Low-power compact continuous-wave stimulated emission depletion microscopy

Stimulated emission depletion (STED) microscopy can break the optical diffraction barrier and provide subdiffraction resolution. According to the STED superresolution imaging principle, the resolution of STED is positively related to the power of the depletion laser. However, high-laser power largely limits the study of living cells or living bodies. Moreover, the high complexity and high cost of conventional pulsed STED microscopy limit the application of this technique. Therefore, this paper describes a simple continuous-wave STED (CW-STED) system constructed on a 45 × 60 cm breadboard and combined with digitally enhanced (DE) technology; low-power superresolution imaging is realized, which has the advantages of reducing system complexity and cost. The low-system complexity, low cost, and low-power superresolution imaging features of CW-STED have great potential to advance the application of STED microscopy in biological research.     

Nanoscale resolution in GFP-based microscopy

We report attainment of subdiffraction resolution using stimulated emission depletion (STED) microscopy with GFP-labeled samples. The approximately 70 nm lateral resolution attained in this study is demonstrated by imaging GFP-labeled viruses and the endoplasmic reticulum (ER) of a mammalian cell. Our results mark the advent of nanoscale biological microscopy with genetically encoded markers.     

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in <80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.     

Live-Cell Superresolution Imaging by Pulsed STED Two-Photon Excitation Microscopy

Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of one-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of many small subcellular compartments of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality that overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. Here, we describe the design and operation of a superresolution two-photon microscope using pulsed excitation and STED lasers. We examine the depth dependence of STED imaging in acute tissue slices and find enhancement of 2P resolution ranging from approximately fivefold at 20 μm to approximately twofold at 90-μm deep. The depth dependence of resolution is found to be consistent with the depth dependence of depletion efficiency, suggesting resolution is limited by STED laser propagation through turbid tissue. Finally, we achieve live imaging of dendritic spines with 60-nm resolution and demonstrate that our technique allows accurate quantification of neuronal morphology up to 30-μm deep in living brain tissue.     

STED microscopy with continuous wave beams

We report stimulated emission depletion (STED) fluorescence microscopy with continuous wave (CW) laser beams. Lateral fluorescence confinement from the scanning focal spot delivered a resolution of 29-60 nm in the focal plane, corresponding to a 5-8-fold improvement over the diffraction barrier. Axial spot confinement increased the axial resolution by 3.5-fold. We observed three-dimensional (3D) subdiffraction resolution in 3D image stacks. Viable for fluorophores with low triplet yield, the use of CW light sources greatly simplifies the implementation of this concept of far-field fluorescence nanoscopy.     

Limited Intermixing of Synaptic Vesicle Components upon Vesicle Recycling

Synaptic vesicles recycle repeatedly in order to maintain synaptic transmission. We have previously proposed that upon exocytosis the vesicle components persist as clusters, which would be endocytosed as whole units. It has also been proposed that the vesicle components diffuse into the plasma membrane and are then randomly gathered into new vesicles. We found here that while strong stimulation (releasing the entire recycling pool) causes the diffusion of the vesicle marker synaptotagmin out of synaptic boutons, moderate stimulation (releasing ～19% of all vesicles) is followed by no measurable diffusion. In agreement with this observation, synaptotagmin molecules labeled with different fluorescently tagged antibodies did not appear to mix upon vesicle recycling, when investigated by subdiffraction resolution stimulated emission depletion (STED) microscopy. Finally, as protein diffusion from vesicles has been mainly observed using molecules tagged with pH‐sensitive green fluorescent protein (pHluorin), we have also investigated the membrane patterning of several native and pHluorin‐tagged proteins. While the native proteins had a clustered distribution, the GFP‐tagged ones were diffused in the plasma membrane. We conclude that synaptic vesicle components intermix little, at least under moderate stimulation, possibly because of the formation of clusters in the plasma membrane. We suggest that several pHluorin‐tagged vesicle proteins are less well integrated in clusters.     

STED-SPIM: Stimulated emission depletion improves sheet illumination microscopy resolution

We demonstrate the first, to our knowledge, integration of stimulated emission depletion (STED) with selective plane illumination microscopy (SPIM). Using this method, we were able to obtain up to 60% improvements in axial resolution with lateral resolution enhancements in control samples and zebrafish embryos. The integrated STED-SPIM method combines the advantages of SPIM with the resolution enhancement of STED, and thus provides a method for fast, high-resolution imaging with >100 μm deep penetration into biological tissue.     

A new filtering technique for removing anti‐Stokes emission background in gated CW‐STED microscopy

Stimulated emission depletion (STED) microscopy is a prominent approach of super‐resolution optical microscopy, which allows cellular imaging with so far unprecedented unlimited spatial resolution. The introduction of time‐gated detection in STED microscopy significantly reduces the (instantaneous) intensity required to obtain sub‐diffraction spatial resolution. If the time‐gating is combined with a STED beam operating in continuous wave (CW), a cheap and low labour demand implementation is obtained, the so called gated CW‐STED microscope. However, time‐gating also reduces the fluorescence signal which forms the image. Thereby, background sources such as fluorescence emission excited by the STED laser (anti‐Stokes fluorescence) can reduce the effective resolution of the system. We propose a straightforward method for subtraction of anti‐Stokes background. The method hinges on the uncorrelated nature of the anti‐Stokes emission background with respect to the wanted fluorescence signal. The specific importance of the method towards the combination of two‐photon‐excitation with gated CW‐STED microscopy is demonstrated. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Self-Calibrated Line-Scan STED-FCS to Quantify Lipid Dynamics in Model and Cell Membranes

Only a limited number of noninvasive techniques are available to directly measure the dynamic behavior of lipids in model and cell membranes. Here, we explored whether a commercial instrument could be used for fluorescence correlation spectroscopy (FCS) under pulsed stimulated emission depletion (STED). To overcome issues with photobleaching and poor distinction between confocal and STED signals, we implemented resonant line-scan STED with filtered FCS, which has the additional benefit of autocalibrating the dimensions of the point-spread function and obtaining spatially resolved molecular mobility at subdiffraction resolution. With supported lipid bilayers, we achieved a detection spot radius of 40 nm, although at the expense of decreased molecular brightness. We also used this approach to map the dynamics of Atto646N-labeled sphingomyelin and phosphatidylethanolamine in the plasma membrane. Despite the reliability of the method and the demonstration that photobleaching and the photophysical properties of the dye did not influence diffusion measurements, we found great heterogeneities even within one cell. For both lipids, regions of high local density correlated with slow molecular diffusion, indicating trapping of Atto646N-labeled lipids. Future studies with new dyes are needed to reveal the origin of the trapping.     

Two-color STED microscopy of living synapses using a single laser-beam pair

The advent of superresolution microscopy has opened up new research opportunities into dynamic processes at the nanoscale inside living biological specimens. This is particularly true for synapses, which are very small, highly dynamic, and embedded in brain tissue. Stimulated emission depletion (STED) microscopy, a recently developed laser-scanning technique, has been shown to be well suited for imaging living synapses in brain slices using yellow fluorescent protein as a single label. However, it would be highly desirable to be able to image presynaptic boutons and postsynaptic spines, which together form synapses, using two different fluorophores. As STED microscopy uses separate laser beams for fluorescence excitation and quenching, incorporation of multicolor imaging for STED is more difficult than for conventional light microscopy. Although two-color schemes exist for STED microscopy, these approaches have several drawbacks due to their complexity, cost, and incompatibility with common labeling strategies and fluorophores. Therefore, we set out to develop a straightforward method for two-color STED microscopy that permits the use of popular green-yellow fluorescent labels such as green fluorescent protein, yellow fluorescent protein, Alexa Fluor 488, and calcein green. Our new (to our knowledge) method is based on a single-excitation/STED laser-beam pair to simultaneously excite and quench pairs of these fluorophores, whose signals can be separated by spectral detection and linear unmixing. We illustrate the potential of this approach by two-color superresolution time-lapse imaging of axonal boutons and dendritic spines in living organotypic brain slices.     

Nanoscopy of Filamentous Actin in Cortical Dendrites of a Living Mouse

We demonstrate superresolution fluorescence microscopy (nanoscopy) of protein distributions in a mammalian brain in vivo. Stimulated emission depletion microscopy reveals the morphology of the filamentous actin in dendritic spines down to 40 μm in the molecular layer of the visual cortex of an anesthetized mouse. Consecutive recordings at 43–70 nm resolution reveal dynamical changes in spine morphology.The postsynaptic part of most excitatory synapses in the brain is formed by dendritic spines, which are small protrusions along the dendrites that are highly dynamic during development, but also undergo morphological changes in adulthood (1,2). A prime candidate for regulating these dynamics is the neuronal actin network (3). Filamentous (F-) actin is also important for anchoring postsynaptic receptors and modulating synaptic activities, e.g., through the organization of the postsynaptic density (3). Clearly, the actin dynamics of dendritic spines is best studied in vivo, e.g., in a living mouse, and with confocal and multiphoton microscopy because these techniques can provide three-dimensional optical sectioning several 100 μm inside brain tissue (4). However, because necks of dendritic spines are on the 50–150-nm scale, their details are beyond the 250–400-nm resolution afforded by these diffraction-limited techniques. Fortunately, the diffraction resolution barrier of lens-based fluorescence microscopy has recently been overcome by causing the fluorophores of nearby features to emit sequentially (5). One of the techniques relying on this principle, stimulated emission depletion (STED) microscopy, has recently resolved dendritic spines in the cortex of a living mouse (6). In that initial, in vivo superresolution study, the dendrites were only volume-labeled, and consequently, the spatial arrangements of specific cytoskeletal proteins could not be imaged. On the other hand, F-actin has actually been imaged in living brain slices (7), but in vivo imaging of these structures has not yet been attained.Compared to other superresolution or nanoscopy techniques, STED microscopy bears a number of advantages for imaging spines in the living brain. Implemented as a beam scanning confocal microscope, STED nanoscopy offers optical sectioning and measurements at greater depth. In addition, motion artifacts of the dynamic structures can be minimized by fast scanning. And last but not least, STED can be performed with standard fluorescent proteins. Therefore, we here apply STED nanoscopy to noninvasively uncover the actin cytoskeleton in the living mouse brain. In particular, we show that the 43–70-nm resolution obtained by STED visualizes rearrangements of the dendritic spines in vivo.We took on the challenge of labeling the actin cytoskeleton in the living mouse cortex. We utilized Lifeact-EYFP, a fusion protein consisting of a small peptide and the yellow fluorescent protein EYFP, which directly binds to F-actin without disturbing its polymerization (8). The labeling itself was accomplished by viral infection. To this end, adeno-associated viral particles (AAV) of serotype 2, facilitated by the neuron specific human synapsin promoter hSYN (9) and Semliki Forest viruses (SFV), were created to express Lifeact-EYFP in neurons. For virus injection, the mouse was anesthetized and the head was fixed in a model No. SG-4N head holder (Narishige International USA, East Meadow, NY). A 5-mm incision of the skin of the head enabled drilling a 0.5-mm-diameter hole into the skull. The hole was positioned 0.5 mm outside the prospective imaging center in the visual cortex. The AAVs were injected with a micropipette connected to a pressure generator (Tooheyspritzer; Toohey Company, Fairfield, NJ). Thus, we were able to inject ∼750 nL of concentrated AAV at an angle of 30° over a time of ∼5 min to the layer of pyramidal cells in the prospective imaging center. After injection and 5-min pause, the pipette was retracted with a 5-min break at the half-way point to allow the virus to diffuse into the tissue. The skin was closed with three stitches and the mouse kept on a heating plate in an anesthetic recovery box until wake-up.After 10 days the mouse was prepared for in vivo STED nanoscopy, according to Berning et al. (6) (see also the Supporting Material). At this point, the skin had completely healed and the mouse showed no sign of obvious behavioral abnormality. Optical access was provided by a glass-sealed hole of ∼2 mm in diameter, exposing the visual cortex (Fig. 1 a). STED nanoscopy was performed with an upright beam-scanning microscope similar to that described by Berning et al. (6), with short optical paths and good vibration-damping (Fig. 1 b and see the Supporting Material). The coaligned excitation and STED beams were focused onto the mouse brain using a 1.3 numerical-aperture glycerol immersion lens. The correction collar of the lens allowed compensation of spherical aberrations arising from focusing beneath the brain surface (7).Open in a separate windowFigure 1STED nanoscopy of the dendritic filamentous (F-) actin cytoskeleton in the visual cortex of a living mouse. (a) Clear view of the visual cortex through an optical window. (b) Upright STED imaging of the anesthetized mouse. (c) Dendritic F-actin in the molecular layer of the visual cortex at 4, 25, and 40-μm depths. Maximum intensity projection of a stack of five (xy) images taken in 500-nm axial (z) distances. (Right) Line profile at the marked positions; average of five lines of the raw data and Lorentz fit with full width at half-maximum (FWHM); all image data are raw.Fig. 1 c shows representative parts of dendrites in the molecular layer of the visual cortex. The combination of Lifeact-EYFP labeling and superresolution displayed the dendritic actin of the living mouse neuron in unprecedented detail. Most spines have an actin-rich bulbous end, i.e., a spine head. Sometimes, the dendrite shows small areas with high actin enrichment, which presumably constitute the beginning of filopodia outgrowths (see Fig. S1 in the Supporting Material). The STED image quality was maintained down to a depth of 40 μm below the cover glass. The actin filaments in the spine neck were 43–70-nm thin (see Fig. S2), which can also be interpreted as an upper estimate (poorest value) for the resolution obtained by STED. Note that the images were not processed after recording. All dendrites appeared normal, i.e., in comparison with the morphology of volume-labeled pyramidal cells of transgenic mice (6). The STED beam average laser power was 34 mW. For somewhat greater laser power, we occasionally saw swelling of the dendrites but they were never destroyed. The maximum applicable power depends on the thickness of the dendrite and most likely on the presence of mitochondria as well.Next, we raised the expression level of Lifeact-EYFP by replacing AAV with SFV infection (7,10). We injected 750 nL of SFV (see the Supporting Material) analog to the AAV protocol and allowed the mouse to wake up and recover. After one day, we recorded in vivo STED nanoscopy images of the visual cortex. The labeling was sparser than with the AAV, i.e., fewer cells expressed Lifeact-EYFP, but the signal was brighter and highly specific to neurons. Fig. 2 shows a STED image of a part of a dendrite in the visual cortex at depth <10 μm. The actin label is brighter in the spine heads than in the body of the dendrite, showing that Lifeact-EYFP is primarily attached to F-actin. STED recording over 12 min revealed morphological changes in the actin cytoskeleton. No changes were observed after fixation (see Fig. S4). Bleaching-corrected brightness changes in the spine head inherently reflect density changes in the actin network. In contrast to AAV, SFV shuts down host cell protein synthesis, which leads to cell death after >24 h (11,12); this was not improved by the less cytotoxic SFV(PD) variant (11). Therefore, we recorded in vivo nanoscopy images one day after viral transduction where most dendrites looked healthy. To confirm the viral transduction and verify the subtype of the infected neurons, we perfused the mouse with paraformaldehyde and imaged the brain slices of the region of interest (see Fig. S5). Whereas the AAV labeled mainly neurons of the pyramidal layer, the SFV infected sparsely neurons from all layers of the cortex.Open in a separate windowFigure 2Actin rearrangement in dendritic spines at 60-nm subdiffraction spatial resolution. Image stacks reveal dynamic changes of actin in the spines. (Arrows) Shape changes of spine heads. Maximum intensity projection of five slices of 500-nm axial (z) separation; all data are raw. Average power at back-aperture of objective lens: 2.4 μW excitation and 38-mW STED.The 4–5-fold lateral resolution improvement of STED over standard confocal and multiphoton microscopy is not sufficient to resolve single actin fibers, as with platinum replica electron microscopy (13). Future refinements of both labeling and STED imaging should make this goal achievable. The resolution along the optical (z) axis was kept diffraction-limited (∼500 nm) so that the total illumination dose remained small. At depth >40 μm, scattering and aberrations compromise the image quality. Nonetheless, the molecular layer of the sensory cortex is a highly interesting target for functional optical nanoscopy, because it is the site of the first stage of cortical sensory processing.In summary, STED microscopy can be applied to study subcellular protein structures at 43–70-nm resolution down to 40 μm in the brain of a living mammal. Specifically, we showed that the dynamic actin network responsible for the morphologic plasticity in the brain can be superresolved in the living mouse. Extending in vivo STED microscopy to other protein assemblies as well as to other cell types should provide basic insights into the working principles of the brain.     

isoSTED microscopy with water-immersion lenses and background reduction

Fluorescence microscopy is an excellent tool to gain knowledge on cellular structures and biochemical processes. Stimulated emission depletion (STED) microscopy provides a resolution in the range of a few 10 nm at relatively fast data acquisition. As cellular structures can be oriented in any direction, it is of great benefit if the microscope exhibits an isotropic resolution. Here, we present an isoSTED microscope that utilizes water-immersion objective lenses and enables imaging of cellular structures with an isotropic resolution of better than 60 nm in living samples at room temperature and without CO₂ supply or another pH control. This corresponds to a reduction of the focal volume by far more than two orders of magnitude as compared to confocal microscopy. The imaging speed is in the range of 0.8 s/μm³. Because fluorescence signal can only be detected from a diffraction-limited volume, a background signal is inevitably observed at resolutions well beyond the diffraction limit. Therefore, we additionally present a method that allows us to identify this unspecific background signal and to remove it from the image.     

STED microscopy of living cells – new frontiers in membrane and neurobiology

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.     

Increasing fluorescence lifetime for resolution improvement in stimulated emission depletion nanoscopy

Super‐resolution microscopy (SRM) has had a substantial impact on the biological sciences due to its ability to observe tiny objects less than 200 nm in size. Stimulated emission depletion (STED) microscopy represents a major category of these SRM techniques that can achieve diffraction‐unlimited resolution based on a purely optical modulation of fluorescence behaviors. Here, we investigated how the laser beams affect fluorescence lifetime in both confocal and STED imaging modes. The results showed that with increasing illumination time, the fluorescence lifetime in two kinds of fluorescent microspheres had an obvious change in STED imaging mode, compared with that in confocal imaging mode. As a result, the reduction of saturation intensity induced by the increase of fluorescence lifetime can improve the STED imaging resolution at the same depletion power. The phenomenon was also observed in Star635P‐labeled human Nup153 in fixed HeLa cells, which can be treated as a reference for the synthesis of fluorescent labels with the sensitivity to the surrounding environment for resolution improvement in STED nanoscopy.      

Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy

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 ∼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.     

STED microscopy with optimized labeling density reveals 9-fold arrangement of a centriole protein

Super-resolution fluorescence microscopy can achieve resolution beyond the optical diffraction limit, partially closing the gap between conventional optical imaging and electron microscopy for elucidation of subcellular architecture. The centriole, a key component of the cellular control and division machinery, is 250 nm in diameter, a spatial scale where super-resolution methods such as stimulated emission depletion (STED) microscopy can provide previously unobtainable detail. We use STED with a resolution of 60 nm to demonstrate that the centriole distal appendage protein Cep164 localizes in nine clusters spaced around a ring of ～300 nm in diameter, and quantify the influence of the labeling density in STED immunofluorescence microscopy. We find that the labeling density dramatically influences the observed number, size, and brightness of labeled Cep164 clusters, and estimate the average number of secondary antibody labels per cluster. The arrangements are morphologically similar in centrioles of both proliferating cells and differentiated multiciliated cells, suggesting a relationship of this structure to function. Our STED measurements in single centrioles are consistent with results obtained by electron microscopy, which involve ensemble averaging or very different sample preparation conditions, suggesting that we have arrived at a direct measurement of a centriole protein by careful optimization of the labeling density.     

STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects

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.     

RESOLFT Nanoscopy of Fixed Cells Using a Z-Domain Based Fusion Protein for Labelling

RESOLFT super-resolution microscopy allows subdiffraction resolution imaging of living cells using low intensities of light. It relies on the light-driven switching of reversible switchable fluorescent proteins (RSFPs). So far, RESOLFT imaging was restricted to living cells, because chemical fixation typically affects the switching characteristics of RSFPs. In this study we created a fusion construct (FLASR) consisting of the RSFP rsEGFP2 and the divalent form of the antibody binding Z domain from protein A. FLASR can be used analogous to secondary antibodies in conventional immunochemistry, facilitating simple and robust sample preparation. We demonstrate RESOLFT super-resolution microscopy on chemically fixed mammalian cells. The approach may be extended to other super-resolution approaches requiring fluorescent proteins in an aqueous environment.     

Bioothogonally applicable, π-extended rhodamines for super-resolution microscopy imaging for intracellular proteins

A set of new, bioorthogonally applicable tetrazine and polarity modulated double fluorogenic π-extended rhodamine probes were synthesized. Fluorogenicity and cell labeling experiments suggest that combination of the two quenching mechanisms allows low background labeling schemes even for probes with poor reactivity based fluorogenicity. Two of the new probes were tested in biological labeling schemes of intracellular proteins both in fixed and live cells. The labeled cells were subsequently subjected to confocal and STED imaging. These studies revealed that the rhodaindanes tested are membrane permeable, can stand the challenging environment of live cells and suitable for bioorthogonal, site-specific labeling of intracellular proteins. Furthermore, we found that both probes are suitable for subdiffraction imaging of the labeled structures using STED microscopy.     

Applications of STED fluorescence nanoscopy in unravelling nanoscale structure and dynamics of biological systems

Fluorescence microscopy, especially confocal microscopy, has revolutionized the field of biological imaging. Breaking the optical diffraction barrier of conventional light microscopy, through the advent of super-resolution microscopy, has ushered in the potential for a second revolution through unprecedented insight into nanoscale structure and dynamics in biological systems. Stimulated emission depletion (STED) microscopy is one such super-resolution microscopy technique which provides real-time enhanced-resolution imaging capabilities. In addition, it can be easily integrated with well-established fluorescence-based techniques such as fluorescence correlation spectroscopy (FCS) in order to capture the structure of cellular membranes at the nanoscale with high temporal resolution. In this review, we discuss the theory of STED and different modalities of operation in order to achieve the best resolution. Various applications of this technique in cell imaging, especially that of neuronal cell imaging, are discussed as well as examples of application of STED imaging in unravelling structure formation on biological membranes. Finally, we have discussed examples from some of our recent studies on nanoscale structure and dynamics of lipids in model membranes, due to interaction with proteins, as revealed by combination of STED and FCS techniques.