin of anomalous diffusion in bilayer membranes. We use supported lipid bilayers as a model system and demonstrate that the label does not influence time-dependent diffusion in the small particle limit (≤40 nm). By tracking the motion of the ganglioside lipid GM1 bound to the cholera toxin B subunit for different substrates and lipid tail properties, we show that molecular pinning and interleaflet coupling between lipid tail domains on a nanoscopic scale suffice to induce transient immobilization and thereby anomalous subdiffusion on the millisecond time scale.

Schönle A, Von Middendorff C, Ringemann C, Hell SW, Eggeling C. 2014. Monitoring triplet state dynamics with fluorescence correlation spectroscopy: bias and correction. Microsc Res Tech, 77 (7), pp. 528-536. | Show Abstract | Read more

A marker's dark triplet state is of great importance in fluorescence microscopy: It serves as a means to switch off fluorescent markers and is thus the enabling element for several super-resolution methods. On the other hand, intersystem-crossing to the electronic dark triplet state strongly reduces the fluorescence yield in conventional fluorescence microscopy. The ability to determine the kinetic parameters of transitions into the triplet state is thus of great importance and because fluorescence correlation spectroscopy (FCS) can be applied without disturbing the system under study, it is one of the preferred methods to do so. However, conventional FCS observations of triplet dynamics suffer from bias due to the spatially inhomogeneous irradiance profile of the excitation laser. Herein, we present a novel method to correct this bias and verify it by analyzing both Monte Carlo simulated and experimental data of the organic dye Rhodamine 110 in aqueous solution for both continuous-wave and pulsed excitation. Importantly, our approach can be readily generalized to most other FCS experiments that determine intensity dependent kinetic parameters.

Sahl SJ, Leutenegger M, Hell SW, Eggeling C. 2014. High-Resolution Tracking of Single-Molecule Diffusion in Membranes by Confocalized and Spatially Differentiated Fluorescence Photon Stream Recording ChemPhysChem, 15 (4), pp. 771-783. | Show Abstract | Read more

The performance of a method is assessed which allows for the spatiotemporal tracking of single dye-labeled molecules during two-dimensional (2D) diffusional transits through the focal area of a modified confocal microscope. In addition to facilitating the observation of molecular diffusion paths at the shot-noise limit of bright organic emitters with spatial and temporal precisions of ∼10-20 nm and <0.5 ms, respectively, the direct access to the complete stream of detected photons is beneficial for characterizing nanoscale details such as transient pausing (binding). We discuss technical aspects of this approach, along with results from its application to measuring lipid membrane dynamics in live mammalian cells. Presented topics include a discussion of the advantages of the single-photon collection mode and instrument as well as computational considerations for the localization process. A proof-of-principle experiment shows that optical nanoscopy by stochastic single-molecule switching and position readout could be implementable in parallel with such fast molecular tracking. This would allow direct access to contextual imaging data of local cytoskeletal structural elements or localized longer-lived protein assemblies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sahl SJ, Leutenegger M, Hell SW, Eggeling C. 2014. High-resolution tracking of single-molecule diffusion in membranes by confocalized and spatially differentiated fluorescence photon stream recording. Chemphyschem, 15 (4), pp. 771-783. | Show Abstract | Read more

The performance of a method is assessed which allows for the spatiotemporal tracking of single dye-labeled molecules during two-dimensional (2D) diffusional transits through the focal area of a modified confocal microscope. In addition to facilitating the observation of molecular diffusion paths at the shot-noise limit of bright organic emitters with spatial and temporal precisions of ∼10-20 nm and <0.5 ms, respectively, the direct access to the complete stream of detected photons is beneficial for characterizing nanoscale details such as transient pausing (binding). We discuss technical aspects of this approach, along with results from its application to measuring lipid membrane dynamics in live mammalian cells. Presented topics include a discussion of the advantages of the single-photon collection mode and instrument as well as computational considerations for the localization process. A proof-of-principle experiment shows that optical nanoscopy by stochastic single-molecule switching and position readout could be implementable in parallel with such fast molecular tracking. This would allow direct access to contextual imaging data of local cytoskeletal structural elements or localized longer-lived protein assemblies.

Guzmán C, Šolman M, Ligabue A, Blaževitš O, Andrade DM, Reymond L, Eggeling C, Abankwa D. 2014. The efficacy of Raf kinase recruitment to the GTPase H-ras depends on H-ras membrane conformer-specific nanoclustering. J Biol Chem, 289 (14), pp. 9519-9533. | Show Abstract | Read more

Solution structures and biochemical data have provided a wealth of mechanistic insight into Ras GTPases. However, information on how much the membrane organization of these lipid-modified proteins impacts on their signaling is still scarce. Ras proteins are organized into membrane nanoclusters, which are necessary for Ras-MAPK signaling. Using quantitative conventional and super-resolution fluorescence methods, as well as mathematical modeling, we investigated nanoclustering of H-ras helix α4 and hypervariable region mutants that have different bona fide conformations on the membrane. By following the emergence of conformer-specific nanoclusters in the plasma membrane of mammalian cells, we found that conformers impart distinct nanoclustering responses depending on the cytoplasmic levels of the nanocluster scaffold galectin-1. Computational modeling revealed that complexes containing H-ras conformers and galectin-1 affect both the number and lifetime of nanoclusters and thus determine the specific Raf effector recruitment. Our results show that mutations in Ras can affect its nanoclustering response and thus allosterically effector recruitment and downstream signaling. We postulate that cancer- and developmental disease-linked mutations that are associated with the Ras membrane conformation may exhibit so far unrecognized Ras nanoclustering and therefore signaling alterations.

Honigmann A, Sadeghi S, Keller J, Hell SW, Eggeling C, Vink R. 2014. A lipid bound actin meshwork organizes liquid phase separation in model membranes. Elife, 3 (3), pp. e01671. | Show Abstract | Read more

The eukaryotic cell membrane is connected to a dense actin rich cortex. We present FCS and STED experiments showing that dense membrane bound actin networks have severe influence on lipid phase separation. A minimal actin cortex was bound to a supported lipid bilayer via biotinylated lipid streptavidin complexes (pinning sites). In general, actin binding to ternary membranes prevented macroscopic liquid-ordered and liquid-disordered domain formation, even at low temperature. Instead, depending on the type of pinning lipid, an actin correlated multi-domain pattern was observed. FCS measurements revealed hindered diffusion of lipids in the presence of an actin network. To explain our experimental findings, a new simulation model is proposed, in which the membrane composition, the membrane curvature, and the actin pinning sites are all coupled. Our results reveal a mechanism how cells may prevent macroscopic demixing of their membrane components, while at the same time regulate the local membrane composition. DOI: http://dx.doi.org/10.7554/eLife.01671.001.

Lauterbach MA, Eggeling C. 2014. Foundations of STED microscopy Neuromethods, 86 pp. 41-71. | Show Abstract | Read more

This chapter presents the foundations of STED microscopy with a comparison to its generalization Resolft microscopy and to stochastic imaging methods (PALM, STORM, FPALM, and alike). The first section reviews the advantages of optical microscopy, explains the diffraction limit, and shows how the classical resolution limit was finally broken. It also reviews some of the achievements in super-resolution imaging. The second section explains in depth the principle of STED microscopy and highlights some special STED modalities like the use of continuous wave lasers, time-gating, fast imaging with up to 200 frames per second, and combination with fluorescence correlation spectroscopy. The third section treats some aspects of resolution in the presence of noise, especially in the scope of high-resolution imaging. © 2014 Springer Science+Business Media, LLC.

Lauterbach MA, Eggeling C. 2014. Foundations of STED microscopy Neuromethods, 86 pp. 41-71. | Show Abstract | Read more

This chapter presents the foundations of STED microscopy with a comparison to its generalization Resolft microscopy and to stochastic imaging methods (PALM, STORM, FPALM, and alike). The first section reviews the advantages of optical microscopy, explains the diffraction limit, and shows how the classical resolution limit was finally broken. It also reviews some of the achievements in super-resolution imaging. The second section explains in depth the principle of STED microscopy and highlights some special STED modalities like the use of continuous wave lasers, time-gating, fast imaging with up to 200 frames per second, and combination with fluorescence correlation spectroscopy. The third section treats some aspects of resolution in the presence of noise, especially in the scope of high-resolution imaging. © 2014 Springer Science+Business Media, LLC.

Honigmann A, Mueller V, Ta H, Schoenle A, Sezgin E, Hell SW, Eggeling C. 2014. Scanning STED-FCS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells. Nat Commun, 5 pp. 5412. | Show Abstract | Read more

The interaction of lipids and proteins plays an important role in plasma membrane bioactivity, and much can be learned from their diffusion characteristics. Here we present the combination of super-resolution STED microscopy with scanning fluorescence correlation spectroscopy (scanning STED-FCS, sSTED-FCS) to characterize the spatial and temporal heterogeneity of lipid interactions. sSTED-FCS reveals transient molecular interaction hotspots for a fluorescent sphingolipid analogue. The interaction sites are smaller than 80 nm in diameter and lipids are transiently trapped for several milliseconds in these areas. In comparison, newly developed fluorescent phospholipid and cholesterol analogues with improved phase-partitioning properties show more homogenous diffusion, independent of the preference for liquid-ordered or disordered membrane environments. Our results do not support the presence of nanodomains based on lipid-phase separation in the basal membrane of our cultured nonstimulated cells, and show that alternative interactions are responsible for the strong local trapping of our sphingolipid analogue.

Eggeling C, Heilemann M. 2014. Editorial overview: Molecular imaging Current Opinion in Chemical Biology, 20 (1), pp. v-vii. | Read more

Saka SK, Honigmann A, Eggeling C, Hell SW, Lang T, Rizzoli SO. 2014. Multi-protein assemblies underlie the mesoscale organization of the plasma membrane. Nat Commun, 5 pp. 4509. | Show Abstract | Read more

Most proteins have uneven distributions in the plasma membrane. Broadly speaking, this may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins. The latter hypothesis has been difficult to test in the past. Here, we introduce several approaches based on click chemistry, through which we study the distribution of membrane proteins in living cells, as well as in membrane sheets. We found that the plasma membrane proteins form multi-protein assemblies that are long lived (minutes), and in which protein diffusion is restricted. The formation of the assemblies is dependent on cholesterol. They are separated and anchored by the actin cytoskeleton. Specific proteins are preferentially located in different regions of the assemblies, from their cores to their edges. We conclude that the assemblies constitute a basic mesoscale feature of the membrane, which affects the patterning of most membrane proteins, and possibly also their activity.

Eggeling C, Heilemann M. 2014. Editorial overview: Molecular imaging. Curr Opin Chem Biol, 20 pp. v-vii. | Read more

Schönle A, Von Middendorff C, Ringemann C, Hell SW, Eggeling C. 2014. Monitoring triplet state dynamics with fluorescence correlation spectroscopy: Bias and correction Microscopy Research and Technique, 77 (7), pp. 528-536. | Show Abstract | Read more

A marker's dark triplet state is of great importance in fluorescence microscopy: It serves as a means to switch off fluorescent markers and is thus the enabling element for several super-resolution methods. On the other hand, intersystem-crossing to the electronic dark triplet state strongly reduces the fluorescence yield in conventional fluorescence microscopy. The ability to determine the kinetic parameters of transitions into the triplet state is thus of great importance and because fluorescence correlation spectroscopy (FCS) can be applied without disturbing the system under study, it is one of the preferred methods to do so. However, conventional FCS observations of triplet dynamics suffer from bias due to the spatially inhomogeneous irradiance profile of the excitation laser. Herein, we present a novel method to correct this bias and verify it by analyzing both Monte Carlo simulated and experimental data of the organic dye Rhodamine 110 in aqueous solution for both continuous-wave and pulsed excitation. Importantly, our approach can be readily generalized to most other FCS experiments that determine intensity dependent kinetic parameters. © 2014 Wiley Periodicals, Inc.

Solanko LM, Honigmann A, Midtiby HS, Lund FW, Brewer JR, Dekaris V, Bittman R, Eggeling C, Wüstner D. 2013. Membrane orientation and lateral diffusion of BODIPY-cholesterol as a function of probe structure. Biophys J, 105 (9), pp. 2082-2092. | Show Abstract | Read more

Cholesterol tagged with the BODIPY fluorophore via the central difluoroboron moiety of the dye (B-Chol) is a promising probe for studying intracellular cholesterol dynamics. We synthesized a new BODIPY-cholesterol probe (B-P-Chol) with the fluorophore attached via one of its pyrrole rings to carbon-24 of cholesterol (B-P-Chol). Using two-photon fluorescence polarimetry in giant unilamellar vesicles and in the plasma membrane (PM) of living intact and actin-disrupted cells, we show that the BODIPY-groups in B-Chol and B-P-Chol are oriented perpendicular and almost parallel to the bilayer normal, respectively. B-Chol is in all three membrane systems much stronger oriented than B-P-Chol. Interestingly, we found that the lateral diffusion in the PM was two times slower for B-Chol than for B-P-Chol, although we found no difference in lateral diffusion in model membranes. Stimulated emission depletion microscopy, performed for the first time, to our knowledge, with fluorescent sterols, revealed that the difference in lateral diffusion of the BODIPY-cholesterol probes was not caused by anomalous subdiffusion, because diffusion of both analogs in the PM was free but not hindered. Our combined measurements show that the position and orientation of the BODIPY moiety in cholesterol analogs have a severe influence on lateral diffusion specifically in the PM of living cells.

Cited:

82

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Chmyrov A, Keller J, Grotjohann T, Ratz M, D'Este E, Jakobs S, Eggeling C, Hell SW. 2013. Nanoscopy with more than 100,000 'doughnuts' Nature Methods, 10 (8), pp. 737-740. | Show Abstract | Read more

We show that nanoscopy based on the principle called RESOLFT (reversible saturable optical fluorescence transitions) or nonlinear structured illumination can be effectively parallelized using two incoherently superimposed orthogonal standing light waves. The intensity minima of the resulting pattern act as 'doughnuts', providing isotropic resolution in the focal plane and making pattern rotation redundant. We super-resolved living cells in 120 μm × 100 μm-sized fields of view in <1 s using 116,000 such doughnuts. © 2013 Nature America, Inc. All rights reserved.

Chmyrov A, Keller J, Grotjohann T, Ratz M, d'Este E, Jakobs S, Eggeling C, Hell SW. 2013. Nanoscopy with more than 100,000 'doughnuts'. Nat Methods, 10 (8), pp. 737-740. | Show Abstract | Read more

We show that nanoscopy based on the principle called RESOLFT (reversible saturable optical fluorescence transitions) or nonlinear structured illumination can be effectively parallelized using two incoherently superimposed orthogonal standing light waves. The intensity minima of the resulting pattern act as 'doughnuts', providing isotropic resolution in the focal plane and making pattern rotation redundant. We super-resolved living cells in 120 μm × 100 μm-sized fields of view in <1 s using 116,000 such doughnuts.

Cited:

28

Scopus

Eggeling C, Willig KI, Barrantes FJ. 2013. STED microscopy of living cells - New frontiers in membrane and neurobiology Journal of Neurochemistry, 126 (2), pp. 203-212. | Show Abstract | Read more

Recent developments in fluorescence far-field microscopy such as STED microscopy have accomplished observation of the living cell with a spatial resolution far below the diffraction limit. Here, we briefly review the current approaches to super-resolution optical microscopy and present the implementation of STED microscopy for novel insights into live cell mechanisms, with a focus on neurobiology and plasma membrane dynamics. © 2013 International Society for Neurochemistry.

Cited:

65

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Honigmann A, Van Den Bogaart G, Iraheta E, Risselada HJ, Milovanovic D, Mueller V, Müllar S, Diederichsen U et al. 2013. Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment Nature Structural and Molecular Biology, 20 (6), pp. 679-686. | Show Abstract | Read more

Synaptic-vesicle exocytosis is mediated by the vesicular Ca 2+ sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca 2+ through PIP2. This interaction allows both Ca 2+ -binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca 2+ triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca 2+ influx bringing the vesicle membrane close enough for membrane fusion. © 2013 Nature America, Inc. All rights reserved.

Honigmann A, van den Bogaart G, Iraheta E, Risselada HJ, Milovanovic D, Mueller V, Müllar S, Diederichsen U et al. 2013. Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment. Nat Struct Mol Biol, 20 (6), pp. 679-686. | Show Abstract | Read more

Synaptic-vesicle exocytosis is mediated by the vesicular Ca(2+) sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca(2+) through PIP2. This interaction allows both Ca(2+)-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca(2+) triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca(2+) influx bringing the vesicle membrane close enough for membrane fusion.

Eggeling C, Willig KI, Barrantes FJ. 2013. STED microscopy of living cells--new frontiers in membrane and neurobiology. J Neurochem, 126 (2), pp. 203-212. | Show Abstract | Read more

Recent developments in fluorescence far-field microscopy such as STED microscopy have accomplished observation of the living cell with a spatial resolution far below the diffraction limit. Here, we briefly review the current approaches to super-resolution optical microscopy and present the implementation of STED microscopy for novel insights into live cell mechanisms, with a focus on neurobiology and plasma membrane dynamics.

Lukinavičius G, Umezawa K, Olivier N, Honigmann A, Yang G, Plass T, Mueller V, Reymond L et al. 2013. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat Chem, 5 (2), pp. 132-139. | Show Abstract | Read more

The ideal fluorescent probe for bioimaging is bright, absorbs at long wavelengths and can be implemented flexibly in living cells and in vivo. However, the design of synthetic fluorophores that combine all of these properties has proved to be extremely difficult. Here, we introduce a biocompatible near-infrared silicon-rhodamine probe that can be coupled specifically to proteins using different labelling techniques. Importantly, its high permeability and fluorogenic character permit the imaging of proteins in living cells and tissues, and its brightness and photostability make it ideally suited for live-cell super-resolution microscopy. The excellent spectroscopic properties of the probe combined with its ease of use in live-cell applications make it a powerful new tool for bioimaging.

Chmyrov A, Keller J, Grotjohann T, Eggeling C, Hell SW. 2013. Resolft nanoscopy in living cells at high speed Optical Molecular Probes, Imaging and Drug Delivery, OMP 2013, | Show Abstract

Among various superresolution methods, RESOLFT nanoscopy has advantages in compatibility with live cell imaging via utilization of genetically encoded reversely-switchable fluorescent proteins (rsFPs). Recent developments in rsFPs allow nanoscopy in living cells at high speed. ©Optics in the Life Sciences Congress Technical Digest.

Honigmann A, Mueller V, Hell SW, Eggeling C. 2013. STED microscopy detects and quantifies liquid phase separation in lipid membranes using a new far-red emitting fluorescent phosphoglycerolipid analogue. Faraday Discuss, 161 pp. 77-89. | Show Abstract | Read more

We have developed a bright, photostable, and far-red emitting fluorescent phosphoglycerolipid analogue to probe diffusion characteristics of lipids in membranes. The lipid analogue consists of a saturated (C18) phosphoethanolamine and a hydrophilic far-red emitting fluorescent dye (KK114) that is tethered to the head group by a long polyethylenglycol linker. In contrast to reported far-red emitting fluorescent lipid analogues, this one partitions predominantly into liquid ordered domains of phase-separated ternary bilayers. We performed fluorescence correlation spectroscopy with a super-resolution STED microscope (STED-FCS) to measure the lateral diffusion of the new lipid analogue in the liquid ordered (Lo) and disordered (Ld) phase. On a mica support, we observed micrometer large phases and found that the lipid analogue diffuses freely on all tested spatial scales (40-250 nm) in both the Ld and Lo phase with diffusion coefficients of 1.8 microm2 s(-1) and 0.7 microm2 s(-1) respectively. This indicates that the tight molecular packing of the Lo phase mainly slows down the diffusion rather than causing anomalous sub-diffusion. The same ternary mixture deposited on acid-cleaned glass forms Lo nanodomains of < 40 nm to 300 nm in diameter as only revealed by STED microscopy, which demonstrates the severe influence of interactions with the substrate on the sizes of domains in membranes. When averaging over different positions, STEd-FCS measurements on such glass supported membranes displayed anomalous sub-diffusion. This anomaly can be attributed to a transient partitioning of the lipid analogue into the nano-domains, where diffusion is slowed down. Our results suggest that STED-FCS in combination with a Lo-partitioning fluorescent lipid analogue can directly probe the presence of Lo nano-domains, which in the future should allow the study of potential lipid rafts in live-cell membranes.

Vicidomini G, Schönle A, Ta H, Han KY, Moneron G, Eggeling C, Hell SW. 2013. STED nanoscopy with time-gated detection: theoretical and experimental aspects. PLoS One, 8 (1), pp. e54421. | Show Abstract | Read more

In a stimulated emission depletion (STED) microscope the region in which fluorescence markers can emit spontaneously shrinks with continued STED beam action after a singular excitation event. This fact has been recently used to substantially improve the effective spatial resolution in STED nanoscopy using time-gated detection, pulsed excitation and continuous wave (CW) STED beams. We present a theoretical framework and experimental data that characterize the time evolution of the effective point-spread-function of a STED microscope and illustrate the physical basis, the benefits, and the limitations of time-gated detection both for CW and pulsed STED lasers. While gating hardly improves the effective resolution in the all-pulsed modality, in the CW-STED modality gating strongly suppresses low spatial frequencies in the image. Gated CW-STED nanoscopy is in essence limited (only) by the reduction of the signal that is associated with gating. Time-gated detection also reduces/suppresses the influence of local variations of the fluorescence lifetime on STED microscopy resolution.

Mueller V, Honigmann A, Ringemann C, Medda R, Schwarzmann G, Eggeling C. 2013. FCS in STED microscopy: studying the nanoscale of lipid membrane dynamics. Methods Enzymol, 519 pp. 1-38. | Show Abstract | Read more

Details of molecular membrane dynamics in living cells such as lipid-protein interactions or the incorporation of molecules into lipid "rafts" are often hidden to the observer because of the limited spatial resolution of conventional far-field optical microscopy. Fortunately, the superior spatial resolution of far-field stimulated-emission-depletion (STED) nanoscopy allows gaining new insights. Applying fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes free from anomalous molecular diffusion due to transient binding, as for the diffusion of fluorescent phosphoglycero- and sphingolipid analogs in the plasma membrane of living cells. STED-FCS data recorded at different environmental conditions and on different lipid analogs reveal molecular details of the observed nanoscale trapping. Dependencies on the molecular structure of the lipids point to the distinct connectivity of the various lipids to initiate or assist cellular signaling events, but also outline strong differences to the characteristics of liquid-ordered and disordered phase separation in model membranes. STED-FCS is a highly sensitive and exceptional tool to study the membrane organization by introducing a new class of nanoscale biomolecular studies.

Bally M, Rydell GE, Zahn R, Nasir W, Eggeling C, Breimer ME, Svensson L, Höök F, Larson G. 2012. Norovirus GII.4 virus-like particles recognize galactosylceramides in domains of planar supported lipid bilayers. Angew Chem Int Ed Engl, 51 (48), pp. 12020-12024. | Show Abstract | Read more

A sticky situation: Domain-dependent recognition of the glycosphingolipid galactosylceramide by norovirus-like particles (see picture; red/yellow) is shown using supported lipid bilayers (purple) as model membranes. Optimal ligand presentation is found to promote strong binding to GalCer. This presentation can be found at the edges of the glycosphingolipid-enriched domains (green) and binding is repressed in the absence of these domains. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Testa I, Urban NT, Jakobs S, Eggeling C, Willig KI, Hell SW. 2012. Nanoscopy of living brain slices with low light levels. Neuron, 75 (6), pp. 992-1000. | Show Abstract | Read more

Lens-based fluorescence microscopy, which has long been limited in resolution to about 200 nanometers by diffraction, is rapidly evolving into a nanoscale imaging technique. Here, we show that the superresolution fluorescence microscopy called RESOLFT enables comparatively fast and continuous imaging of sensitive, nanosized features in living brain tissue. Using low-intensity illumination to switch photochromic fluorescent proteins reversibly between a fluorescent and a nonfluorescent state, we increased the resolution more than 3-fold over that of confocal microscopy in all dimensions. Dendritic spines located 10-50 μm deep inside living organotypic hippocampal brain slices were recorded for hours without signs of degradation. Using a fast-switching protein increased the imaging speed 50-fold over reported RESOLFT schemes, which in turn enabled the recording of spontaneous and stimulated changes of dendritic actin filaments and spine morphology occurring on time scales from seconds to hours.

Sezgin E, Levental I, Grzybek M, Schwarzmann G, Mueller V, Honigmann A, Belov VN, Eggeling C, Coskun U, Simons K, Schwille P. 2012. Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes. Biochim Biophys Acta, 1818 (7), pp. 1777-1784. | Show Abstract | Read more

Several simplified membrane models featuring coexisting liquid disordered (Ld) and ordered (Lo) lipid phases have been developed to mimic the heterogeneous organization of cellular membranes, and thus, aid our understanding of the nature and functional role of ordered lipid-protein nanodomains, termed "rafts". In spite of their greatly reduced complexity, quantitative characterization of local lipid environments using model membranes is not trivial, and the parallels that can be drawn to cellular membranes are not always evident. Similarly, various fluorescently labeled lipid analogs have been used to study membrane organization and function in vitro, although the biological activity of these probes in relation to their native counterparts often remains uncharacterized. This is particularly true for raft-preferring lipids ("raft lipids", e.g. sphingolipids and sterols), whose domain preference is a strict function of their molecular architecture, and is thus susceptible to disruption by fluorescence labeling. Here, we analyze the phase partitioning of a multitude of fluorescent raft lipid analogs in synthetic Giant Unilamellar Vesicles (GUVs) and cell-derived Giant Plasma Membrane Vesicles (GPMVs). We observe complex partitioning behavior dependent on label size, polarity, charge and position, lipid headgroup, and membrane composition. Several of the raft lipid analogs partitioned into the ordered phase in GPMVs, in contrast to fully synthetic GUVs, in which most raft lipid analogs mis-partitioned to the disordered phase. This behavior correlates with the greatly enhanced order difference between coexisting phases in the synthetic system. In addition, not only partitioning, but also ligand binding of the lipids is perturbed upon labeling: while cholera toxin B binds unlabeled GM1 in the Lo phase, it binds fluorescently labeled GMI exclusively in the Ld phase. Fluorescence correlation spectroscopy (FCS) by stimulated emission depletion (STED) nanoscopy on intact cellular plasma membranes consistently reveals a constant level of confined diffusion for raft lipid analogs that vary greatly in their partitioning behavior, suggesting different physicochemical bases for these phenomena.

Leutenegger M, Ringemann C, Lasser T, Hell SW, Eggeling C. 2012. Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS). Opt Express, 20 (5), pp. 5243-5263. | Show Abstract | Read more

We characterize a novel fluorescence microscope which combines the high spatial discrimination of a total internal reflection epi-fluorescence (epi-TIRF) microscope with that of stimulated emission depletion (STED) nanoscopy. This combination of high axial confinement and dynamic-active lateral spatial discrimination of the detected fluorescence emission promises imaging and spectroscopy of the structure and function of cell membranes at the macro-molecular scale. Following a full theoretical description of the sampling volume and the recording of images of fluorescent beads, we exemplify the performance and limitations of the TIRF-STED nanoscope with particular attention to the polarization state of the laser excitation light. We demonstrate fluorescence correlation spectroscopy (FCS) with the TIRF-STED nanoscope by observing the diffusion of dye molecules in aqueous solutions and of fluorescent lipid analogs in supported lipid bilayers in the presence of background signal. The nanoscope reduced the out-of-focus background signal. A lateral resolution down to 40-50 nm was attained which was ultimately limited by the low lateral signal-to-background ratio inherent to the confocal epi-TIRF scheme. Together with the estimated axial confinement of about 55 nm, our TIRF-STED nanoscope achieved an almost isotropic and less than 1 attoliter small all-optically induced measurement volume.

Vicidomini G, Moneron G, Eggeling C, Rittweger E, Hell SW. 2012. STED with wavelengths closer to the emission maximum. Opt Express, 20 (5), pp. 5225-5236. | Show Abstract | Read more

In stimulated emission depletion (STED) nanoscopy the wavelength of the STED beam is usually tuned towards the red tail of the emission maximum of the fluorophore. Shifting the STED wavelength closer to the emission peak, i.e. towards the blue region, favorably increases the stimulated emission cross-section. However, this blue-shifting also increases the probability to excite fluorophores that have remained in their ground state, compromising the image contrast. Here we present a method to exploit the higher STED efficiency of blue-shifted STED beams while maintaining the contrast in the image. The method is exemplified by imaging immunolabeled features in mammalian cells with an up to 3-fold increased STED efficiency compared to that encountered in standard STED nanoscopy implementations.

Han KY, Wildanger D, Rittweger E, Meijer J, Pezzagna S, Hell SW, Eggeling C. 2012. Dark state photophysics of nitrogen–vacancy centres in diamond New Journal of Physics, 14 (12), pp. 123002-123002. | Show Abstract | Read more

Nitrogen-vacancy (NV) colour centres in diamond are attractive fluorescence emitters owing to their unprecedented photostability and superior applicability to spin manipulation and sub-diffraction far-field optical microscopy. However, some applications are limited by the co-occurrence of dark state population and optical excitation. In this paper, we use fluorescence microscopy and correlation spectroscopy on single negatively charged NV centres in type IIa bulk diamond to unravel the population kinetics of a >100 s long-lived dark state. The bright-dark state interconversion rates show a quadratic dependence on the applied laser intensity, which implies that higher excited states are involved. Depopulation of the dark state becomes less effective at wavelengths above 532 nm, resulting in a complete fluorescence switch-off at wavelengths >600 nm. This switch is reversible by the addition of shorter wavelengths. This behaviour can be explained by a model consisting of three dark and three bright states of different excitation levels, with the most efficient interconversion via the respective higher excited states. This model accounts for the nonlinear dark state and photoswitching kinetics, as well as for the decrease of the NV's fluorescence lifetime with excitation intensity and the strong dependence of fluorescence emission on excitation intensity. Unfortunately, our data do not give enough insight to allow us to assign the different states to specific electronic states known from the literature. Nevertheless, our observations allowed us to improve the recording of fluorescence images of single NV centres with sub-diffraction spatial resolution but they also have important implications for studying their spin states. © IOP Publishing Ltd and Deutsche Physikalische Gesellschaft.

Grotjohann T, Testa I, Reuss M, Brakemann T, Eggeling C, Hell SW, Jakobs S. 2012. rsEGFP2 enables fast RESOLFT nanoscopy of living cells. Elife, 1 (1), pp. e00248. | Show Abstract | Read more

The super-resolution microscopy called RESOLFT relying on fluorophore switching between longlived states, stands out by its coordinate-targeted sequential sample interrogation using low light levels. While RESOLFT has been shown to discern nanostructures in living cells, the reversibly photoswitchable green fluorescent protein (rsEGFP) employed in these experiments was switched rather slowly and recording lasted tens of minutes. We now report on the generation of rsEGFP2 providing faster switching and the use of this protein to demonstrate 25-250 times faster recordings.DOI:http://dx.doi.org/10.7554/eLife.00248.001.

Honigmann A, Eggeling C, Schulze M, Lepert A. 2012. Super-resolution STED microscopy advances with yellow CW OPSL LASER FOCUS WORLD, 48 (1), pp. 75-79. | Show Abstract

Researchers have a growing need to push optical microscopy beyond the diffraction limit to answer key questions in biology, and stimulated emission depletion (STED) has proven to be a fluorescence imaging technique that can accomplish this goal. Biologists are currently seeking to connect molecular behavior to macroscopic behavior, determining how cells signal with each other, and how signaling at the cellular/organism level is then relayed back to DNA/RNA level control to regulate single genes. A STED nanoscope uses two laser beams. The first is the excitation laser, which as in confocal microscopy is usually focused to a near-diffraction-limited spot within a fluorescently labeled sample. The excitation wavelength of this laser is chosen to match the absorption peak of the target fluorophore. When applying a high enough STED laser power above a certain threshold, all the excited fluorophores in the path of the STED beam emit at the STED wavelength making them unavailable for fluorescence.

Grotjohann T, Testa I, Leutenegger M, Bock H, Urban NT, Lavoie-Cardinal F, Willig KI, Eggeling C, Jakobs S, Hell SW. 2011. Diffraction-unlimited all-optical imaging and writing with a photochromic GFP. Nature, 478 (7368), pp. 204-208. | Show Abstract | Read more

Lens-based optical microscopy failed to discern fluorescent features closer than 200 nm for decades, but the recent breaking of the diffraction resolution barrier by sequentially switching the fluorescence capability of adjacent features on and off is making nanoscale imaging routine. Reported fluorescence nanoscopy variants switch these features either with intense beams at defined positions or randomly, molecule by molecule. Here we demonstrate an optical nanoscopy that records raw data images from living cells and tissues with low levels of light. This advance has been facilitated by the generation of reversibly switchable enhanced green fluorescent protein (rsEGFP), a fluorescent protein that can be reversibly photoswitched more than a thousand times. Distributions of functional rsEGFP-fusion proteins in living bacteria and mammalian cells are imaged at <40-nanometre resolution. Dendritic spines in living brain slices are super-resolved with about a million times lower light intensities than before. The reversible switching also enables all-optical writing of features with subdiffraction size and spacings, which can be used for data storage.

Mueller V, Ringemann C, Honigmann A, Schwarzmann G, Medda R, Leutenegger M, Polyakova S, Belov VN, Hell SW, Eggeling C. 2011. STED nanoscopy reveals molecular details of cholesterol- and cytoskeleton-modulated lipid interactions in living cells. Biophys J, 101 (7), pp. 1651-1660. | Show Abstract | Read more

Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeleton-dependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol- and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes.

Vicidomini G, Moneron G, Han KY, Westphal V, Ta H, Reuss M, Engelhardt J, Eggeling C, Hell SW. 2011. Sharper low-power STED nanoscopy by time gating. Nat Methods, 8 (7), pp. 571-573. | Show Abstract | Read more

Applying pulsed excitation together with time-gated detection improves the fluorescence on-off contrast in continuous-wave stimulated emission depletion (CW-STED) microscopy, thus revealing finer details in fixed and living cells using moderate light intensities. This method also enables super-resolution fluorescence correlation spectroscopy with CW-STED beams, as demonstrated by quantifying the dynamics of labeled lipid molecules in the plasma membrane of living cells.

Brakemann T, Stiel AC, Weber G, Andresen M, Testa I, Grotjohann T, Leutenegger M, Plessmann U et al. 2011. A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching. Nat Biotechnol, 29 (10), pp. 942-947. | Show Abstract | Read more

Photoswitchable fluorescent proteins have enabled new approaches for imaging cells, but their utility has been limited either because they cannot be switched repeatedly or because the wavelengths for switching and fluorescence imaging are strictly coupled. We report a bright, monomeric, reversibly photoswitchable variant of GFP, Dreiklang, whose fluorescence excitation spectrum is decoupled from that for optical switching. Reversible on-and-off switching in living cells is accomplished at illumination wavelengths of ∼365 nm and ∼405 nm, respectively, whereas fluorescence is elicited at ∼515 nm. Mass spectrometry and high-resolution crystallographic analysis of the same protein crystal in the photoswitched on- and off-states demonstrate that switching is based on a reversible hydration/dehydration reaction that modifies the chromophore. The switching properties of Dreiklang enable far-field fluorescence nanoscopy in living mammalian cells using both a coordinate-targeted and a stochastic single molecule switching approach.

Leutenegger M, Eggeling C, Hell SW. 2010. Analytical description of STED microscopy performance. Opt Express, 18 (25), pp. 26417-26429. | Show Abstract | Read more

Stimulated emission depletion (STED) resolves fluorescent features that are closer than the far-field optical diffraction limit by applying a spatially modulated light field keeping all but one of these features dark consecutively. For estimating the efficiency of transient fluorophore darkening, we developed analytical equations considering the spatio-temporal intensity profile of the STED beam. These equations provide a quick analysis and optimization of the resolution and contrast to be gained under various conditions, such as continuous wave or pulsed STED beams having different pulse durations. Particular emphasis is placed on fluorescence fluctuation methods such as correlation spectroscopy (FCS) using STED.

Testa I, Wurm CA, Medda R, Rothermel E, von Middendorf C, Fölling J, Jakobs S, Schönle A, Hell SW, Eggeling C. 2010. Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength. Biophys J, 99 (8), pp. 2686-2694. | Show Abstract | Read more

Current far-field fluorescence nanoscopes provide subdiffraction resolution by exploiting a mechanism of fluorescence inhibition. This mechanism is implemented such that features closer than the diffraction limit emit separately when simultaneously exposed to excitation light. A basic mechanism for such transient fluorescence inhibition is the depletion of the fluorophore ground state by transferring it (via a triplet) in a dark state, a mechanism which is workable in most standard dyes. Here we show that microscopy based on ground state depletion followed by individual molecule return (GSDIM) can effectively provide multicolor diffraction-unlimited resolution imaging of immunolabeled fixed and SNAP-tag labeled living cells. Implemented with standard labeling techniques, GSDIM is demonstrated to separate up to four different conventional fluorophores using just two detection channels and a single laser line. The method can be expanded to even more colors by choosing optimized dichroic mirrors and selecting marker molecules with negligible inhomogeneous emission broadening.

Bierwagen J, Testa I, Fölling J, Wenzel D, Jakobs S, Eggeling C, Hell SW. 2010. Far-field autofluorescence nanoscopy. Nano Lett, 10 (10), pp. 4249-4252. | Show Abstract | Read more

We demonstrate far-field optical imaging at the nanoscale with unlabeled samples. Subdiffraction resolution images of autofluorescent samples are obtained by depleting the ground state of natural fluorophores by transferring them to a metastable dark state and simultaneously localizing those fluorophores that are transiently returning. Our approach is based on the insight that nanoscopy methods relying on stochastic single-molecule switching require only a single fluorescence on-off cycle to yield an image, a condition fulfilled by various biomolecules. The method is exemplified by recording label-free nanoscopy images of thylakoid membranes of spinach chloroplasts.

Han KY, Kim SK, Eggeling C, Hell SW. 2010. Metastable dark States enable ground state depletion microscopy of nitrogen vacancy centers in diamond with diffraction-unlimited resolution. Nano Lett, 10 (8), pp. 3199-3203. | Show Abstract | Read more

Current far-field optical nanoscopy schemes overcome the diffraction barrier by ensuring that adjacent features assume different states upon detection. Ideally, the transition between these states can be repeated endlessly and, if performed optically, with low levels of light. Here we report such optical switching, realized by pairing the luminescent triplet and a long-lived dark state of diamond color centers, enabling their imaging with a resolution >10 times beyond the diffraction barrier (<20 nm).

Honigmann A, Walter C, Erdmann F, Eggeling C, Wagner R. 2010. Characterization of horizontal lipid bilayers as a model system to study lipid phase separation. Biophys J, 98 (12), pp. 2886-2894. | Show Abstract | Read more

Artificial lipid membranes are widely used as a model system to study single ion channel activity using electrophysiological techniques. In this study, we characterize the properties of the artificial bilayer system with respect to its dynamics of lipid phase separation using single-molecule fluorescence fluctuation and electrophysiological techniques. We determined the rotational motions of fluorescently labeled lipids on the nanosecond timescale using confocal time-resolved anisotropy to probe the microscopic viscosity of the membrane. Simultaneously, long-range mobility was investigated by the lateral diffusion of the lipids using fluorescence correlation spectroscopy. Depending on the solvent used for membrane preparation, lateral diffusion coefficients in the range D(lat) = 10-25 mum(2)/s and rotational diffusion coefficients ranging from D(rot) = 2.8 - 1.4 x 10(7) s(-1) were measured in pure liquid-disordered (L(d)) membranes. In ternary mixtures containing saturated and unsaturated phospholipids and cholesterol, liquid-ordered (L(o)) domains segregated from the L(d) phase at 23 degrees C. The lateral mobility of lipids in L(o) domains was around eightfold lower compared to those in the L(d) phase, whereas the rotational mobility decreased by a factor of 1.5. Burst-integrated steady-state anisotropy histograms, as well as anisotropy imaging, were used to visualize the rotational mobility of lipid probes in phase-separated bilayers. These experiments and fluorescence correlation spectroscopy measurements at different focal diameters indicated a heterogeneous microenvironment in the L(o) phase. Finally, we demonstrate the potential of the optoelectro setup to study the influence of lipid domains on the electrophysiological properties of ion channels. We found that the electrophysiological activity of gramicidin A (gA), a well-characterized ion-channel-forming peptide, was related to lipid-domain partitioning. During liquid-liquid phase separation, gA was largely excluded from L(o) domains. Simultaneously, the number of electrically active gA dimers increased due to the increased surface density of gA in the L(d) phase.

Brakemann T, Weber G, Andresen M, Groenhof G, Stiel AC, Trowitzsch S, Eggeling C, Grubmüller H, Hell SW, Wahl MC, Jakobs S. 2010. Molecular basis of the light-driven switching of the photochromic fluorescent protein Padron. J Biol Chem, 285 (19), pp. 14603-14609. | Show Abstract | Read more

Reversibly switchable fluorescent proteins can be repeatedly photoswitched between a fluorescent and a nonfluorescent state by irradiation with the light of two different wavelengths. The molecular basis of the switching process remains a controversial topic. Padron0.9 is a reversibly switchable fluorescent protein with "positive" switching characteristics, exhibiting excellent spectroscopic properties. Its chromophore is formed by the amino acids Cys-Tyr-Gly. We obtained high resolution x-ray structures of Padron0.9 in both the fluorescent and the nonfluorescent states and used the structural information for molecular dynamics simulations. We found that in Padron0.9 the chromophore undergoes a cis-trans isomerization upon photoswitching. The molecular dynamics simulations clarified the protonation states of the amino acid residues within the chromophore pocket that influence the protonation state of the chromophore. We conclude that a light driven cis-trans isomerization of the chromophore appears to be the fundamental switching mechanism in all photochromic fluorescent proteins known to date. Distinct absorption cross-sections for the switching wavelengths in the fluorescent and the nonfluorescent state are not essential for efficient photochromism in fluorescent proteins, although they may facilitate the switching process.

Sahl SJ, Leutenegger M, Hilbert M, Hell SW, Eggeling C. 2010. Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids. Proc Natl Acad Sci U S A, 107 (15), pp. 6829-6834. | Show Abstract | Read more

We describe an optical method capable of tracking a single fluorescent molecule with a flexible choice of high spatial accuracy (approximately 10-20 nm standard deviation or approximately 20-40 nm full-width-at-half-maximum) and temporal resolution (< 1 ms). The fluorescence signal during individual passages of fluorescent molecules through a spot of excitation light allows the sequential localization and thus spatio-temporal tracking of the molecule if its fluorescence is collected on at least three separate point detectors arranged in close proximity. We show two-dimensional trajectories of individual, small organic dye labeled lipids diffusing in the plasma membrane of living cells and directly observe transient events of trapping on < 20 nm spatial scales. The trapping is cholesterol-assisted and much more pronounced for a sphingo- than for a phosphoglycero-lipid, with average trapping times of approximately 15 ms and < 4 ms, respectively. The results support previous STED nanoscopy measurements and suggest that, at least for nontreated cells, the transient interaction of a single lipid is confined to macromolecular dimensions. Our experimental approach demonstrates that fast molecular movements can be tracked with minimal invasion, which can reveal new important details of cellular nano-organization.

Mitronova GY, Belov VN, Bossi ML, Wurm CA, Meyer L, Medda R, Moneron G, Bretschneider S, Eggeling C, Jakobs S, Hell SW. 2010. New fluorinated rhodamines for optical microscopy and nanoscopy. Chemistry, 16 (15), pp. 4477-4488. | Show Abstract | Read more

New photostable rhodamine dyes represented by the compounds 1 a-r and 3-5 are proposed as efficient fluorescent markers with unique combination of structural features. Unlike rhodamines with monoalkylated nitrogen atoms, N',N-bis(2,2,2-trifluoroethyl) derivatives 1 e, 1 i, 1 j, 3-H and 5 were found to undergo sulfonation of the xanthene fragment at the positions 4' and 5'. Two fluorine atoms were introduced into the positions 2' and 7' of the 3',6'-diaminoxanthene fragment in compounds 1 a-d, 1 i-l and 1 m-r. The new rhodamine dyes may be excited with λ=488 or 514 nm light; most of them emit light at λ=512-554 nm (compounds 1 q and 1r at λ=576 and 589 nm in methanol, respectively) and have high fluorescence quantum yields in solution (up to 98 %), relatively long excited-state lifetimes (>3 ns) and are resistant against photobleaching, especially at high laser intensities, as is usually applied in confocal microscopy. Sulfonation of the xanthene fragment with 30 % SO3 in H2SO4 is compatible with the secondary amide bond (rhodamine-CON(Me)CH2CH2COOH) formed with MeNHCH2CH2COOCH3 to providing the sterically unhindered carboxylic group required for further (bio)conjugation reactions. After creating the amino reactive sites, the modified derivatives may be used as fluorescent markers and labels for (bio)molecules in optical microscopy and nanoscopy with very-high light intensities. Further, the new rhodamine dyes are able to pass the plasma membrane of living cells, introducing them as potential labels for recent live-cell-tag approaches. We exemplify the excellent performance of the fluorinated rhodamines in optical microscopy by fluorescence correlation spectroscopy (FCS) and stimulated emission depletion (STED) nanoscopy experiments.

Kolmakov K, Belov VN, Bierwagen J, Ringemann C, Müller V, Eggeling C, Hell SW. 2010. Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy. Chemistry, 16 (1), pp. 158-166. | Show Abstract | Read more

Fluorescent markers emitting in the red are extremely valuable in biological microscopy since they minimize cellular autofluorescence and increase flexibility in multicolor experiments. Novel rhodamine dyes excitable with 630 nm laser light and emitting at around 660 nm have been developed. The new rhodamines are very photostable and have high fluorescence quantum yields of up to 80 %, long excited state lifetimes of 3.4 ns, and comparatively low intersystem-crossing rates. They perform very well both in conventional and in subdiffraction-resolution microscopy such as STED (stimulated emission depletion) and GSDIM (ground-state depletion with individual molecular return), as well as in single-molecule-based experiments such as fluorescence correlation spectroscopy (FCS). Syntheses of lipophilic and hydrophilic derivatives starting from the same chromophore-containing scaffold are described. Introduction of two sulfo groups provides high solubility in water and a considerable rise in fluorescence quantum yield. The attachment of amino or thiol reactive groups allows the dyes to be used as fluorescent markers in biology. Dyes deuterated at certain positions have narrow and symmetrical molecular mass distribution patterns, and are proposed as new tags in MS or LC-MS for identification and quantification of various substance classes (e.g., amines and thiols) in complex mixtures. High-resolution GSDIM images and live-cell STED-FCS experiments on labeled microtubules and lipids prove the versatility of the novel probes for modern fluorescence microscopy and nanoscopy.

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Kolmakov K, Belov VN, Wurm CA, Harke B, Leutenegger M, Eggeling C, Hell SW. 2010. A Versatile Route to Red-Emitting Carbopyronine Dyes for Optical Microscopy and Nanoscopy EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, 2010 (19), pp. 3593-3610. | Show Abstract | Read more

Biological microscopy favors photostable fluorescent markers with large fluorescence quantum yields, low dark triplet state population, good biocompatibility and absorption and emission maxima in the near-infrared, where cellular autofluorescence is minimized. In the present study, carbopyronines absorbing around 640 nm and emitting at around 660 nm, with a low intersystem crossing rate (kisc ≈ 0.5×106 s-1) and excellent properties for cellular imaging were synthesized. A general synthetic route to carbopyronines with functional groups variable in the final steps of the synthesis or in the resulting fluorescent dye is presented. Possessing two 2-me-thoxyethyl groups, the parent dye is soluble in water and most organic solvents. Demethylation of the dye or its precursors is straightforward, clean, and furnishes compounds with one or two 2-hydroxyethyl groups, which can be used for further transformations. Modifications in the linker-containing carboxy group are also possible. A multistep synthesis of the dye starting from a simple precursor and utilizing a single temporary protective group is described. The presented approach may be further applied to the design of caged carbopyronines. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Ringemann C, Harke B, von Middendorff C, Medda R, Honigmann A, Wagner R, Leutenegger M, Schoenle A, Hell SW, Eggeling C. 2009. Exploring single-molecule dynamics with fluorescence nanoscopy NEW JOURNAL OF PHYSICS, 11 (10), pp. 103054-103054. | Read more

Han KY, Willig KI, Rittweger E, Jelezko F, Eggeling C, Hell SW. 2009. Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light. Nano Lett, 9 (9), pp. 3323-3329. | Show Abstract | Read more

Charged nitrogen-vacancy (NV) color centers in diamond are excellent luminescence sources for far-field fluorescence nanoscopy by stimulated emission depletion (STED). Here we show that these photostable color centers can be visualized by STED using simple continuous-wave or high repetition pulsed lasers (76 MHz) at wavelengths >700 nm for STED. Furthermore, we show that NV centers can be imaged in three dimensions (3D) inside the diamond crystal and present single-photon signatures of single color centers recorded in high density samples, demonstrating a new recording scheme for STED and related far-field nanoscopy approaches. Finally, we exemplify the potential of using nanodiamonds containing NV centers as luminescence tags in STED microscopy. Our results offer new experimental avenues in nanooptics, nanotechnology, and the life sciences.

Eggeling C, Ringemann C, Mueller V, Medda R, Schwarzmann G, Sandhoff K, Hein B, Schoenle A, Hell SW. 2009. Studying nanoscale membrane dynamics by sub-diffraction far-field fluorescence STED microscopy ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 238

Donnert G, Eggeling C, Hell SW. 2009. Triplet-relaxation microscopy with bunched pulsed excitation. Photochem Photobiol Sci, 8 (4), pp. 481-485. | Show Abstract | Read more

Obtaining high signal levels in fluorescence microscopy is usually spoiled by the concomitant population of the dark (triplet) state of the marker, which is often followed by photobleaching. Recently, we introduced the triplet relaxation (T-Rex) modality in fluorescence microscopy which led to a major increase in total signal and dye photostability. The idea behind T-Rex is to avoid the illumination of fluorophores in the triplet state, e.g. by using pulsed excitation with interpulse time distances that are long enough for the triplet state to relax between two pulses. While our previous implementation came at the expense of extended recording, here we investigate pulsed excitation patterns for T-Rex illumination implying shorter total recording times. In particular, we balance signal enhancement and imaging speed by exciting with bunches of quickly succeeding pulses that are separated by dark periods for triplet relaxation. Taking the green fluorescent protein and the organic dye Atto532 as examples, we observe the dependence of photobleaching and total fluorescence gain on the number of pulses within a bunch. Reaching almost T-Rex conditions this excitation scheme mimics fast scanning of the illumination beam and has the potential to improve a whole range of analytical tools that suffer from photobleaching and low signal levels.

Eggeling C, Ringemann C, Medda R, Schwarzmann G, Sandhoff K, Polyakova S, Belov VN, Hein B, von Middendorff C, Schönle A, Hell SW. 2009. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature, 457 (7233), pp. 1159-1162. | Show Abstract | Read more

Cholesterol-mediated lipid interactions are thought to have a functional role in many membrane-associated processes such as signalling events. Although several experiments indicate their existence, lipid nanodomains ('rafts') remain controversial owing to the lack of suitable detection techniques in living cells. The controversy is reflected in their putative size of 5-200 nm, spanning the range between the extent of a protein complex and the resolution limit of optical microscopy. Here we demonstrate the ability of stimulated emission depletion (STED) far-field fluorescence nanoscopy to detect single diffusing (lipid) molecules in nanosized areas in the plasma membrane of living cells. Tuning of the probed area to spot sizes approximately 70-fold below the diffraction barrier reveals that unlike phosphoglycerolipids, sphingolipids and glycosylphosphatidylinositol-anchored proteins are transiently ( approximately 10-20 ms) trapped in cholesterol-mediated molecular complexes dwelling within <20-nm diameter areas. The non-invasive optical recording of molecular time traces and fluctuation data in tunable nanoscale domains is a powerful new approach to study the dynamics of biomolecules in living cells.

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Ringemann C, Harke B, Von Middendorff C, Medda R, Honigmann A, Wagner R, Leutenegger M, Schönle A, Hell SW, Eggeling C. 2009. Exploring single-molecule dynamics with fluorescence nanoscopy New Journal of Physics, 11 | Show Abstract | Read more

The study of molecular dynamics at the single-molecule level with fluorescence correlation spectroscopy (FCS) and far-field optics has contributed greatly to the functional understanding of complex systems. Unfortunately, such studies are restricted to length scales of >200 nm because diffraction does not allow further reduction of the measurement volume. This sets an upper limit on the applicable concentration of fluorescently labeled molecules and even more importantly, averages out details of nanoscale dynamics. By combining FCS and fluorescence intensity distribution analysis (FIDA) with sub-diffraction- resolution stimulated emission depletion (STED) nanoscopy, we remove this restriction and obtain open measurement volumes of nanoscale dimensions which are tunable in size. As a consequence, single-molecule studies can now be extended to nanoscale dynamics and may be applied to much larger, often endogenous concentrations. In solution, low-brightness signal from axial out-of-focus volume shells was taken into account by using both FCS and FIDA in conjunction to analyze the data. In two-dimensional systems, such as lipid membranes, the background is greatly reduced and measurements feature excellent signal-to-noise ratios. Measurement foci of down to 30 nm in diameter directly reveal anomalous diffusion of lipids in the plasma membrane of living cells and allow for the determination of on/off rates of the binding of lipids to other membrane constituents. Such important insight into the prominent biological question of lipid membrane organization or 'lipid rafts' shows that combining fluctuation analysis with STED-engineered ultra-small measurement volumes is a viable and powerful new approach to probing molecular dynamics on the nanoscale. © IOP Publishing Ltd and Deutsche Physikalische Gesellschaft.

Rittweger E, Han KY, Irvine SE, Wildanger JD, Eggeling C, Hell SW. 2009. Far-field optical nanoscopy of diamond color centers CLEO/Europe - EQEC 2009 - European Conference on Lasers and Electro-Optics and the European Quantum Electronics Conference, | Read more

Polyakova SM, Belov VN, Yan SF, Eggeling C, Ringemann C, Schwarzmann G, de Meijere A, Hell SW. 2009. New GM1 Ganglioside Derivatives for Selective Single and Double Labelling of the Natural Glycosphingolipid Skeleton EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, 2009 (30), pp. 5162-5177. | Show Abstract | Read more

Selective single and double labelling of the natural ganglioside GM1. enables one to introduce various markers into different parts of the glycosphingolipid molecule without changing the natural skeleton. To that end, N-Fmoc-2amino-, N-Fmoc-18-amino- and S-(ethoxythiocarbonyl)-18mercaptostearic acids have been prepared, and. coupled with the primary amino group in the sphingosine part of lyso-GM1 and. deAc-deAcyl-GM1. gangliosides. The products of these coupling reactions - building blocks 16a, 16b, 16c, 26 and 27 - may be used for the synthesis of GM1 derivatives with one or two fluorescent dye moieties or other labels of various polarities. Examples of various labelling strategies, using hydrophilic and lipophilic photostable fluorescent dyes, have been made available. The GM1. derivatives 17a, 22a and 23c labelled with the fluorescent dye ATTO 647N or the doubly labelled derivative 25b can be used as probes in fluorescence correlation spectroscopy (in conventional, microscopy or stimulated emission depletion nanoscopy) to study the diffusion of lipid analogues in model or live cell membranes. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.

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Rittweger E, Han KY, Irvine SE, Eggeling C, Hell SW. 2009. STED microscopy reveals crystal colour centres with nanometric resolution NATURE PHOTONICS, 3 (3), pp. 144-147. | Show Abstract | Read more

Because they have spin states that can be optically polarized and detected, fluorescent nitrogen vacancies in diamond have considerable potential for applications in quantum cryptography and computation, as well as for nanoscale magnetic imaging and biolabelling. However, their optical detection and control are hampered by the diffraction resolution barrier of far-field optics. Here, we show that stimulated emission depletion (STED) microscopy is capable of imaging nitrogen-vacancy centres with nanoscale resolution and ngström precision using focused light. The far-field optical control of the population of their excited state at the nanoscale expands the versatility of these centres and demonstrates the suitability of STED microscopy to image dense colour centres in crystals. Nitrogen-vacancy defects show great potential as tags for far-field optical nanoscopy because they exhibit nearly ideal STED without bleaching. Measured point-spread functions of 5.8nm in width demonstrate an all-physics-based far-field optical resolving power exceeding the wavelength of light by two orders of magnitude.

Testa I, Schönle A, von Middendorff C, Geisler C, Medda R, Wurm CA, Stiel AC, Jakobs S et al. 2008. Nanoscale separation of molecular species based on their rotational mobility. Opt Express, 16 (25), pp. 21093-21104. | Show Abstract | Read more

We combine far-field fluorescence nanoscopy through serialized recording of switchable emitters with polarization-sensitive fluorescence detection. In addition to imaging with nanoscale spatial resolution, this technique allows determination of the fluorescence anisotropy of each detected dipole emitter and thus an estimate of its rotational mobility. Sub-populations of fluorescent markers can thus be separated based on their interaction with the sample. We applied this new functional nanoscopy to imaging of living mammalian cells.

Fölling J, Bossi M, Bock H, Medda R, Wurm CA, Hein B, Jakobs S, Eggeling C, Hell SW. 2008. Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods, 5 (11), pp. 943-945. | Show Abstract | Read more

We introduce far-field fluorescence nanoscopy with ordinary fluorophores based on switching the majority of them to a metastable dark state, such as the triplet, and calculating the position of those left or those that spontaneously returned to the ground state. Continuous widefield illumination by a single laser and a continuously operating camera yielded dual-color images of rhodamine- and fluorescent protein-labeled (living) samples, proving a simple yet powerful super-resolution approach.

Stiel AC, Andresen M, Bock H, Hilbert M, Schilde J, Schönle A, Eggeling C, Egner A, Hell SW, Jakobs S. 2008. Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy. Biophys J, 95 (6), pp. 2989-2997. | Show Abstract | Read more

Reversibly switchable fluorescent proteins (RSFPs) are GFP-like proteins that may be repeatedly switched by irradiation with light from a fluorescent to a nonfluorescent state, and vice versa. They can be utilized as genetically encodable probes and bear large potential for a wide array of applications, in particular for new protein tracking schemes and subdiffraction resolution microscopy. However, the currently described monomeric RSFPs emit only blue-green or green fluorescence; the spectral window for their use is thus rather limited. Using a semirational engineering approach based on the crystal structure of the monomeric nonswitchable red fluorescent protein mCherry, we generated rsCherry and rsCherryRev. These two novel red fluorescent RSFPs exhibit fluorescence emission maxima at approximately 610 nm. They display antagonistic switching modes, i.e., in rsCherry irradiation with yellow light induces the off-to-on transition and blue light the on-to-off transition, whereas in rsCherryRev the effects of the switching wavelengths are reversed. We demonstrate time-lapse live-cell subdiffraction microscopy by imaging rsCherryRev targeted to the endoplasmic reticulum utilizing the switching and localization of single molecules.

Andresen M, Stiel AC, Fölling J, Wenzel D, Schönle A, Egner A, Eggeling C, Hell SW, Jakobs S. 2008. Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy. Nat Biotechnol, 26 (9), pp. 1035-1040. | Show Abstract | Read more

Fluorescent proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state are important for innovative microscopy schemes, such as protein tracking, fluorescence resonance energy transfer imaging, sub-diffraction resolution microscopy and others. However, all available monomeric reversibly switchable fluorescent proteins (RSFPs) have similar properties and switching characteristics, thereby limiting their use. Here, we introduce two bright green fluorescent RSFPs, bsDronpa and Padron, generated by extensive mutagenesis of the RSFP Dronpa, with unique absorption and switching characteristics. Whereas bsDronpa features a broad absorption spectrum extending into the UV, Padron displays a switching behavior that is reversed to that of all green fluorescent RSFPs known to date. These two RSFPs enable live-cell fluorescence microscopy with multiple labels using a single detection color, because they can be distinguished by photoswitching. Furthermore, we demonstrate dual-color fluorescence microscopy with sub-diffraction resolution using bsDronpa and Dronpa whose emission maxima are separated by <20 nm.

Bossi M, Fölling J, Belov VN, Boyarskiy VP, Medda R, Egner A, Eggeling C, Schönle A, Hell SW. 2008. Multicolor far-field fluorescence nanoscopy through isolated detection of distinct molecular species. Nano Lett, 8 (8), pp. 2463-2468. | Show Abstract | Read more

By combining the photoswitching and localization of individual fluorophores with spectroscopy on the single molecule level, we demonstrate simultaneous multicolor imaging with low crosstalk and down to 15 nm spatial resolution using only two detection color channels. The applicability of the method to biological specimens is demonstrated on mammalian cells. The combination of far-field fluorescence nanoscopy with the recording of a single switchable molecular species at a time opens up a new class of functional imaging techniques.

Ringemann C, Schönle A, Giske A, von Middendorff C, Hell SW, Eggeling C. 2008. Enhancing fluorescence brightness: effect of reverse intersystem crossing studied by fluorescence fluctuation spectroscopy. Chemphyschem, 9 (4), pp. 612-624. | Show Abstract | Read more

Experiments based on fluorescence detection are limited by the population of the fluorescence marker's long-lived dark triplet state, leading to pronounced photobleaching reactions and blinking which reduces the average fluorescence signal obtained per time interval. By irradiation with a second, red-shifted laser line, we initiate reverse intersystem crossing (ReISC) which enhances the fluorescence signal of common fluorophores up to a factor of 14. The reverse intersystem crossing from the triplet state back to the singlet system is achieved by photoexcitation to higher-excited triplet states, which are, however, prone to photobleaching. We gain insights into the competing pathways of ReISC and photobleaching. The relative efficiencies of these two pathways and the triplet lifetime determine the achievable fluorescence enhancement, which varies strongly with the choice of dye, excitation irradiance and wavelength, and with environmental conditions. The study of ReISC not only results in a better understanding of a fluorescent label's photophysics, but the method is a possible approach to optimize fluorescence emission in experiments, where signal strength is a critical parameter.

Fölling J, Belov V, Riedel D, Schönle A, Egner A, Eggeling C, Bossi M, Hell SW. 2008. Fluorescence nanoscopy with optical sectioning by two-photon induced molecular switching using continuous-wave lasers. Chemphyschem, 9 (2), pp. 321-326. | Show Abstract | Read more

During the last decade far-field fluorescence microscopy methods have evolved that have resolution far below the wavelength of light. To outperform the limiting role of diffraction, all these methods, in one way or another, switch the ability of a molecule to emit fluorescence. Here we present a novel rhodamine amide that can be photoswitched from a nonfluorescent to a fluorescent state by absorption of one or two photons from a continuous-wave laser beam. This bright marker enables strict control of on/off switching and provides single-molecule localization precision down to 15 nm in the focal plane. Two-photon induced nonlinear photoswitching of this marker with continuous-wave illumination offers optical sectioning with simple laser equipment. Future synthesis of similar compounds holds great promise for cost-effective fluorescence nanoscopy with noninvasive optical sectioning.

Eggeling C, Hilbert M, Bock H, Ringemann C, Hofmann M, Stiel AC, Andresen M, Jakobs S, Egner A, Schönle A, Hell SW. 2007. Reversible photoswitching enables single-molecule fluorescence fluctuation spectroscopy at high molecular concentration. Microsc Res Tech, 70 (12), pp. 1003-1009. | Show Abstract | Read more

We demonstrate that photoswitchable markers enable fluorescence fluctuation spectroscopy at high molecular concentration. Reversible photoswitching allows precise control of the density of fluorescing entities, because the equilibrium between the fluorescent ON- and the dark OFF-state can be shifted through optical irradiation at a specific wavelength. Depending on the irradiation intensity, the concentration of the ON-state markers can be up to 1,000 times lower than the actual concentration of the labeled molecular entity. Photoswitching expands the range of single-molecule detection based experiments such as fluorescence fluctuation spectroscopy to large entity concentrations in the micromolar range.

Egner A, Geisler C, von Middendorff C, Bock H, Wenzel D, Medda R, Andresen M, Stiel AC et al. 2007. Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys J, 93 (9), pp. 3285-3290. | Show Abstract | Read more

We demonstrate nanoscale resolution in far-field fluorescence microscopy using reversible photoswitching and localization of individual fluorophores at comparatively fast recording speeds and from the interior of intact cells. These advancements have become possible by asynchronously recording the photon bursts of individual molecular switching cycles. We present images from the microtubular network of an intact mammalian cell with a resolution of 40 nm.

Neubauer H, Gaiko N, Berger S, Schaffer J, Eggeling C, Tuma J, Verdier L, Seidel CA, Griesinger C, Volkmer A. 2007. Orientational and dynamical heterogeneity of rhodamine 6G terminally attached to a DNA helix revealed by NMR and single-molecule fluorescence spectroscopy. J Am Chem Soc, 129 (42), pp. 12746-12755. | Show Abstract | Read more

The comparison of Förster resonance energy transfer (FRET) efficiencies between two fluorophores covalently attached to a single protein or DNA molecule is an elegant approach for deducing information about their structural and dynamical heterogeneity. For a more detailed structural interpretation of single-molecule FRET assays, information about the positions as well as the dynamics of the dye labels attached to the biomolecule is important. In this work, Rhodamine 6G (2-[3'-(ethylamino)-6'-(ethylimino)-2',7'-dimethyl-6'H-xanthen-9'-yl]-benzoic acid) bound to the 5'-end of a 20 base pair long DNA duplex is investigated by both single-molecule multiparameter fluorescence detection (MFD) experiments and NMR spectroscopy. Rhodamine 6G is commonly employed in nucleic acid research as a FRET dye. MFD experiments directly reveal the equilibrium of the dye bound to DNA between three heterogeneous environments, which are characterized by distinct fluorescence lifetime and intensity distributions as a result of different guanine-dye excited-state electron transfer interactions. Sub-ensemble fluorescence autocorrelation analysis shows the highly dynamic character of the dye-DNA interactions ranging from nano- to milliseconds and species-specific triplet relaxation times. Two-dimensional NMR spectroscopy corroborates this information by the determination of the detailed geometric structures of the dye-nucleobase complex and their assignment to each population observed in the single-molecule fluorescence experiments. From both methods, a consistent and detailed molecular description of the structural and dynamical heterogeneity is obtained.

Sieber JJ, Willig KI, Kutzner C, Gerding-Reimers C, Harke B, Donnert G, Rammner B, Eggeling C, Hell SW, Grubmüller H, Lang T. 2007. Anatomy and dynamics of a supramolecular membrane protein cluster. Science, 317 (5841), pp. 1072-1076. | Show Abstract | Read more

Most plasmalemmal proteins organize in submicrometer-sized clusters whose architecture and dynamics are still enigmatic. With syntaxin 1 as an example, we applied a combination of far-field optical nanoscopy, biochemistry, fluorescence recovery after photobleaching (FRAP) analysis, and simulations to show that clustering can be explained by self-organization based on simple physical principles. On average, the syntaxin clusters exhibit a diameter of 50 to 60 nanometers and contain 75 densely crowded syntaxins that dynamically exchange with freely diffusing molecules. Self-association depends on weak homophilic protein-protein interactions. Simulations suggest that clustering immobilizes and conformationally constrains the molecules. Moreover, a balance between self-association and crowding-induced steric repulsions is sufficient to explain both the size and dynamics of syntaxin clusters and likely of many oligomerizing membrane proteins that form supramolecular structures.

Andresen M, Stiel AC, Trowitzsch S, Weber G, Eggeling C, Wahl MC, Hell SW, Jakobs S. 2007. Structural basis for reversible photoswitching in Dronpa. Proc Natl Acad Sci U S A, 104 (32), pp. 13005-13009. | Show Abstract | Read more

Dronpa is a novel GFP-like fluorescent protein with exceptional light-controlled switching properties. It may be reversibly switched between a fluorescent on-state and a nonfluorescent off-state by irradiation with light. To elucidate the molecular basis of the switching mechanism, we generated reversibly switchable Dronpa protein crystals. Using these crystals we determined the elusive dark-state structure of Dronpa at 1.95-A resolution. We found that the photoswitching results in a cis-trans isomerization of the chromophore accompanied by complex structural rearrangements of four nearby amino acid residues. Because of this cascade of intramolecular events, the chromophore is exposed to distinct electrostatic surface potentials, which are likely to influence the protonation equilibria at the chromophore. We suggest a comprehensive model for the light-induced switching mechanism, connecting a cascade of structural rearrangements with different protonation states of the chromophore.

Bretschneider S, Eggeling C, Hell SW. 2007. Breaking the diffraction barrier in fluorescence microscopy by optical shelving. Phys Rev Lett, 98 (21), pp. 218103. | Show Abstract | Read more

We report the breaking of the diffraction resolution barrier in far-field fluorescence microscopy by transiently shelving the fluorophore in a metastable dark state. Using a relatively modest light intensity of several kW/cm(2) in a focal distribution featuring a local zero, we confine the fluorescence emission to a spot whose diameter is a fraction of the wavelength of light. Nanoscale far-field optical resolution down to 50 nm is demonstrated by imaging microtubules in a mammalian cell and proteins on the plasma membrane of a neuron. The presence of dark states in virtually any fluorescent molecule opens up a new venue for far-field microscopy with resolution that is no longer limited by diffraction.

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Bretschneider S, Eggeling C, Hell SW. 2007. Breaking the diffraction barrier in fluorescence microscopy by optical shelving PHYSICAL REVIEW LETTERS, 98 (21), | Read more

Donnert G, Keller J, Wurm CA, Rizzoli SO, Westphal V, Schönle A, Jahn R, Jakobs S, Eggeling C, Hell SW. 2007. Two-color far-field fluorescence nanoscopy. Biophys J, 92 (8), pp. L67-L69. | Show Abstract | Read more

We demonstrate two-color fluorescence microscopy with nanoscale spatial resolution by applying stimulated emission depletion on fluorophores differing in their absorption and emission spectra. Green- and red-emitting fluorophores are selectively excited and quenched using dedicated beam pairs. The stimulated emission depletion beams deliver a lateral resolution of <30 nm and 65 nm for the green and the red color channel, respectively. The approximately 5 nm alignment accuracy of the two images establishes the precision with which differently labeled proteins are correlated in space. Colocalized nanoscopy is demonstrated with endosomal protein patterns and by resolving nanoclusters of a mitochondrial outer membrane protein, Tom20, in relation with the F(1)F(0)ATP synthase. The joint improvement of resolution and colocalization demonstrates the emerging potential of far-field fluorescence nanoscopy to study the spatial organization of macromolecules in cells.

Schwentker MA, Bock H, Hofmann M, Jakobs S, Bewersdorf J, Eggeling C, Hell SW. 2007. Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching. Microsc Res Tech, 70 (3), pp. 269-280. | Show Abstract | Read more

Subdiffraction fluorescence imaging is presented in a parallelized wide-field arrangement exploiting the principle of reversible saturable/switchable optical transitions (RESOLFT). The diffraction barrier is overcome by photoswitching ensembles of the label protein asFP595 between a nonfluorescent off- and a fluorescent on-state. Relying on ultralow continuous-wave intensities, reversible protein switching facilitates parallelized fast image acquisition. The RESOLFT principle is implemented by illuminating with intensity distributions featuring zero intensity lines that are further apart than the conventional Abbe resolution limit. The subdiffraction resolution is verified by recording live Escherichia coli bacteria labeled with asFP595. The obtained resolution of 50 nm ( approximately lambda/12) is limited only by the spectroscopic properties of the proteins and the imperfections of the optical implementation, but not on principle grounds.

Stiel AC, Trowitzsch S, Weber G, Andresen M, Eggeling C, Hell SW, Jakobs S, Wahl MC. 2007. 1.8 A bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants. Biochem J, 402 (1), pp. 35-42. | Show Abstract | Read more

RSFPs (reversibly switchable fluorescent proteins) may be repeatedly converted between a fluorescent and a non-fluorescent state by irradiation and have attracted widespread interest for many new applications. The RSFP Dronpa may be switched with blue light from a fluorescent state into a non-fluorescent state, and back again with UV light. To obtain insight into the underlying molecular mechanism of this switching, we have determined the crystal structure of the fluorescent equilibrium state of Dronpa. Its bicyclic chromophore is formed spontaneously from the Cys62-Tyr63-Gly64 tripeptide. In the fluorescent state, it adopts a slightly non-coplanar cis conformation within the interior of a typical GFP (green fluorescent protein) b-can fold. Dronpa shares some structural features with asFP595, another RSFP whose chromophore has previously been demonstrated to undergo a cis-trans isomerization upon photoswitching. Based on the structural comparison with asFP595, we have generated new Dronpa variants with an up to more than 1000-fold accelerated switching behaviour. The mutations which were introduced at position Val157 or Met159 apparently reduce the steric hindrance for a cis-trans isomerization of the chromophore, thus lowering the energy barrier for the blue light-driven on-to-off transition. The findings reported in the present study support the view that a cis-trans isomerization is one of the key events common to the switching mechanism in RSFPs.

Widengren J, Chmyrov A, Eggeling C, Löfdahl PA, Seidel CA. 2007. Strategies to improve photostabilities in ultrasensitive fluorescence spectroscopy. J Phys Chem A, 111 (3), pp. 429-440. | Show Abstract | Read more

Given the particular importance of dye photostability for single-molecule and fluorescence fluctuation spectroscopy investigations, refined strategies were explored for how to chemically retard dye photobleaching. These strategies will be useful for fluorescence correlation spectroscopy (FCS), fluorescence-based confocal single-molecule detection (SMD) and related techniques. In particular, the effects on the addition of two main categories of antifading compounds, antioxidants (n-propyl gallate, nPG, ascorbic acid, AA) and triplet state quenchers (mercaptoethylamine, MEA, cyclo-octatetraene, COT), were investigated, and the relevant rate parameters involved were determined for the dye Rhodamine 6G. Addition of each of the compound categories resulted in significant improvements in the fluorescence brightness of the monitored fluorescent molecules in FCS measurements. For antioxidants, we identify the balance between reduction of photoionized fluorophores on the one hand and that of intact fluorophores on the other as an important guideline for what concentrations to be added for optimal fluorescence generation in FCS and SMD experiments. For nPG/AA, this optimal concentration was found to be in the lower micromolar range, which is considerably less than what has previously been suggested. Also, for MEA, which is a compound known as a triplet state quencher, it is eventually its antioxidative properties and the balance between reduction of fluorophore cation radicals and that of intact fluorophores that defines the optimal added concentration. Interestingly, in this optimal concentration range the triplet state quenching is still far from sufficient to fully minimize the triplet populations. We identify photoionization as the main mechanism of photobleaching within typical transit times of fluorescent molecules through the detection volume in a confocal FCS or SMD instrument (<1-20 ms), and demonstrate its generation via both one- and multistep excitation processes. Apart from reflecting a major pathway for photobleaching, our results also suggest the exploitation of the photoinduced ionization and the subsequent reduction by antioxidants for biomolecular monitoring purposes and as a possible switching mechanism with applications in high-resolution microscopy.

Donnert G, Eggeling C, Hell SW. 2007. Major signal increase in fluorescence microscopy through dark-state relaxation. Nat Methods, 4 (1), pp. 81-86. | Show Abstract | Read more

We report a substantial signal gain in fluorescence microscopy by ensuring that transient molecular dark states with lifetimes >1 micros, such as the triplet state relax between two molecular absorption events. For GFP and Rhodamine dye Atto532, we observed a 5-25-fold increase in total fluorescence yield before molecular bleaching when strong continuous-wave or high-repetition-rate pulsed illumination was replaced with pulses featuring temporal pulse separation >1 micros. The signal gain was observed both for one- and two-photon excitation. Obeying dark or triplet state relaxation in the illumination process signifies a major step toward imaging with low photobleaching and strong fluorescence fluxes.

Fölling J, Belov V, Kunetsky R, Medda R, Schönle A, Egner A, Eggeling C, Bossi M, Hell SW. 2007. Photochromic rhodamines provide nanoscopy with optical sectioning. Angew Chem Int Ed Engl, 46 (33), pp. 6266-6270. | Show Abstract | Read more

Exciting developments: Switching individual photochromic and fluorescent rhodamine amides enables 3D far-field optical microscopy with nanoscale resolution, excellent signal-to-noise ratio, and fast acquisition times. The rhodamine amides can be switched on using two photons, which enables 3D detailed imaging of thick and densely stained samples (such as 5-μm silica beads (see image) and living cells) to be constructed. (Figure Presented). © 2007 Wiley-VCH Verlag GmbH & Co. KGaA.

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Geisler C, Schoenle A, von Middendorff C, Bock H, Eggeling C, Egner A, Hell SW. 2007. Resolution of lambda/10 in fluorescence microscopy using fast single molecule photo-switching APPLIED PHYSICS A-MATERIALS SCIENCE & PROCESSING, 88 (2), pp. 223-226. | Show Abstract | Read more

We demonstrate nanoscale resolution in far-field optical microscopy based on photo-switching of molecules. By enabling, recording and disabling fluorescence from individual labels sequentially, the detection volume is reduced to the size of a single molecule and the diffraction limit is broken. Images of nanostructures milled into a coverslip and tagged by fluorescent proteins could be recorded at 50 nm resolution. Due to the fast and asynchronous image acquisition protocol used in these experiments, we were able to reduce acquisition times to ∼ 2.5 min, which is two orders of magnitude lower than in previous implementations. © Springer-Verlag 2007.

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Bock H, Geisler C, Wurm CA, Von Middendorff C, Jakobs S, Schoenle A, Egner A, Hell SW, Eggeling C. 2007. Two-color far-field fluorescence nanoscopy based on photoswitchable emitters APPLIED PHYSICS B-LASERS AND OPTICS, 88 (2), pp. 161-165. | Show Abstract | Read more

We demonstrate two-color far-field fluorescence microscopy with nanoscale spatial resolution based on the photoswitching of individual fluorescent markers. By enabling, recording, and disabling the emission of the reversibly switchable fluorescent protein rsFastLime and of the organic fluorophore cyanine5, we recorded two-color nanoscale images inside whole cells. The position of individual emitters was determined with a typical accuracy of 20 nm, which largely constitutes the lateral resolution of the system. Photoswitching in two-color colocalization experiments represents a major step towards the application of far-field fluorescence nanoscopy to the study of (biological) samples on the macromolecular level. © 2007 Springer-Verlag.

Donnert G, Keller J, Medda R, Andrei MA, Rizzoli SO, Lührmann R, Jahn R, Eggeling C, Hell SW. 2006. Macromolecular-scale resolution in biological fluorescence microscopy. Proc Natl Acad Sci U S A, 103 (31), pp. 11440-11445. | Show Abstract | Read more

We demonstrate far-field fluorescence microscopy with a focal-plane resolution of 15-20 nm in biological samples. The 10- to 12-fold multilateral increase in resolution below the diffraction barrier has been enabled by the elimination of molecular triplet state excitation as a major source of photobleaching of a number of dyes in stimulated emission depletion microscopy. Allowing for relaxation of the triplet state between subsequent excitation-depletion cycles yields an up to 30-fold increase in total fluorescence signal as compared with reported stimulated emission depletion illumination schemes. Moreover, it enables the reduction of the effective focal spot area by up to approximately 140-fold below that given by diffraction. Triplet-state relaxation can be realized either by reducing the repetition rate of pulsed lasers or by increasing the scanning speed such that the build-up of the triplet state is effectively prevented. This resolution in immunofluorescence imaging is evidenced by revealing nanoscale protein patterns on endosomes, the punctuated structures of intermediate filaments in neurons, and nuclear protein speckles in mammalian cells with conventional optics. The reported performance of diffraction-unlimited fluorescence microscopy opens up a pathway for addressing fundamental problems in the life sciences.

Leutenegger M, Blom H, Widengren J, Eggeling C, Gösch M, Leitgeb RA, Lasser T. 2006. Dual-color total internal reflection fluorescence cross-correlation spectroscopy. J Biomed Opt, 11 (4), pp. 040502. | Show Abstract | Read more

We present the development and first application of a novel dual-color total internal reflection (TIR) fluorescence system for single-molecule coincidence analysis and fluorescence cross-correlation spectroscopy (FCCS). As a performance analysis, we measured a synthetic DNA-binding assay, demonstrating this dual-color TIR-FCCS approach to be a suitable method for measuring coincidence assays such as biochemical binding, fusion, or signal transduction at solid/liquid interfaces. Due to the very high numerical aperture of the epi-illumination configuration, our setup provides a very high fluorescence collection efficiency resulting in a two- to three-fold increase in molecular brightness compared to conventional confocal FCCS. Further improvements have been achieved through global analysis of the spectroscopic data.

Eggeling C, Widengren J, Brand L, Schaffer J, Felekyan S, Seidel CA. 2006. Analysis of photobleaching in single-molecule multicolor excitation and Förster resonance energy transfer measurements. J Phys Chem A, 110 (9), pp. 2979-2995. | Show Abstract | Read more

Dye photobleaching is a major constraint of fluorescence readout within a range of applications. In this study, we investigated the influence of photobleaching in fluorescence experiments applying multicolor laser as well as Förster resonance energy transfer (FRET) mediated excitation using several red-emitting dyes frequently used in multicolor experiments or as FRET acceptors. The chosen dyes (cyanine 5 (Cy5), MR121, Alexa660, Alexa680, Atto647N, Atto655) have chemically distinct chromophore systems and can be excited at 650 nm. Several fluorescence analysis techniques have been applied to detect photobleaching and to disclose the underlying photophysics, all of which are based on single-molecule detection: (1) fluorescence correlation spectroscopy (FCS) of bulk solutions, (2) fluorescence cross-correlation of single-molecule trajectories, and (3) multiparameter fluorescence detection (MFD) of single-molecule events. The maximum achievable fluorescence signals as well as the survival times of the red dyes were markedly reduced under additional laser irradiation in the range of 500 nm. Particularly at excitation levels at or close to saturation, the 500 nm irradiation effectively induced transitions to higher excited electronic states on already excited dye molecules, leading to a pronounced bleaching reactivity. A theoretical model for the observed laser irradiance dependence of the fluorescence brightness of a Cy5 FRET acceptor dye has been developed introducing the full description of the underlying photophysics. The model takes into account acceptor as well as donor photobleaching from higher excited electronic states, population of triplet states, and energy transfer to both the ground and excited states of the acceptor dye. Also, photoinduced reverse intersystem crossing via higher excited triplet states is included, which was found to be very efficient for Cy5 attached to DNA. Comparing continuous wave (cw) and pulsed donor excitation, a strong enhancement of acceptor photobleaching by a factor of 5 was observed for the latter. Thus, in the case of fluorescence experiments utilizing multicolor pulsed laser excitation, the application of the appropriate timing of synchronized green and red laser pulses in an alternating excitation mode can circumvent excessive photobleaching. Moreover, important new single-molecule analysis diagnosis tools are presented: (1) For the case of excessive acceptor photobleaching, cross-correlation analysis of single-molecule trajectories of the fluorescence signal detected in the donor and acceptor detection channels and vice versa shows an anticorrelated exponential decay and growth, respectively. (2) The time difference, Tg - Tr, of the mean observation times of all photons detected for the donor and acceptor detection channels within a single-molecule fluorescence burst allows one to identify and exclude molecules with an event of acceptor photobleaching. The presented single-molecule analysis methods can be constrained to, for example, FRET-active subpopulations, reducing bias from FRET-inactive molecules. The observations made are of strong relevance for and demand a careful choice of laser action in multicolor and FRET experiments, in particular when performed at or close to saturation.

Blom H, Kastrup L, Eggeling C. 2006. Fluorescence fluctuation spectroscopy in reduced detection volumes. Curr Pharm Biotechnol, 7 (1), pp. 51-66. | Show Abstract | Read more

Fluorescence fluctuation spectroscopy is a versatile technique applied to in vitro and in vivo investigations of biochemical processes such as interactions, mobilities or densities with high specifity and sensitivity. The prerequisite of this dynamical fluorescence technique is to have, at a time, only few fluorescent molecules in the detection volume in order to generate significant fluorescence fluctuations. For usual confocal fluorescence microscopy this amounts to a useful concentration in the nanomolar range. The concentration of many biomolecules in living cell or on cell membranes is, however, often quite high, usually in the micro- to the millimolar range. To allow fluctuation spectroscopy and track intracellular interaction or localization of single fluorescently labeled biomolecules in such crowded environments, development of detection volumes with nanoscale resolution is necessary. As diffraction prevents this in the case of light microscopy, new (non-invasive) optical concepts have been developed. In this mini-review article we present recent advancements, implemented to decrease the detection volume below that of normal fluorescence microscopy. Especially, their combination with fluorescence fluctuation spectroscopy is emphasized.

Hofmann M, Eggeling C, Jakobs S, Hell SW. 2005. Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc Natl Acad Sci U S A, 102 (49), pp. 17565-17569. | Show Abstract | Read more

Fluorescence microscopy is indispensable in many areas of science, but until recently, diffraction has limited the resolution of its lens-based variant. The diffraction barrier has been broken by a saturated depletion of the marker's fluorescent state by stimulated emission, but this approach requires picosecond laser pulses of GW/cm2 intensity. Here, we demonstrate the surpassing of the diffraction barrier in fluorescence microscopy with illumination intensities that are eight orders of magnitude smaller. The subdiffraction resolution results from reversible photoswitching of a marker protein between a fluorescence-activated and a nonactivated state, whereby one of the transitions is accomplished by means of a spatial intensity distribution featuring a zero. After characterizing the switching kinetics of the used marker protein asFP595, we demonstrate the current capability of this RESOLFT (reversible saturable optical fluorescence transitions) type of concept to resolve 50-100 nm in the focal plane. The observed resolution is limited only by the photokinetics of the protein and the perfection of the zero. Our results underscore the potential to finally achieve molecular resolution in fluorescence microscopy by technical optimization.

Eggeling C, Jäger S, Winkler D, Kask P. 2005. Comparison of different fluorescence fluctuation methods for their use in FRET assays: monitoring a protease reaction. Curr Pharm Biotechnol, 6 (5), pp. 351-371. | Show Abstract | Read more

We compare the accuracy of a variety of Fluorescence Fluctuation Spectroscopy (FFS) methods for the study of Förster Resonance Energy Transfer (FRET) assays. As an example, the cleavage of a doubly labeled, FRET-active peptide substrate by the protease Trypsin is monitored and analyzed using methods based on fluorescence intensity, Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Intensity Distribution Analysis (FIDA). The presented fluorescence data are compared to High-Pressure Liquid Chromatography (HPLC) data obtained from the same assay. The HPLC analysis discloses general disadvantages of the FRET approach, such as incomplete labeling and the need for aliquots. However, the simultaneous use of two photon detectors monitoring the fluorescence signal of both labels significantly improves the analysis. In particular, the two global analysis tools Two-Dimensional Fluorescence Intensity Distribution Analysis (2D-FIDA) and Two-Color Global Fluorescence Correlation Spectroscopy (2CG-FCS) highlight the potential of a combination of FFS and FRET. While conventional FIDA and FCS auto- or cross-correlation analysis leaves the user with drawbacks inherent in two-color and FRET applications, these effects are overcome by the global analysis on the molecular level. Furthermore, it is advantageous to analyze the unnormalized as opposed to the normalized correlation data when combining any fluorescence correlation method with FRET, since the analysis of the unnormalized data introduces more accuracy and is less sensitive to the experimental drawbacks.

Andresen M, Wahl MC, Stiel AC, Gräter F, Schäfer LV, Trowitzsch S, Weber G, Eggeling C, Grubmüller H, Hell SW, Jakobs S. 2005. Structure and mechanism of the reversible photoswitch of a fluorescent protein. Proc Natl Acad Sci U S A, 102 (37), pp. 13070-13074. | Show Abstract | Read more

Proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state bear enormous potential in diverse fields, such as data storage, in vivo protein tracking, and subdiffraction resolution light microscopy. However, these proteins could hitherto not live up to their full potential because the molecular switching mechanism is not resolved. Here, we clarify the molecular photoswitching mechanism of asFP595, a green fluorescent protein (GFP)-like protein that can be transferred from a nonfluorescent "off" to a fluorescent "on" state and back again, by green and blue light, respectively. To this end, we establish reversible photoswitching of fluorescence in whole protein crystals and show that the switching kinetics in the crystal is identical with that in solution. Subsequent x-ray analysis demonstrated that upon the absorption of a green photon, the chromophore isomerizes from a trans (off) to a cis (on) state. Molecular dynamics calculations suggest that isomerization occurs through a bottom hula twist mechanism with concomitant rotation of both bonds of the chromophoric methine ring bridge. This insight into the switching mechanism should facilitate the targeted design of photoswitchable proteins. Reversible photoswitching of the protein chromophore system within intact crystals also constitutes a step toward the use of fluorescent proteins in three-dimensional data recording.

Eggeling C, Kask P, Winkler D, Jäger S. 2005. Rapid analysis of Forster resonance energy transfer by two-color global fluorescence correlation spectroscopy: trypsin proteinase reaction. Biophys J, 89 (1), pp. 605-618. | Show Abstract | Read more

In this study we introduce the combination of two-color global fluorescence correlation spectroscopy (2CG-FCS) and Förster resonance energy transfer (FRET) as a very powerful combination for monitoring biochemical reactions on the basis of single molecule events. 2CG-FCS, which is a new variation emerging from the family of fluorescence correlation spectroscopy, globally analyzes the simultaneously recorded auto- and cross-correlation data from two photon detectors monitoring the fluorescence emission of different colors. Overcoming the limitations inherent in mere auto- and cross-correlation analysis, 2CG-FCS is sensitive in resolving and quantifying fluorescent species that differ in their diffusion characteristics and/or their molecular brightness either in one or both detection channels. It is able to account for effects that have often been considered as sources of severe artifacts in two-color and FRET measurements, the most prominent artifacts comprising photobleaching, cross talk, or concentration variations in sample preparation. Because of its very high statistical accuracy, the combination of FRET and 2CG-FCS is suited for high-throughput applications such as drug screening. Employing beam scanning during data acquisition even further enhances this capability and allows measurement times of <2 s. The improved performance in monitoring a FRET sample was verified by following the protease cleavage reaction of a FRET-active peptide. The FRET-inactive subpopulation of uncleaved substrate could be correctly assigned, revealing a substantial portion of inactive or missing acceptor label. The results were compared to those obtained by two-dimensional fluorescence intensity distribution analysis.

Kastrup L, Blom H, Eggeling C, Hell SW. 2005. Fluorescence fluctuation spectroscopy in subdiffraction focal volumes. Phys Rev Lett, 94 (17), pp. 178104. | Show Abstract | Read more

We establish fluorescence fluctuation spectroscopy (FFS) with nanoscale detection volumes generated by stimulated emission depletion. Our method applies fluorescence correlation spectroscopy and fluorescence intensity distribution analysis to extract molecular information about mobilities and fluorescence emission in solution. The combination of correlation analysis with that of photon intensity distributions reveals a fivefold squeezing of the detection volume over current diffraction-limited systems, which is in full agreement with the simultaneously demonstrated 25-fold reduction in (axial) focal transit time. Our method significantly extends the potential of far-field FFS, including for the noninvasive investigation of molecular reactions at higher concentrations.

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Kastrup L, Blom H, Eggeling C, Hell SW. 2005. Fluorescence fluctuation spectroscopy in subdiffraction focal volumes PHYSICAL REVIEW LETTERS, 94 (17), | Read more

Eggeling C, Volkmer A, Seidel CA. 2005. Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy. Chemphyschem, 6 (5), pp. 791-804. | Show Abstract | Read more

Under high-excitation irradiance conditions in one- and two-photon induced fluorescence microscopy, the photostability of fluorescent dyes is of crucial importance for the detection sensitivity of single molecules and for the contrast in fluorescence imaging. Herein, we report on the dependence of photobleaching on the excitation conditions, using the dye Rhodamine 6G as a typical example. The different excitation modes investigated include 1) one-photon excitation into the first-excited singlet state in the range of 500 to 528 nm by continuous wave and picosecond-pulsed lasers and 2) two- and one-photon excitation to higher-excited singlet states at 800 and 350 nm, respectively, by femtosecond pulses. Experimental strategies are presented, which allow resolving the photophysics. From single-molecule trajectories and fluorescence correlation spectroscopy, as well as with a simple theoretical model based on steady-state solutions of molecular rate equation analysis, we determined the underlying photobleaching mechanisms and quantified the photokinetic parameters describing the dependence of the fluorescence signal on the excitation irradiance. The comparison with experimental data and an exact theoretical model show that only minor deviations between the different theoretical approaches can be observed for high-pulsed excitation irradiances. It is shown that fluorescence excitation is in all cases limited by photolysis from higher-excited electronic states. In contrast to picosecond-pulsed excitation, this is extremely severe for both one- and two-photon excitation with femtosecond pulses. Furthermore, the photostability of the higher-excited electronic states is strongly influenced by environmental conditions, such as polarity and temperature.

Eggeling C. 2004. Nanotechnology and single molecules. Chemphyschem, 5 (10), pp. 1483-1487. | Show Abstract | Read more

Single molecules on the move: The fourth international symposium on physics, chemistry and biology with single molecules in Staffelstein, Germany, witnessed many new insights and plans for the future developments of this research field. All kinds of experimental techniques such as scanning tunnelling, atomic force, or optical microscopy are involved. The immense progress in the field of nanotechnology opens up ways for the realization of "nanovisions" such as working nanomotors and molecular optoelectronic devices and circuits (for example, see picture).

Gribbon P, Schaertl S, Wickenden M, Williams G, Grimley R, Stuhmeier F, Preckel H, Eggeling C et al. 2004. Experiences in implementing uHTS--cutting edge technology meets the real world. Curr Drug Discov Technol, 1 (1), pp. 27-35. | Show Abstract | Read more

Driven by growing corporate compound files, the demands of target biology, and attempts to cut cost, the number of solutions to HTS has spiralled. In quick succession new assay technologies and screening platforms are appearing on the market, with the promise of screening faster than ever in low volume high density formats whilst providing high quality data. Within this world of rapid change, Pfizer has applied cutting edge technology to HTS by introducing screening in 1 microl formats utilising single molecule detection technology. Instead of resource intensive in-house development, Pfizer entered into a collaboration with Evotec OAI / Evotec Technologies and introduced their Mark-II EVOscreen platform. In this article we will outline the benefits of the approach taken at Pfizer, Sandwich, and introduce the Mark-II EVOscreen platform, illustrating the potential but also possible pitfalls of HTS miniaturisation.

Jäger S, Brand L, Eggeling C. 2003. New fluorescence techniques for high-throughput drug discovery. Curr Pharm Biotechnol, 4 (6), pp. 463-476. | Show Abstract | Read more

The rapid increase of compound libraries as well as new targets emerging from the Human Genome Project require constant progress in pharmaceutical research. An important tool is High-Throughput Screening (HTS), which has evolved as an indispensable instrument in the pre-clinical target-to-IND (Investigational New Drug) discovery process. HTS requires machinery, which is able to test more than 100,000 potential drug candidates per day with respect to a specific biological activity. This calls for certain experimental demands especially with respect to sensitivity, speed, and statistical accuracy, which are fulfilled by using fluorescence technology instrumentation. In particular the recently developed family of fluorescence techniques, FIDA (Fluorescence Intensity Distribution Analysis), which is based on confocal single-molecule detection, has opened up a new field of HTS applications. This report describes the application of these new techniques as well as of common fluorescence techniques--such as confocal fluorescence lifetime and anisotropy--to HTS. It gives experimental examples and presents advantages and disadvantages of each method. In addition the most common artifacts (auto-fluorescence or quenching by the drug candidates) emerging from the fluorescence detection techniques are highlighted and correction methods for confocal fluorescence read-outs are presented, which are able to circumvent this deficiency.

Eggeling C, Brand L, Ullmann D, Jäger S. 2003. Highly sensitive fluorescence detection technology currently available for HTS. Drug Discov Today, 8 (14), pp. 632-641. | Show Abstract | Read more

Homogeneous fluorescence methods are providing an important tool for HTS technologies. A wide range of different techniques have been established on the market, with read-outs ranging from total fluorescence intensity to statistical analysis of fluorescence fluctuations for biochemical assays or fluorescence imaging techniques for cellular systems. Each method has its own advantages and limitations, which have to be accounted for when designing a specific assay. Here, recently developed fluorescence techniques and some of their applications, with a particular focus on sensitivity, are summarized and their principles are presented.

Eggeling C, Gall K, Palo K, Kask P, Brand L. 2003. Confocal fluorescence techniques in industrial application MANIPULATION AND ANALYSIS OF BIOMOLECULES, CELLS AND TISSUES, 4962 pp. 101-109. | Show Abstract | Read more

The FCS+plus family of evaluation tools for confocal fluorescence spectroscopy, which was developed during recent years, offers a comprehensive view to a series of fluorescence properties. Originating in fluorescence correlation spectroscopy (PCS) and using similar experimental equipment, a system of signal processing methods such as fluorescence intensity distribution analysis (FIDA) was created to analyze in detail the fluctuation behavior of fluorescent particles within a small area of detection. Giving simultaneous access to molecular parameters like concentration, translational and rotational diffusion, molecular brightness, and multicolor coincidence, this portfolio was enhanced by more traditional techniques of fluorescence lifetime as well as time-resolved anisotropy determination. The cornerstones of the FCS+plus methodology will be shortly described. The inhibition of a phosphatase enzyme activity gives a comprehensive industrial application that demonstrates FCS+plus versatility and its potential for pharmaceutical drug discovery.

Palo K, Brand L, Eggeling C, Jäger S, Kask P, Gall K. 2002. Fluorescence intensity and lifetime distribution analysis: toward higher accuracy in fluorescence fluctuation spectroscopy. Biophys J, 83 (2), pp. 605-618. | Show Abstract | Read more

Fluorescence fluctuation methods such as fluorescence correlation spectroscopy and fluorescence intensity distribution analysis (FIDA) have proven to be versatile tools for studying molecular interactions with single molecule sensitivity. Another well-known fluorescence technique is the measurement of the fluorescence lifetime. Here, we introduce a method that combines the benefits of both FIDA and fluorescence lifetime analysis. It is based on fitting the two-dimensional histogram of the number of photons detected in counting time intervals of given width and the sum of excitation to detection delay times of these photons. Referred to as fluorescence intensity and lifetime distribution analysis (FILDA), the technique distinguishes fluorescence species on the basis of both their specific molecular brightness and the lifetime of the excited state and is also able to determine absolute fluorophore concentrations. The combined information yielded by FILDA results in significantly increased accuracy compared to that of FIDA or fluorescence lifetime analysis alone. In this paper, the theory of FILDA is elaborated and applied to both simulated and experimental data. The outstanding power of this technique in resolving different species is shown by quantifying the binding of calmodulin to a peptide ligand, thus indicating the potential for application of FILDA to similar problems in the life sciences.

Aicher B, Eggeling C, Brand L, Schaertl S, Funsch B, Kirchhoff S, Beringer B, Siemers A, Petersen Y, Winkler D, Gall K. 2002. Confocal fluorescence lifetime analysis revisited - A novel readout for assay development and screening applications BIOPHYSICAL JOURNAL, 82 (1), pp. 434A-435A.

Gall K, Palo K, Brand L, Eggeling C, Kask P. 2002. Fluorescence intensity and lifetime distribution analysis: A new and highly sensitive fluorescence fluctuation method BIOPHYSICAL JOURNAL, 82 (1), pp. 355A-355A.

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Eggeling C, Schaffer J, Seidel CAM, Korte J, Brehm G, Schneider S, Schrof W. 2001. Homogeneity, transport, and signal properties of single Ag particles studied by single-molecule surface-enhanced resonance Raman scattering JOURNAL OF PHYSICAL CHEMISTRY A, 105 (15), pp. 3673-3679. | Read more

Eggeling C, Berger S, Brand L, Fries JR, Schaffer J, Volkmer A, Seidel CA. 2001. Data registration and selective single-molecule analysis using multi-parameter fluorescence detection. J Biotechnol, 86 (3), pp. 163-180. | Show Abstract | Read more

A general strategy to identify and quantify sample molecules in dilute solution employing a new spectroscopic method for data registration and specific burst analysis denoted as multi-parameter fluorescence detection (MFD) was recently developed. While keeping the experimental advantage of monitoring single molecules diffusing through the microscopic open volume element of a confocal epi-illuminated set-up as in experiments of fluorescence correlation spectroscopy, MFD uses pulsed excitation and time-correlated single-photon counting to simultaneously monitor the evolution of the four-dimensional fluorescence information (intensity, F; lifetime, tau; anisotropy, r; and spectral range, lambda(r)) in real time and allows for exclusion of extraneous events for subsequent analysis. In this review, the versatility of this technique in confocal fluorescence spectroscopy will be presented by identifying freely diffusing single dyes via their characteristic fluorescence properties in homogenous assays, resulting in significantly reduced misclassification probabilities. Major improvements in background suppression are demonstrated by time-gated autocorrelation analysis of fluorescence intensity traces extracted from MFD data. Finally, applications of MFD to real-time conformational dynamics studies of fluorescence labeled oligonucleotides will be presented.

Seidel CAM, Schweinberger E, Berger S, Eggeling C, Schaffer J, Widengren J, Felekyan S. 2001. Analysis of molecular structure and dynamics by selective single-molecule fluorescence spectroscopy. ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 221 pp. U239-U239.

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Eggeling C, Schaffer J, Seidel CAM, Korte J, Brehm G, Schneider S, Schrof W. 2001. Homogeneity, transport, and signal properties of single ag particles studied by single-molecule surface-enhanced resonance raman scattering Journal of Physical Chemistry A, 105 (15), pp. 3678-3679. | Show Abstract

We extended the sensitivity of Raman correlation spectroscopy in solution to the single-molecule level by applying surface- and resonance-enhanced Raman scattering (SERRS) combined with time-gated, confocal signal detection. The brightness of the SERRS signal of single Rhodamine 6G molecules adsorbed on a single silver nanoparticle is comparable to fluorescence. Rare event analysis reveals the existence of few particles with simultaneous SERRS and fluorescence signal. The observation of a dynamic exchange between heterogeneous binding sites is supported by the existence of multiple SERRS brightnesses in the signal intensity distribution and by signal fluctuations in the 60 μs time range detected by autocorrelation analysis. Finally, polarization-dependent SERRS autocorrelation curves and single-particle analysis allowed us to measure individual rotational diffusion times and to directly analyze the heterogeneity of the ensemble in solution. © 2001 American Chemical Society.

Seidel CAM, Berger S, Eggeling C, Schaffer J, Schweinberger E, Widengren J, Goody R, Kensch O, Rothwell P. 2000. Dynamics of DNA and HIV-1 reverse transcriptase-DNA complexes studied by four-dimensional single-molecule fluorescence spectroscopy BIOPHYSICAL JOURNAL, 78 (1), pp. 402A-402A.

Volkmer A, Eggeling C, Schaffer J, Berger S, Seidel CAM. 2000. Real-time monitoring and statistical analysis of single oligonucleotide dynamics in aqueous solution. BIOPHYSICAL JOURNAL, 78 (1), pp. 385A-385A.

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Schaffer J, Volkmer A, Eggeling C, Subramaniam V, Striker G, Seidel CAM. 1999. Identification of single molecules in aqueous solution by time-resolved fluorescence anisotropy JOURNAL OF PHYSICAL CHEMISTRY A, 103 (3), pp. 331-336. | Read more

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Schaflfer J, Volkmer A, Eggeling C, Subramaniam V, Striker G, Seidel CAM. 1999. Identification of Single Molecules in Aqueous Solution by Time-Resolved Fluorescence Anisotropy Journal of Physical Chemistry A, 103 (3), | Show Abstract

Using a confocal epi-illuminated microscope with a polarizing beam splitter and dual-channel detection of single-molecule fluorescence induced by pulsed laser excitation, a new application of the three-dimensional, real-time spectroscopic technique BIFL (burst integrated fluorescence lifetime) is introduced. BIFL allows simultaneous registration of fluorescence intensity, lifetime, and anisotropy. It is shown to be well-suited to identify the freely diffusing fluorescent molecule Rhodamine 123 and the Enhanced Yellow Fluorescent Protein via their characteristic fluorescence anisotropy using a time-resolved analysis. Furthermore, data analysis is discussed and rotational correlation times of single molecules are determined. Applications for multidimensional single-molecule identification are outlined.

Volkmer A, Schaffer J, Eggeling C, Fries JR, Berger S, Subramaniam B, Striker G, Seidel CAM. 1999. Multidimensional time-resolved fluorescence spectroscopy of single molecules in aqueous solution BIOPHYSICAL JOURNAL, 76 (1), pp. A260-A260.

Börsch M, Turina P, Eggeling C, Fries JR, Seidel CA, Labahn A, Gräber P. 1998. Conformational changes of the H+-ATPase from Escherichia coli upon nucleotide binding detected by single molecule fluorescence. FEBS Lett, 437 (3), pp. 251-254. | Show Abstract | Read more

Using a confocal fluorescence microscope with an avalanche photodiode as detector, we studied the fluorescence of the tetramethylrhodamine labeled F1 part of the H+-ATPase from Escherichia coli, EF1, carrying the gammaT106-C mutation [Aggeler, J.A. and Capaldi, R.A. (1992) J. Biol. Chem. 267, 21355-21359] in aqueous solution upon excitation with a mode-locked argon ion laser at 528 nm. The diffusion of the labeled EF1 through the confocal volume gives rise to photon bursts, which were analyzed with fluorescence correlation spectroscopy, resulting in a diffusion coefficient of 3.3 x 10(-7) cm2 s(-1). In the presence of nucleotides the diffusion coefficient increases by about 15%. This effect indicates a change of the shape and/or the volume of the enzyme upon binding of nucleotides, i.e. fluorescence correlation spectroscopy with single EF1 molecules allows the detection of conformational changes.

Eggeling C, Widengren J, Rigler R, Seidel CA. 1998. Photobleaching of Fluorescent Dyes under Conditions Used for Single-Molecule Detection:  Evidence of Two-Step Photolysis. Anal Chem, 70 (13), pp. 2651-2659. | Show Abstract | Read more

The photostability of fluorescent dyes is of crucial importance for the statistical accuracy of single-molecule detection (SMD) and for the image quality of scanning confocal microscopy. Concurrent results for the photostability were obtained by two different experimental techniques. First, the photostabilities of several coumarin and rhodamine derivatives in aqueous solution were obtained by monitoring the steady-state fluorescence decay in a quartz cell. Furthermore, an epi-illuminated microscope, continuous wave (CW) excitation at 514.5 nm, and fluorescence correlation spectroscopy (FCS) with a newly developed theory were used to study the photobleaching characteristics of rhodamines under conditions used for SMD. Depending on the rhodamine structure, the probability of photobleaching, p(b), is in the order of 10(-)(6)-10(-)(7) for irradiances below 10(3) W/cm(2). However, a considerable increase of p(b) for irradiances above this level was observed which can only be described by photobleaching reactions from higher excited states (two-step photolysis). In view of these observations, the probability of photobleaching, p(b), as well as a closed expression of its dependence on the CW excitation irradiance considering a five-level molecular electronic state model with the possibility of photobleaching from higher excited electronic states, is derived. From this model, optimal conditions for SMD with respect to the number of emitted fluorescence photons and to the signal-to-background ratio are discussed, taking into account both saturation and photobleaching. The additional photobleaching due to two-step photolysis limits the applicable irradiance.

Eggeling C, Fries JR, Brand L, Günther R, Seidel CA. 1998. Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy. Proc Natl Acad Sci U S A, 95 (4), pp. 1556-1561. | Show Abstract | Read more

A recently developed, real-time spectroscopic technique, burst-integrated fluorescence lifetime (BIFL), is shown to be well suited for monitoring the individual molecular conformational dynamics of a single molecule diffusing through the microscopic, open measurement volume (approximately 10 fl) of a confocal epi-illuminated set-up. In a highly diluted aqueous solution of 20-mer oligonucleotide strand of DNA duplex labeled with the environment-sensitive fluorescent dye tetramethylrhodamine (TMR), fluorescence bursts indicating traces of individual molecules are registered and further subjected to selective burst analysis. The two-dimensional BIFL data allow the identification and detection of different temporally resolved conformational states. A complementary autocorrelation analysis was performed on the time-dependent fluctuations in fluorescence lifetime and intensity. The consistent results strongly support the hypothesized three-state model of the conformational dynamics of the TMR-DNA duplex with a polar, a nonpolar, and a quenching environment of TMR.

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Fries JR, Brand L, Eggeling C, Kollner M, Seidel CAM. 1998. Quantitative identification of different single molecules by selective time-resolved confocal fluorescence spectroscopy JOURNAL OF PHYSICAL CHEMISTRY A, 102 (33), pp. 6601-6613. | Show Abstract | Read more

Using a confocal epi-illuminated microscope together with a pulsed laser, new applications of the recently developed, real-time spectroscopic technique BIFL (burst integrated fluorescence lifetime) are introduced. BIFL registers two different types of information on every detected photon with regard to the macroscopic time scale of a measurement and to the fluorescence lifetime. Thus, it is shown to be well suited to identify freely diffusing single dye molecules via their characteristic fluorescence lifetime. This allows for selective counting of dye molecules in an open volume element and opens up the possibility to quantify the relative concentration of the dye molecules, using a recently derived theoretical model, which analyzes the obtained burst size distribution of a sample survey. A closed theory is presented to calculate the probability of a specific dye to cause a fluorescence burst containing a certain number of detected photons. It considers the distribution of the excitation irradiance over the detection volume together with saturation effects of the fluorescence and of the detection electronics, the probability of different transit times through the detection volume, and the probability of multimolecule events. Using BIFL together with selective counting, the concentration of two dyes, Rhodamine B and Rhodamine 6G, in separate solutions and in a mixture were determined. The obtained results are consistent with the applied dye concentrations and with simultaneous measurements by fluorescence correlation spectroscopy (FCS). The introduced method is an appropriate tool for the complete characterization and quantitative analysis of a highly diluted sample in homogeneous assays.

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Zander C, Sauer M, Drexhage KH, Ko DS, Schulz A, Wolfrum J, Brand L, Eggeling C, Seidel CAM. 1996. Detection and characterization of single molecules in aqueous solution APPLIED PHYSICS B-LASERS AND OPTICS, 63 (5), pp. 517-523. | Show Abstract

Using a confocal microscope with a single-photon avalanche photodiode as detector, we studied photon bursts of single Rhodamine 6G (R6G) and Rhodamin B-zwitterion (RB) molecules in aqueous solution by excitation of the lowest excited singlet state S1 with a frequency-doubled titanium:sapphire laser. Multichannel scaler traces, the fluorescence autocorrelation function and fluorescence decay times determined by time-correlated single-photon counting have been measured simultaneously. The time-resolved fluorescence signals were analyzed with a maximum likelihood estimator. Fluorescence lifetime patterns in steps of 100 ps were generated by convolution with the excitation pulse. The lifetime of the S1 state was derived from the Kullback-Leibler minimum discrimination information. We are able to demonstrate for the first time identification of two different single dye molecules via their characteristic fluorescence lifetimes of 1.79 ± 0.33 ns (RB) and 3.79 ± 0.38 ns (R6G) in aqueous solution.

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Eggeling C, Brand L, Seidel CAM. 1997. Laser‐induced fluorescence of coumarin derivatives in aqueous solution: Photochemical aspects for single molecule detection Bioimaging, 5 (3), pp. 105-115. | Show Abstract | Read more

The efficiency of detecting a single fluorescent coumarin dye molecule in aqueous solution by one-photon excitation (OPE) at 350 nm as well as by coherent two-photon excitation (TPE) at 700 nm is studied. The photostability, which is crucial for single molecule detection (SMD), is determined at a low irradiance for various coumarin derivatives using a 'cell-bleaching' method. The yields of photobleaching for these coumarins in aqueous solution are in the order of 10-3 to 10-4. Thus, most of the dyes are sufficiently stable to allow SMD. However, for SMD in a fluorescence microscope a high quasi-CW irradiance (at least 104 W cm-2) is necessary for efficient OPE by a pulsed, frequency doubled titanium:sapphire laser. Detailed investigations on the dye Coumarin-120 using fluorescence correlation spectroscopy (FCS), different repetition rates of the laser and transient absorption spectroscopy (TRABS) gave clear evidence that OPE at a high irradiance results in two-step photolysis via the first electronic excited singlet and triplet state, S1 and T1, producing dye radical ions and solvated electrons. Hence, this additional photobleaching pathway limits the applicable irradiance for OPE. Using coherent TPE for single molecule detection, saturation of the fluorescence was observed for a high quasi-CW irradiance (108 W cm-2), which may also be caused by photobleaching. Furthermore, TPE is deteriorated by other competing nonlinear processes (e.g. continuum generation in the solvent), which only occur above a threshold irradiance (7 x 107 W cm-2). Nevertheless, TPE allows an efficient detection of single Coumarin-120 molecules in water. Using a maximum likelihood estimator, we are also able to identify single dye molecules via their characteristic fluorescence lifetime of 4.8 ± 1.2 ns.

Zander C, Sauer M, Drexhage KH, Wolfrum J, Brand L, Eggeling C, Seidel CAM. 1997. Lifetime-identification of single molecules in aqueous solution ADVANCES IN FLUORESCENCE SENSING TECHNOLOGY III, 2980 pp. 107-114. | Show Abstract | Read more

Photon bursts of single rhodamine 6G and rhodamine B molecules in aqueous solution were studied by excitation with a frequency-doubled titanium: sapphire laser. Multichannel scalar traces, fluorescence correlation functions and fluorescence decays determined by time- correlated single-photon counting have been measured simultaneously. The time-resolved fluorescence signals were analyzed with a maximum likelihood estimator. With the setup described it is possible to distinguish single dye molecules of different kind via their characteristic fluorescence lifetimes of 1.79 +/- 0.33 ns for rhodamine B-zwitterion and 3.79 +/- 0.38 ns for rhodamine 6G. ©2005 Copyright SPIE - The International Society for Optical Engineering.

Zander C, Brand L, Eggeling C, Drexhage KH, Seidel CAM. 1997. Single-molecule detection by two-photon excitation of fluorescence ADVANCES IN FLUORESCENCE SENSING TECHNOLOGY III, 2980 pp. 552-558. | Show Abstract | Read more

Using a mode-locked titanium: sapphire laser at 700 nm for two-photon excitation we studied fluorescence bursts from individual coumarin 120 molecules in water and triacetin. Fluorescence lifetimes and multichannel scaler traces have been measured simultaneously. Due to the fact that scattered excitation light as well as Raman scattered photons can be suppressed by a short-pass filter a very low background level was achieved. To identify the fluorophore by its characteristic fluorescence lifetime the time-resolved fluorescence signals were analyzed by a maximum likelihood estimator. The obtained average fluorescence lifetimes (tau) av equals 4.8 +/- 1.2 ns for coumarin 120 in water and (tau) av equals 3.3 +/- 0.6 for coumarin 120 in triacetin are in good agreement with results obtained from separate measurements at higher concentrations. ©2005 Copyright SPIE - The International Society for Optical Engineering.

Brand L, Eggeling C, Seidel CAM. 1997. Single-molecule detection of coumarin-120 NUCLEOSIDES & NUCLEOTIDES, 16 (5-6), pp. 551-556. | Show Abstract | Read more

Two-photon excitation with a mode-locked titanium: sapphire laser at 700 nm and confocal fluorescence microscopy have been used to detect single Coumarin-120 (C-120) molecules. The dye C-120 is quenched by the nucleobases, if coupled to nucleotides, resuiting in nucleobase-specific fluorescence lifetimes. This suggests applications in current projects for ultrasensitive DNA characterization.

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Brand L, Eggeling C, Zander C, Drexhage KH, Seidel CAM. 1997. Single-molecule identification of Coumarin-120 by time-resolved fluorescence detection: Comparison of one- and two-photon excitation in solution JOURNAL OF PHYSICAL CHEMISTRY A, 101 (24), pp. 4313-4321. | Show Abstract | Read more

Using two-photon excitation (TPE) at 700 nm as well as one-photon excitation (OPE) at 350 nm, we applied confocal fluorescence microscopy to detect single Coumarin-120 molecules in the solvents water and triacetin. To study the behavior of Coumarin-120 under different excitation conditions, fluorescence lifetimes, multichannel scaler traces, and autocorrelation curves have been measured simultaneously. A signal-to-background ratio of 1300 was achieved for TPE due to a very low background level. The detection efficiency of TPE is limited by other competing nonlinear processes, in particular continuum generation in the solvent. The applicable laser intensity for OPE is limited by two-step photolysis of the dye as shown by fluorescence correlation spectroscopy (FCS). The time-resolved fluorescence signals were analyzed by a maximum likelihood estimator to identify the fluorophore through its characteristic fluorescence lifetime. The average fluorescence lifetimes 4.8 ± 1.2 ns in water and 3.3 ± 0.6 ns in triacetin are in good agreement with results obtained from separate measurements at higher concentrations.

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Chmyrov A, Keller J, Grotjohann T, Ratz M, D'Este E, Jakobs S, Eggeling C, Hell SW. 2013. Nanoscopy with more than 100,000 'doughnuts' Nature Methods, 10 (8), pp. 737-740. | Show Abstract | Read more

We show that nanoscopy based on the principle called RESOLFT (reversible saturable optical fluorescence transitions) or nonlinear structured illumination can be effectively parallelized using two incoherently superimposed orthogonal standing light waves. The intensity minima of the resulting pattern act as 'doughnuts', providing isotropic resolution in the focal plane and making pattern rotation redundant. We super-resolved living cells in 120 μm × 100 μm-sized fields of view in <1 s using 116,000 such doughnuts. © 2013 Nature America, Inc. All rights reserved.

Grotjohann T, Testa I, Leutenegger M, Bock H, Urban NT, Lavoie-Cardinal F, Willig KI, Eggeling C, Jakobs S, Hell SW. 2011. Diffraction-unlimited all-optical imaging and writing with a photochromic GFP. Nature, 478 (7368), pp. 204-208. | Show Abstract | Read more

Lens-based optical microscopy failed to discern fluorescent features closer than 200 nm for decades, but the recent breaking of the diffraction resolution barrier by sequentially switching the fluorescence capability of adjacent features on and off is making nanoscale imaging routine. Reported fluorescence nanoscopy variants switch these features either with intense beams at defined positions or randomly, molecule by molecule. Here we demonstrate an optical nanoscopy that records raw data images from living cells and tissues with low levels of light. This advance has been facilitated by the generation of reversibly switchable enhanced green fluorescent protein (rsEGFP), a fluorescent protein that can be reversibly photoswitched more than a thousand times. Distributions of functional rsEGFP-fusion proteins in living bacteria and mammalian cells are imaged at <40-nanometre resolution. Dendritic spines in living brain slices are super-resolved with about a million times lower light intensities than before. The reversible switching also enables all-optical writing of features with subdiffraction size and spacings, which can be used for data storage.

Mueller V, Ringemann C, Honigmann A, Schwarzmann G, Medda R, Leutenegger M, Polyakova S, Belov VN, Hell SW, Eggeling C. 2011. STED nanoscopy reveals molecular details of cholesterol- and cytoskeleton-modulated lipid interactions in living cells. Biophys J, 101 (7), pp. 1651-1660. | Show Abstract | Read more

Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeleton-dependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol- and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes.

Vicidomini G, Moneron G, Han KY, Westphal V, Ta H, Reuss M, Engelhardt J, Eggeling C, Hell SW. 2011. Sharper low-power STED nanoscopy by time gating. Nat Methods, 8 (7), pp. 571-573. | Show Abstract | Read more

Applying pulsed excitation together with time-gated detection improves the fluorescence on-off contrast in continuous-wave stimulated emission depletion (CW-STED) microscopy, thus revealing finer details in fixed and living cells using moderate light intensities. This method also enables super-resolution fluorescence correlation spectroscopy with CW-STED beams, as demonstrated by quantifying the dynamics of labeled lipid molecules in the plasma membrane of living cells.

Sahl SJ, Leutenegger M, Hilbert M, Hell SW, Eggeling C. 2010. Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids. Proc Natl Acad Sci U S A, 107 (15), pp. 6829-6834. | Show Abstract | Read more

We describe an optical method capable of tracking a single fluorescent molecule with a flexible choice of high spatial accuracy (approximately 10-20 nm standard deviation or approximately 20-40 nm full-width-at-half-maximum) and temporal resolution (< 1 ms). The fluorescence signal during individual passages of fluorescent molecules through a spot of excitation light allows the sequential localization and thus spatio-temporal tracking of the molecule if its fluorescence is collected on at least three separate point detectors arranged in close proximity. We show two-dimensional trajectories of individual, small organic dye labeled lipids diffusing in the plasma membrane of living cells and directly observe transient events of trapping on < 20 nm spatial scales. The trapping is cholesterol-assisted and much more pronounced for a sphingo- than for a phosphoglycero-lipid, with average trapping times of approximately 15 ms and < 4 ms, respectively. The results support previous STED nanoscopy measurements and suggest that, at least for nontreated cells, the transient interaction of a single lipid is confined to macromolecular dimensions. Our experimental approach demonstrates that fast molecular movements can be tracked with minimal invasion, which can reveal new important details of cellular nano-organization.

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Ringemann C, Harke B, von Middendorff C, Medda R, Honigmann A, Wagner R, Leutenegger M, Schoenle A, Hell SW, Eggeling C. 2009. Exploring single-molecule dynamics with fluorescence nanoscopy NEW JOURNAL OF PHYSICS, 11 (10), pp. 103054-103054. | Read more

Eggeling C, Ringemann C, Medda R, Schwarzmann G, Sandhoff K, Polyakova S, Belov VN, Hein B, von Middendorff C, Schönle A, Hell SW. 2009. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature, 457 (7233), pp. 1159-1162. | Show Abstract | Read more

Cholesterol-mediated lipid interactions are thought to have a functional role in many membrane-associated processes such as signalling events. Although several experiments indicate their existence, lipid nanodomains ('rafts') remain controversial owing to the lack of suitable detection techniques in living cells. The controversy is reflected in their putative size of 5-200 nm, spanning the range between the extent of a protein complex and the resolution limit of optical microscopy. Here we demonstrate the ability of stimulated emission depletion (STED) far-field fluorescence nanoscopy to detect single diffusing (lipid) molecules in nanosized areas in the plasma membrane of living cells. Tuning of the probed area to spot sizes approximately 70-fold below the diffraction barrier reveals that unlike phosphoglycerolipids, sphingolipids and glycosylphosphatidylinositol-anchored proteins are transiently ( approximately 10-20 ms) trapped in cholesterol-mediated molecular complexes dwelling within <20-nm diameter areas. The non-invasive optical recording of molecular time traces and fluctuation data in tunable nanoscale domains is a powerful new approach to study the dynamics of biomolecules in living cells.

Fölling J, Bossi M, Bock H, Medda R, Wurm CA, Hein B, Jakobs S, Eggeling C, Hell SW. 2008. Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods, 5 (11), pp. 943-945. | Show Abstract | Read more

We introduce far-field fluorescence nanoscopy with ordinary fluorophores based on switching the majority of them to a metastable dark state, such as the triplet, and calculating the position of those left or those that spontaneously returned to the ground state. Continuous widefield illumination by a single laser and a continuously operating camera yielded dual-color images of rhodamine- and fluorescent protein-labeled (living) samples, proving a simple yet powerful super-resolution approach.

Sieber JJ, Willig KI, Kutzner C, Gerding-Reimers C, Harke B, Donnert G, Rammner B, Eggeling C, Hell SW, Grubmüller H, Lang T. 2007. Anatomy and dynamics of a supramolecular membrane protein cluster. Science, 317 (5841), pp. 1072-1076. | Show Abstract | Read more

Most plasmalemmal proteins organize in submicrometer-sized clusters whose architecture and dynamics are still enigmatic. With syntaxin 1 as an example, we applied a combination of far-field optical nanoscopy, biochemistry, fluorescence recovery after photobleaching (FRAP) analysis, and simulations to show that clustering can be explained by self-organization based on simple physical principles. On average, the syntaxin clusters exhibit a diameter of 50 to 60 nanometers and contain 75 densely crowded syntaxins that dynamically exchange with freely diffusing molecules. Self-association depends on weak homophilic protein-protein interactions. Simulations suggest that clustering immobilizes and conformationally constrains the molecules. Moreover, a balance between self-association and crowding-induced steric repulsions is sufficient to explain both the size and dynamics of syntaxin clusters and likely of many oligomerizing membrane proteins that form supramolecular structures.

Donnert G, Keller J, Medda R, Andrei MA, Rizzoli SO, Lührmann R, Jahn R, Eggeling C, Hell SW. 2006. Macromolecular-scale resolution in biological fluorescence microscopy. Proc Natl Acad Sci U S A, 103 (31), pp. 11440-11445. | Show Abstract | Read more

We demonstrate far-field fluorescence microscopy with a focal-plane resolution of 15-20 nm in biological samples. The 10- to 12-fold multilateral increase in resolution below the diffraction barrier has been enabled by the elimination of molecular triplet state excitation as a major source of photobleaching of a number of dyes in stimulated emission depletion microscopy. Allowing for relaxation of the triplet state between subsequent excitation-depletion cycles yields an up to 30-fold increase in total fluorescence signal as compared with reported stimulated emission depletion illumination schemes. Moreover, it enables the reduction of the effective focal spot area by up to approximately 140-fold below that given by diffraction. Triplet-state relaxation can be realized either by reducing the repetition rate of pulsed lasers or by increasing the scanning speed such that the build-up of the triplet state is effectively prevented. This resolution in immunofluorescence imaging is evidenced by revealing nanoscale protein patterns on endosomes, the punctuated structures of intermediate filaments in neurons, and nuclear protein speckles in mammalian cells with conventional optics. The reported performance of diffraction-unlimited fluorescence microscopy opens up a pathway for addressing fundamental problems in the life sciences.

Hofmann M, Eggeling C, Jakobs S, Hell SW. 2005. Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc Natl Acad Sci U S A, 102 (49), pp. 17565-17569. | Show Abstract | Read more

Fluorescence microscopy is indispensable in many areas of science, but until recently, diffraction has limited the resolution of its lens-based variant. The diffraction barrier has been broken by a saturated depletion of the marker's fluorescent state by stimulated emission, but this approach requires picosecond laser pulses of GW/cm2 intensity. Here, we demonstrate the surpassing of the diffraction barrier in fluorescence microscopy with illumination intensities that are eight orders of magnitude smaller. The subdiffraction resolution results from reversible photoswitching of a marker protein between a fluorescence-activated and a nonactivated state, whereby one of the transitions is accomplished by means of a spatial intensity distribution featuring a zero. After characterizing the switching kinetics of the used marker protein asFP595, we demonstrate the current capability of this RESOLFT (reversible saturable optical fluorescence transitions) type of concept to resolve 50-100 nm in the focal plane. The observed resolution is limited only by the photokinetics of the protein and the perfection of the zero. Our results underscore the potential to finally achieve molecular resolution in fluorescence microscopy by technical optimization.

Eggeling C, Brand L, Ullmann D, Jäger S. 2003. Highly sensitive fluorescence detection technology currently available for HTS. Drug Discov Today, 8 (14), pp. 632-641. | Show Abstract | Read more

Homogeneous fluorescence methods are providing an important tool for HTS technologies. A wide range of different techniques have been established on the market, with read-outs ranging from total fluorescence intensity to statistical analysis of fluorescence fluctuations for biochemical assays or fluorescence imaging techniques for cellular systems. Each method has its own advantages and limitations, which have to be accounted for when designing a specific assay. Here, recently developed fluorescence techniques and some of their applications, with a particular focus on sensitivity, are summarized and their principles are presented.

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