Prof Christian Eggeling
|Technology Exchange:||Cellular immunology, In vivo imaging, Microscopy (Confocal) and Microscopy (Video)|
|Scientific Themes:||Immunology and Molecular, Cell & Systems Biology|
Far-field fluorescence Nanoscopy: (a) Confocal (upper) and STED (lower) images of Abberior Star 635P ...
STED microscopy of membrane interactions. (a) The “raft” principle: Lipids and proteins may interact ...
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, www.nanoscopyoxford.com) 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.
|Prof Simon J Davis||Investigative Medicine Division||University of Oxford||United Kingdom|
|Prof Vincenzo Cerundolo||Investigative Medicine Division||University of Oxford||United Kingdom|
|Prof Veronica J Buckle||Nuffield Division of Clinical Laboratory Sciences||University of Oxford||United Kingdom|
|Prof Graham Ogg||Investigative Medicine Division||University of Oxford||United Kingdom|
|Prof David G Jackson||Investigative Medicine Division||University of Oxford||United Kingdom|
|Dr Kerstin Luhn||Investigative Medicine Division||University of Oxford||United Kingdom|
|Dr Geraldine Gillespie||NDM Research Building||University of Oxford||United Kingdom|
|Dr Sergi Padilla-Parra||Structural Biology||University of Oxford||United Kingdom|
|Prof Jan Rehwinkel||Investigative Medicine Division||University of Oxford||United Kingdom|
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. Hide abstract
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. Hide abstract
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. Hide abstract
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. Hide abstract
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. Hide abstract
NEW JOURNAL OF PHYSICS, 11 (10), pp. 103054-103054. | Read more2009. Exploring single-molecule dynamics with fluorescence nanoscopy
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. Hide abstract
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. Hide abstract
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. Hide abstract
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. Hide abstract
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. Hide abstract
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. Hide abstract
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. Hide abstract