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• W3127872079 abstract "Optical super-resolution microscopy (SRM) has enabled biologists to visualize cellular structures with near-molecular resolution, giving unprecedented access to details about the amounts, sizes, and spatial distributions of macromolecules in the cell. Precisely quantifying these molecular details requires large datasets of high-quality, reproducible SRM images. In this review, we discuss the unique set of challenges facing quantitative SRM, giving particular attention to the shortcomings of conventional specimen preparation techniques and the necessity for optimal labeling of molecular targets. We further discuss the obstacles to scaling SRM methods, such as lengthy image acquisition and complex SRM data analysis. For each of these challenges, we review the recent advances in the field that circumvent these pitfalls and provide practical advice to biologists for optimizing SRM experiments. Optical super-resolution microscopy (SRM) has enabled biologists to visualize cellular structures with near-molecular resolution, giving unprecedented access to details about the amounts, sizes, and spatial distributions of macromolecules in the cell. Precisely quantifying these molecular details requires large datasets of high-quality, reproducible SRM images. In this review, we discuss the unique set of challenges facing quantitative SRM, giving particular attention to the shortcomings of conventional specimen preparation techniques and the necessity for optimal labeling of molecular targets. We further discuss the obstacles to scaling SRM methods, such as lengthy image acquisition and complex SRM data analysis. For each of these challenges, we review the recent advances in the field that circumvent these pitfalls and provide practical advice to biologists for optimizing SRM experiments. IntroductionSome 300 years ago, light microscopy guided the discovery that all living organisms consist of individual cells, pioneering the entire discipline of cell biology. Microscopy has been one of the most important and rapidly advancing laboratory techniques, and countless milestones in biology have coincided with the advent of better, more powerful microscopes. Indeed, it is probably not by chance that the image of a researcher looking through the eyepiece of a microscope has become a universal icon for the natural sciences. The introduction of fluorescence microscopy in the early twentieth century marked a major milestone in biology, allowing scientists to visualize previously invisible cellular structures by tagging them with highly specific fluorescent labels. Nevertheless, as with any optical system, the spatial resolution of fluorescence microscopy is limited by the diffraction (or bending) of light as it passes through the circular aperture of the microscope objective. First described by Ernst Abbe in 1873, the diffraction limit implies that the smallest object that can be resolved by an optical microscope is limited to approximately half the wavelength of the light being used (Abbe, 1873Abbe E. Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung.Arch. Für Mikroskopische Anat. 1873; 9: 413-468Google Scholar), amounting typically to ≥200 nm, or more than twenty times the size of an average protein. In the last few decades, a number of techniques collectively termed “super-resolution microscopy” (SRM) have surpassed this diffraction barrier, extending the capabilities of optical imaging to the nanoscale and yielding unprecedented biological insights (Balzarotti et al., 2017Balzarotti F. Eilers Y. Gwosch K.C. Gynnå A.H. Westphal V. Stefani F.D. Elf J. Hell S.W. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes.Science. 2017; 355: 606-612Google Scholar; Betzig et al., 2006Betzig E. Patterson G.H. Sougrat R. Lindwasser O.W. Olenych S. Bonifacino J.S. Davidson M.W. Lippincott-Schwartz J. Hess H.F. Imaging intracellular fluorescent proteins at nanometer resolution.Science. 2006; 313: 1642-1645Google Scholar; Chen et al., 2015Chen F. Tillberg P.W. Boyden E.S. Expansion microscopy.Science. 2015; 347: 543-548Google Scholar; Gustafsson, 2000Gustafsson M.G.L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. SHORT COMMUNICATION.J. Microsc. 2000; 198: 82-87Google Scholar; Hell and Wichmann, 1994Hell S.W. Wichmann J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy.Opt. Lett. 1994; 19: 780Google Scholar; Hess et al., 2006Hess S.T. Girirajan T.P.K. Mason M.D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.Biophys. J. 2006; 91: 4258-4272Google Scholar; Moerner and Kador, 1989Moerner W.E. Kador L. Optical detection and spectroscopy of single molecules in a solid.Phys. Rev. Lett. 1989; 62: 2535-2538Google Scholar; Rust et al., 2006Rust M.J. Bates M. Zhuang X.W. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).Nat. Methods. 2006; 3: 793-795Google Scholar; Sharonov and Hochstrasser, 2006Sharonov A. Hochstrasser R.M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes.Proc. Natl. Acad. Sci. 2006; 103: 18911-18916Google Scholar). As the field progressed, SRM shifted into the realm of quantitative biology, allowing for the accurate measurement of the abundances, distributions, and nanoscale movements of individual molecules. However, amassing such high-precision information requires more than mere access to super-resolution microscopes. Crucially, special care must be taken to prepare biological samples with minimal distortions and to select labels that are well suited to the chosen SRM approach. In addition, a good working knowledge of the applied technique is necessary to account for inherent limitations during acquisition and analysis, and to successfully scale up experiments to gather large datasets for robust quantifications. In this review, we will discuss the major obstacles to achieving quantitative SRM, particularly emphasizing the importance of optimal sample preparation and labeling. In addition, we will review the unique challenges facing SRM data analysis and the design of high-throughput SRM experiments. Our aim is to discuss possible solutions to these challenges based on both existing approaches in the field, as well as emerging techniques from recent years.An overview of common SRM techniquesThe key to achieving super resolution is minimizing the number of fluorophores that can be simultaneously detected within a diffraction-limited area. Most major SRM approaches achieve this by tampering with the on/off (bright/dark) state of the molecules to separate their emission in space, time, or both. These so-called “functional” SRM techniques broadly fall into one of two categories, namely, coordinated-targeted and stochastic, distinguished by their strategy for modulating fluorescent states. Coordinate-targeted SRM techniques use directed, focused lasers to actively push targeted fluorophores into a dark state. The best known of these techniques is stimulated emission depletion (STED) microscopy, where two superimposed beams are scanned over the sample in tandem: a focused excitation beam to switch the fluorophores on, and a powerful STED beam that depletes fluorophores through stimulated emission (Figure 1). The depletion beam is typically doughnut-shaped, gradually weakening toward its center, where it has ideally zero intensity. As a result, only fluorophores residing at the center of the beam can emit, whereas the rest of the diffraction-limited area remains dark (Hell and Wichmann, 1994Hell S.W. Wichmann J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy.Opt. Lett. 1994; 19: 780Google Scholar). Although commercial STED setups typically achieve a lateral resolution of ∼60 nm, resolutions down to ∼20 nm have been demonstrated in biological samples, and down to single nanometers in fluorescent nitrogen vacancies in diamond (Göttfert et al., 2013Göttfert F. Wurm C.A. Mueller V. Berning S. Cordes V.C. Honigmann A. Hell S.W. Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution.Biophys. J. 2013; 105: L01-L03Google Scholar; Rittweger et al., 2009Rittweger E. Han K.Y. Irvine S.E. Eggeling C. Hell S.W. STED microscopy reveals crystal colour centres with nanometric resolution.Nat. Photon. 2009; 3: 144-147Google Scholar; Wegel et al., 2016Wegel E. Göhler A. Lagerholm B.C. Wainman A. Uphoff S. Kaufmann R. Dobbie I.M. Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: a practical comparison.Sci. Rep. 2016; 6: 1-13Google Scholar). In the axial plane, 3D STED approaches such as isoSTED can achieve a resolution of ∼30 nm (Curdt et al., 2015Curdt F. Herr S.J. Lutz T. Schmidt R. Engelhardt J. Sahl S.J. Hell S.W. isoSTED nanoscopy with intrinsic beam alignment.Opt. Express. 2015; 23: 30891Google Scholar; Hell et al., 2009Hell S.W. Schmidt R. Egner A. Diffraction-unlimited three-dimensional optical nanoscopy with opposing lenses.Nat. Photon. 2009; 3: 381-387Google Scholar). A drawback of STED, however, is the high laser intensities required for efficient depletion of the fluorophores, which limits its applicability to live samples. As alternatives, techniques such as ground state depletion (GSD) and reversible saturable/switchable optically linear (fluorescence) transitions (RESOLFT) can be used, which require appreciably lower laser intensities to deplete the targeted fluorophores by transitioning them to additional “meta-stable” dark states (Bretschneider et al., 2007Bretschneider S. Eggeling C. Hell S.W. Breaking the diffraction barrier in fluorescence microscopy by optical shelving.Phys. Rev. Lett. 2007; 98: 218103Google Scholar; Hell and Kroug, 1995Hell S.W. Kroug M. Ground-state-depletion fluorscence microscopy: a concept for breaking the diffraction resolution limit.Appl. Phys. B Lasers Opt. 1995; 60: 495-497Google Scholar). Recent applications of RESOLFT have achieved resolutions comparable to those of STED with reduced excitation power, making them well suited for live-cell and large volume imaging (Grotjohann et al., 2011Grotjohann T. Testa I. Leutenegger M. Bock H. Urban N.T. Lavoie-Cardinal F. Willig K.I. Eggeling C. Jakobs S. Hell S.W. Diffraction-unlimited all-optical imaging and writing with a photochromic GFP.Nature. 2011; 478: 204-208Google Scholar; Masullo et al., 2018Masullo L.A. Bodén A. Pennacchietti F. Coceano G. Ratz M. Testa I. Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems.Nat. Commun. 2018; 9: 3281Google Scholar).Stochastic SRM techniques, collectively termed “single molecule localization microscopy” (SMLM), do not modulate the fluorophores spatially but rely on the stochastic nature of the on/off state transitions. Upon illuminating the entire sample with a widefield light source, only a small, random fraction of the fluorophores will switch states, ideally no more than a single fluorophore per diffraction-limited area. By repeatedly switching on random subsets of fluorophores, or passively measuring “off” fluorophores as they spontaneously return to their bright state, a super-resolved image of all fluorophore localizations can be ultimately reconstructed (Figure 1). Typically, hundreds to thousands of such sequential measurements are required in order to reconstruct an entire image, making acquisition times significantly longer than in targeted laser-scanning approaches. However, a major advantage of these techniques is that they can largely be realized using conventional wide-field setups and cameras, making them relatively simple and cost-effective. To date, numerous SMLM techniques have been devised, but these are mostly derivatives of two prototypical approaches: photoactivated localization microscopy (PALM) (Betzig et al., 2006Betzig E. Patterson G.H. Sougrat R. Lindwasser O.W. Olenych S. Bonifacino J.S. Davidson M.W. Lippincott-Schwartz J. Hess H.F. Imaging intracellular fluorescent proteins at nanometer resolution.Science. 2006; 313: 1642-1645Google Scholar; Hess et al., 2006Hess S.T. Girirajan T.P.K. Mason M.D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.Biophys. J. 2006; 91: 4258-4272Google Scholar), which relies on photoswitchable proteins, and stochastic optical reconstruction microscopy (STORM), which utilizes organic fluorophores (Rust et al., 2006Rust M.J. Bates M. Zhuang X.W. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).Nat. Methods. 2006; 3: 793-795Google Scholar). The main difference between the various techniques lies in their strategy for achieving the on/off switching and the types of photoswitchable probes that are used. A more recent approach, point accumulation in nanoscale topography (PAINT), does not utilize light-mediated activation but instead makes use of labels that fluoresce upon binding a target structure. By using dyes that transiently bind and quickly dissociate from their targets, a “blinking” effect can be produced, quite similar to that which is observed with photoswitchable dyes (Sharonov and Hochstrasser, 2006Sharonov A. Hochstrasser R.M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes.Proc. Natl. Acad. Sci. 2006; 103: 18911-18916Google Scholar). Alternatively, the bound fluorophores may be irreversibly switched off through photobleaching (Burnette et al., 2011Burnette D.T. Sengupta P. Dai Y. Lippincott-Schwartz J. Kachar B. Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules.Proc. Natl. Acad. Sci. 2011; 108: 21081-21086Google Scholar; Schoen et al., 2011Schoen I. Ries J. Klotzsch E. Ewers H. Vogel V. Binding-activated localization microscopy of DNA structures.Nano Lett. 2011; 11: 4008-4011Google Scholar). Both PALM/STORM and PAINT can reach resolutions on the order of tens of nanometers, but the actual values largely depend on the properties of the fluorophores being used. With sufficiently bright and photostable dyes, resolutions are typically ∼10–50 nm in xy on commercial setups, but can potentially reach single nanometers (Dai et al., 2016Dai M. Jungmann R. Yin P. Optical imaging of individual biomolecules in densely packed clusters.Nat. Nanotechnol. 2016; 11: 798-807Google Scholar; Vaughan et al., 2012Vaughan J.C. Jia S. Zhuang X. Ultrabright photoactivatable fluorophores created by reductive caging.Nat. Methods. 2012; 9: 1181-1184Google Scholar; Wegel et al., 2016Wegel E. Göhler A. Lagerholm B.C. Wainman A. Uphoff S. Kaufmann R. Dobbie I.M. Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: a practical comparison.Sci. Rep. 2016; 6: 1-13Google Scholar). SMLM frequently utilizes total internal reflection fluorescence (TIRF) illumination, where the excitation light is shined on the sample at an angle greater than the critical angle and is totally reflected at the glass/water interface. The reflection creates a thin illumination field (known as an “evanescent wave”) that penetrates the sample superficially, selectively exciting fluorophores near the interface, up to ∼100 nm (Diekmann et al., 2017Diekmann R. Helle Ø.I. Øie C.I. McCourt P. Huser T.R. Schüttpelz M. Ahluwalia B.S. Chip-based wide field-of-view nanoscopy.Nat. Photon. 2017; 11: 322-328Google Scholar). The resolution in z can be further enhanced to ∼50–70 nm with 3D systems such as 3D-STORM or iPALM (Huang et al., 2008Huang B. Jones S.A. Brandenburg B. Zhuang X. Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution.Nat. Methods. 2008; 5: 1047-1052Google Scholar; Lin et al., 2020Lin R. Clowsley A.H. Lutz T. Baddeley D. Soeller C. 3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples.Methods. 2020; 174: 56-71Google Scholar; Shtengel et al., 2009Shtengel G. Galbraith J.A. Galbraith C.G. Lippincott-Schwartz J. Gillette J.M. Manley S. Sougrat R. Waterman C.M. Kanchanawong P. Davidson M.W. et al.Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.Proc. Natl. Acad. Sci. 2009; 106: 3125-3130Google Scholar).Recently, a ground-breaking technique named MINFLUX was introduced, combining the strengths of both coordinate-targeted and stochastic SRM approaches. In MINFLUX, the fluorophores undergo both stochastic on/off switching, as in PALM/STORM, and simultaneous illumination with a doughnut-shaped excitation (rather than depletion) beam (Figure 1). The closer the emitting fluorophore to the zero-intensity center of the beam, the smaller the number of photons it will emit, and thus the fluorophore localizations can be deduced from local emission minima. MINFLUX boasts the highest precision of all SRM techniques to date, achieving resolutions of 1–3 nm in both the lateral and axial planes (Balzarotti et al., 2017Balzarotti F. Eilers Y. Gwosch K.C. Gynnå A.H. Westphal V. Stefani F.D. Elf J. Hell S.W. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes.Science. 2017; 355: 606-612Google Scholar; Gwosch et al., 2020Gwosch K.C. Pape J.K. Balzarotti F. Hoess P. Ellenberg J. Ries J. Hell S.W. MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells.Nat. Methods. 2020; 17: 217-224Google Scholar).An additional approach, structured illumination microscopy (SIM), does not rely on fluorescence states but instead exploits wave optics to construct a super-resolution image. In SIM, optical gratings are used to illuminate the sample with patterned light, resulting in the formation of interference patterns, also known as Moiré fringes. Put simply, these fringes are a mixture between the frequency patterns of light emitted by the sample and the frequency of the illumination pattern, and because the latter is known, the former can be retrieved mathematically. In a SIM measurement, the sample is illuminated multiple times with rotations of the grating pattern at different angles (Figure 1). In total, ∼15 rotations are required to reconstruct an image with a lateral resolution of ∼100 nm (Gustafsson, 2000Gustafsson M.G.L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. SHORT COMMUNICATION.J. Microsc. 2000; 198: 82-87Google Scholar). In the axial plane, the introduction of an additional patterned illumination allows a typical resolution of ∼300–400 nm (Schermelleh et al., 2008Schermelleh L. Carlton P.M. Haase S. Shao L. Winoto L. Kner P. Burke B. Cardoso M.C. Agard D.A. Gustafsson M.G.L. et al.Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy.Science. 2008; 320: 1332-1336Google Scholar). Higher lateral resolutions (<50 nm) can be obtained with the non-linear form of SIM (NL-SIM) that relies on saturation of the fluorophore excited state with a high intensity light to produce a nonlinear response between the excitation and emission intensities (Li et al., 2015Li D. Shao L. Chen B.-C. Zhang X. Zhang M. Moses B. Milkie D.E. Beach J.R. Hammer J.A. Pasham M. et al.Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics.Science. 2015; 349: aab3500Google Scholar).Lastly, expansion microscopy (ExM) presents a creative solution for overcoming the diffraction limit without any need for optical “tricks”, by physically expanding the sample itself (Chen et al., 2015Chen F. Tillberg P.W. Boyden E.S. Expansion microscopy.Science. 2015; 347: 543-548Google Scholar). In ExM, the fluorescently labeled sample is embedded in a swellable polymer gel and the fluorophores are linked to the gel matrix. The sample is digested, and the gel is expanded in all dimensions, physically distancing the fluorescent labels (Figure 1). As a result, super-resolution images can be acquired with conventional, diffraction-limited microscopes and with most conventional dyes. Most variants of ExM expand samples by a factor of ∼4, allowing a lateral resolution of ∼70 nm, and iterative ExM, which includes a second gel step, allows x10 expansion, achieving a resolution of ∼25 nm (Truckenbrodt et al., 2018Truckenbrodt S. Maidorn M. Crzan D. Wildhagen H. Kabatas S. Rizzoli S.O. X10 expansion microscopy enables 25-nm resolution on conventional microscopes.EMBO Rep. 2018; 19: e45836Google Scholar). In contrast to the previously mentioned techniques, ExM is limited to fixed samples only. On a cautionary note, it has been demonstrated that the expansion factor within a single cell may vary and should therefore be validated carefully when performing ExM experiments (Büttner et al., 2020Büttner M. Lagerholm C.B. Waithe D. Galiani S. Schliebs W. Erdmann R. Eggeling C. Reglinski K. Challenges of Using Expansion Microscopy for Super-resolved Imaging of Cellular Organelles. ChemBioChem cbic, 2020: 202000571Google Scholar).Variations on the techniques described earlier as well as the conception of new technologies are continually emerging. Since it is well beyond the scope of this review to describe these here, we refer the reader to more extensive reviews of SRM techniques (Sahl and Hell, 2019Sahl S.J. Hell S.W. High Resolution Imaging in Microscopy and Ophthalmology. Springer International Publishing, 2019Google Scholar; Vangindertael et al., 2018Vangindertael J. Camacho R. Sempels W. Mizuno H. Dedecker P. Janssen K.P.F.F. An introduction to optical super-resolution microscopy for the adventurous biologist.Methods Appl. Fluoresc. 2018; 6: 022003Google Scholar).Super-resolution microscopy paves the way for quantitative biological insightsOver the recent years, improvements in measurement fidelity and novel analyses have allowed biologists to ask not only where a molecule is located but also how many molecules there are. As per their name, SMLM techniques are particularly well suited to tackle this question, because the number of molecules can, in theory, be inferred from the discrete single-molecule localizations. Through careful characterization of different experimental variables such as the labeling efficiency of the probes and fluorophore blinking kinetics (see Challenge 4, below), it becomes possible to estimate the true molecule amounts and stoichiometries. For example, PALM was used to count the number of lipid binding sites on endocytic vesicles in yeast, showing how these vary throughout vesicle maturation (Puchner et al., 2013Puchner E.M. Walter J.M. Kasper R. Huang B. Lim W.A. Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory.Proc. Natl. Acad. Sci. 2013; 110: 16015-16020Google Scholar). In a more recent study, quantitative DNA-PAINT was used to determine the stoichiometry between ryanodine receptors (RyRs) and the RyR inhibitory protein junctophilin-1 on the membranes of cardiomyocytes. The authors found that the ratio of expression between the two proteins is highly heterogeneous, implying that other forms of RyR regulation are likely to exist (Jayasinghe et al., 2018Jayasinghe I. Clowsley A.H. Lin R. Lutz T. Harrison C. Green E. Baddeley D. Di Michele L. Soeller C. True molecular scale visualization of variable clustering properties of ryanodine receptors.Cell Rep. 2018; 22: 557-567Google Scholar). Molecule counting can also be achieved by SRM techniques that do not compute single-molecule localizations. For example, using STED, the number of internalized transferrin receptors could be accurately counted through analysis of photon emission statistics, relying on the idea that fluorophores can only emit a single photon at a given time, and thus a simultaneous detection of multiple photons should indicate the presence of multiple molecules (Ta et al., 2015Ta H. Keller J. Haltmeier M. Saka S.K. Schmied J. Opazo F. Tinnefeld P. Munk A. Hell S.W. Mapping molecules in scanning far-field fluorescence nanoscopy.Nat. Commun. 2015; 6: 7977Google Scholar) (see Challenge 4, below) (Figures 2A-2C).Figure 2Biological insights from quantitative super-resolution microscopy. The scheme shows three types of quantitative SRM analyses and example studies.Show full caption(A–C) Counting molecules and determining molecular stoichiometries. (A) PALM images of PI3P binding sites on endocytic vesicles colocalizing with markers of different maturation stages (top to bottom: clathrin, the GTPase Vps21, the GTPase Ypt7Scale). The images show that increasingly mature vesicles have a higher PI3P content. Scale bar: 100 nm. Adapted with permission from (Puchner et al., 2013Puchner E.M. Walter J.M. Kasper R. Huang B. Lim W.A. Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory.Proc. Natl. Acad. Sci. 2013; 110: 16015-16020Google Scholar). (B) Left panel: STED and confocal images of internalized TfRs (axial summation of 0.9 μm). H is the maximal intensity value (number of photon counts) per pixel. Right panel: 3D molecular map generated by photon statistics of STED and confocal recordings. Inset: isosurfaces of the molecular map (corresponding to the boxed region, encompassing ∼70% of these molecules). The colors represent the number of molecules in the corresponding region. Scale bars: 1 μm. Adapted from (Ta et al., 2015Ta H. Keller J. Haltmeier M. Saka S.K. Schmied J. Opazo F. Tinnefeld P. Munk A. Hell S.W. Mapping molecules in scanning far-field fluorescence nanoscopy.Nat. Commun. 2015; 6: 7977Google Scholar), CC-BY-4.0. (C) Exchange qPAINT image of RyR receptors (red) and JPH2 (green) co-clusters. The ratio between the number of JPH2 and RyR molecules is determined for each cluster. Scale bar: 250 nm. Adapted from (Jayasinghe et al., 2018Jayasinghe I. Clowsley A.H. Lin R. Lutz T. Harrison C. Green E. Baddeley D. Di Michele L. Soeller C. True molecular scale visualization of variable clustering properties of ryanodine receptors.Cell Rep. 2018; 22: 557-567Google Scholar), CC-BY-4.0. (D–F) Spatial organization/clustering of molecules.(D) 2-color PALM image of TCRs (red) co-clustered with the linker for activation of T cells LAT (green). Scale bar: 2 μm. Adapted from (Sherman et al., 2013Sherman E. Barr V. Samelson L.E. Super-resolution characterization of TCR-dependent signaling clusters.Immunol. Rev. 2013; 251: 21-35Google Scholar), with permission from John Wiley and Sons.(E) Examples of AMPA receptors (GluA1/2 subunits) clustered in “nanodomains” inside dendritic spines, imaged by PALM, STED, or PAINT (top to bottom panels, respectively). Scale bar: 1 μm. Adapted from (Nair et al., 2013Nair D. Hosy E. Petersen J.D. Constals A. Giannone G. Choquet D. Sibarita J.-B. Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95.J. Neurosci. 2013; 33: 13204-13224Google Scholar), CC BY-NC-SA 3.0.(F) Right: PALM images of single clusters of Gag HIV assembly sites (red) colocalizing with ESCRT-I subunits Tsg101 (green) from axial and lateral views (top and bottom panels, respectively). Scale bar: 50 nm. Left: 3D single-cluster averaging of Gag and Tsg101 demonstrate the presence of ESCRT subunits within the interior of the HIV Gag lattice. Adapted from (Van Engelenburg et al., 2014Van Engelenburg S.B. Shtengel G. Sengupta P. Waki K. Jarnik M. Ablan S.D. Freed E.O. Hess H.F. Lippincott-Schwartz J. Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits.Science. 2014; 343: 653-656Google Scholar), with permission from AAAS.(G–I) Determining molecular interactions through colocalization analyses. (G) Top: 3D STORM image of RIM1/2 (red) and PSD-95 (blue) nanoclusters, with a pixel size of 10 nm, compared with a widefield composite (bottom corner) with a pixel size of 100 nm. Scale bar: 2 μm. Bottom: enlargement of the boxed region in the original and rotated angles (left and right, respectively). Scale bar: 200 nm. Adapted from (Tang et al., 2016Tang A.-H.H. Chen H. Li T.P. Metzbower S.R. MacGillavry H.D. Blanpied T.A. A trans-synaptic nanocolumn aligns neurotransmitter release to receptors.Nature. 2016; 536: 210-214Google Scholar), with permission from Springer Nature. (H) Left: confocal (top) and 3D SIM (bottom) images of a synapse, labeled for Gephyrin (green), GABAA receptors (red), and the vesicular glutamate transporter VGAT (blue). Scale bars: 1 μm (top) and 500 nm (bottom). Right: 3D reconstruction of a single synapse. Scale bar: 500 nm. Adapted from (Crosby et al., 2019Crosby K.C. Gookin S.E. Garcia J.D. Hahm K.M. Dell’Acqua M.L. Smith K.R. Nanoscale subsynaptic domains underlie the organization of the inhibitory synapse.Cell Rep. 2019; 26: 3284-3297.e3Google Scholar), with permission from Elsevier. (I) Multiplex STORM images showing five different synaptic targets images in the same section (out of 16 targets in total). Left, top to bottom: actin marked by phalloidin; the endosome-associated protein Rab5; Golgi marked by GM130. Right, top to bottom: clathrin marked by CHC17; the endosome-associated protein EEA1; overlay of all five channels. Scale bar: 2 μm. Adapted from (Klevanski et al., 2020Klevanski M. Herrmannsdoerfer F. Sass S. Venkataramani V. Heilemann M. Kuner T. Automated highly multiplexed super-resolution imaging of protein nano-architecture in cells and tissues.Nat. Commun. 2020; 11: 1552Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The improved accuracy of SRM also allows more meticulous investigations of spatial organizatio" @default.
• W3127872079 created "2021-02-15" @default.
• W3127872079 creator A5061167925 @default.
• W3127872079 creator A5085252430 @default.
• W3127872079 date "2021-03-01" @default.
• W3127872079 modified "2023-10-18" @default.
• W3127872079 title "Challenges facing quantitative large-scale optical super-resolution, and some simple solutions" @default.
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