Diffraction Limit Of Light Microscopy Research Articles

Pushing the Resolution Limit of Stimulated Emission Depletion Optical Nanoscopy.

Optical nanoscopy, also known as super-resolution optical microscopy, has provided scientists with the means to surpass the diffraction limit of light microscopy and attain new insights into nanoscopic structures and processes that were previously inaccessible. In recent decades, numerous studies have endeavored to enhance super-resolution microscopy in terms of its spatial (lateral) resolution, axial resolution, and temporal resolution. In this review, we discuss recent efforts to push the resolution limit of stimulated emission depletion (STED) optical nanoscopy across multiple dimensions, including lateral resolution, axial resolution, temporal resolution, and labeling precision. We introduce promising techniques and methodologies building on the STED concept that have emerged in the field, such as MINSTED, isotropic STED, and event-triggered STED, and evaluate their respective strengths and limitations. Moreover, we discuss trade-off relationships that exist in far-field optical microscopy and how they come about in STED optical nanoscopy. By examining the latest developments addressing these aspects, we aim to provide an updated overview of the current state of STED nanoscopy and its potential for future research.

Analyzing the cellular plasma membrane by fast and efficient correlative STED and platinum replica EM

The plasma membrane of mammalian cells links transmembrane receptors, various structural components, and membrane-binding proteins to subcellular processes, allowing inter- and intracellular communication. Therefore, membrane-binding proteins, together with structural components such as actin filaments, modulate the cell membrane in their flexibility, stiffness, and curvature. Investigating membrane components and curvature in cells remains challenging due to the diffraction limit in light microscopy. Preparation of 5–15-nm-thin plasma membrane sheets and subsequent inspection by metal replica transmission electron microscopy (TEM) reveal detailed information about the cellular membrane topology, including the structure and curvature. However, electron microscopy cannot identify proteins associated with specific plasma membrane domains. Here, we describe a novel adaptation of correlative super-resolution light microscopy and platinum replica TEM (CLEM-PREM), allowing the analysis of plasma membrane sheets with respect to their structural details, curvature, and associated protein composition. We suggest a number of shortcuts and troubleshooting solutions to contemporary PREM protocols. Thus, implementation of super-resolution stimulated emission depletion (STED) microscopy offers significant reduction in sample preparation time and reduced technical challenges for imaging and analysis. Additionally, highly technical challenges associated with replica preparation and transfer on a TEM grid can be overcome by scanning electron microscopy (SEM) imaging. The combination of STED microscopy and platinum replica SEM or TEM provides the highest spatial resolution of plasma membrane proteins and their underlying membrane and is, therefore, a suitable method to study cellular events like endocytosis, membrane trafficking, or membrane tension adaptations.

GelMap: intrinsic calibration and deformation mapping for expansion microscopy

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy by physically expanding biological specimen in three dimensions. Nonetheless, using ExM for quantitative or diagnostic applications requires robust quality control methods to precisely determine expansion factors and to map deformations due to anisotropic expansion. Here we present GelMap, a flexible workflow to introduce a fluorescent grid into pre-expanded hydrogels that scales with expansion and reports deformations. We demonstrate that GelMap can be used to precisely determine the local expansion factor and to correct for deformations without the use of cellular reference structures or pre-expansion ground-truth images. Moreover, we show that GelMap aids sample navigation for correlative uses of expansion microscopy. Finally, we show that GelMap is compatible with expansion of tissue and can be readily implemented as a quality control step into existing ExM workflows.

Expansion microscopy in honeybee brains for high-resolution neuroanatomical analyses in social insects

The diffraction limit of light microscopy poses a problem that is frequently faced in structural analyses of social insect brains. With the introduction of expansion microscopy (ExM), a tool became available to overcome this limitation by isotropic physical expansion of preserved specimens. Our analyses focus on synaptic microcircuits (microglomeruli, MG) in the mushroom body (MB) of social insects, high-order brain centers for sensory integration, learning, and memory. MG undergo significant structural reorganizations with age, sensory experience, and during long-term memory formation. However, the changes in subcellular architecture involved in this plasticity have only partially been accessed yet. Using the western honeybee Apis mellifera as an experimental model, we established ExM for the first time in a social insect species and applied it to investigate plasticity in synaptic microcircuits within MG of the MB calyces. Using combinations of antibody staining and neuronal tracing, we demonstrate that this technique enables quantitative and qualitative analyses of structural neuronal plasticity at high resolution in a social insect brain.

Ten-fold Robust Expansion Microscopy.

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. By systematic exploration of the ExM recipe space, we developed a novel ExM method termed Ten-fold Robust Expansion Microscopy (TREx) that, as the original ExM method, requires no specialized equipment or procedures. TREx enables ten-fold expansion of both thick mouse brain tissue sections and cultured human cells, can be handled easily, and enables high-resolution subcellular imaging with a single expansion step. Furthermore, TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.

Plasticity of perisynaptic astroglia during ischemia-induced spreading depolarization.

High astroglial capacity for glutamate and potassium clearance aids in recovering spreading depolarization (SD)-evoked disturbance of ion homeostasis during stroke. Since perisynaptic astroglia cannot be imaged with diffraction-limited light microscopy, nothing is known about the impact of SD on the ultrastructure of a tripartite synapse. We used serial section electron microscopy to assess astroglial synaptic coverage in the sensorimotor cortex of urethane-anesthetized male and female mice during and after SD evoked by transient bilateral common carotid artery occlusion. At the subcellular level, astroglial mitochondria were remarkably resilient to SD compared to dendritic mitochondria that were fragmented by SD. Overall, 482 synapses in `Sham' during `SD' and `Recovery' groups were randomly selected and analyzed in 3D. Perisynaptic astroglia was present at the axon-spine interface (ASI) during SD and after recovery. Astrocytic processes were more likely found at large synapses on mushroom spines after recovery, while the length of the ASI perimeter surrounded by astroglia has also significantly increased at large synapses. These findings suggest that as larger synapses have a bigger capacity for neurotransmitter release during SD, they attract astroglial processes to their perimeter during recovery, limiting extrasynaptic glutamate escape and further enhancing the astrocytic ability to protect synapses in stroke.

Unraveling docking and initiation of mRNA export through the nuclear pore complex.

The nuclear export of mRNA through the nuclear pore complex (NPC) is a process required for the healthy functioning of human cells, making it a critical area of research. However, the geometries of mRNA and the NPC are well below the diffraction limit of light microscopy, thereby presenting significant challenges in evaluating the discrete interactions and dynamics involved in mRNA nuclear export through the native NPC. Recent advances in biotechnology and single-molecule super-resolution light microscopy have enabled researchers to gain granular insight into the specific contributions made by discrete nucleoporins in the nuclear basket of the NPC to the export of mRNA. Specifically, by expanding upon the docking step facilitated by the protein TPR in the nuclear basket as well as identifying NUP153 as being the primary nuclear basket protein initiating export through the central channel of the NPC.

Imaging of surface microdomains on individual extracellular vesicles in 3-D.

Extracellular vesicles (EVs) are secreted from all cell types and are intimately involved in tissue homeostasis. They are being explored as vaccine and gene therapy platforms, as well as potential biomarkers. As their size is below the diffraction limit of light microscopy, direct visualizations have been daunting and single‐particle studies under physiological conditions have been hampered. Here, direct stochastic optical reconstruction microscopy (dSTORM) was employed to visualize EVs in three‐dimensions and to localize molecule clusters such as the tetraspanins CD81 and CD9 on the surface of individual EVs. These studies demonstrate the existence of membrane microdomains on EVs. These were confirmed by Cryo‐EM. Individual particle visualization provided insights into the heterogeneity, structure, and complexity of EVs not previously appreciated

Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx).

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. The maximum resolution increase is limited by the expansion factor of the gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures. We demonstrate that TREx gels expand 10-fold, can be handled easily, and can be applied to both thick mouse brain tissue sections and cultured human cells enabling high-resolution subcellular imaging with a single expansion step. Furthermore, we show that TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small-molecule stains for both total protein and membranes.

Ground state depletion microscopy as a tool for studying microglia-synapse interactions.

Ground state depletion followed by individual molecule return microscopy (GSDIM) has been used in the past to study the nanoscale distribution of protein co‐localization in living cells. We now demonstrate the successful application of GSDIM to archival human brain tissue sections including from Alzheimer's disease cases as well as experimental tissue samples from mouse and zebrafish larvae. Presynaptic terminals and microglia and their cell processes were visualized at a resolution beyond diffraction‐limited light microscopy, allowing clearer insights into their interactions in situ. The procedure described here offers time and cost savings compared to electron microscopy and opens the spectrum of molecular imaging using antibodies and super‐resolution microscopy to the analysis of routine formalin‐fixed paraffin sections of archival human brain. The investigation of microglia–synapse interactions in dementia will be of special interest in this context.

Viral particle imaging by super-resolution fluorescence microscopy

Fluorescence microscopy provides a facile imaging methodology and potentials for imaging viruses in vivo. However, it is limited by the diffraction limit of light microscopy which is above the size of the virus particles. Fluorescence super-resolution (FSR) microscopy circumvents the diffraction limit. These techniques, an extension of fluorescence microscopy and alternative to electron microscopy, established over the past twenty years, have permitted the visualization of some virus components. Here, we summarize the principles of different FSR techniques and single virus tracking, and their current applications to virology studies, focusing on some limitations impeding their widespread use in virus replication and infection mechanism studies in living cells. In the specific context of virology studies and live-cell imaging, the advantages and disadvantages of the most known FSR methods are outlined with a focus on their current limitations such as labeling, imaging speed, photo-bleaching, depth of imaging, and the current attempts to overcome them. Some of the recent demonstrations encompassing the combination of different super-resolution (SR) approaches have provided answers to open questions as an example in the human immunodeficiency virus replication and from these experiences, further progress can be envisaged to reveal other viruses.

A workflow for sizing oligomeric biomolecules based on cryo single molecule localization microscopy.

Single molecule localization microscopy (SMLM) has enormous potential for resolving subcellular structures below the diffraction limit of light microscopy: Localization precision in the low digit nanometer regime has been shown to be achievable. In order to record localization microscopy data, however, sample fixation is inevitable to prevent molecular motion during the rather long recording times of minutes up to hours. Eventually, it turns out that preservation of the sample's ultrastructure during fixation becomes the limiting factor. We propose here a workflow for data analysis, which is based on SMLM performed at cryogenic temperatures. Since molecular dipoles of the fluorophores are fixed at low temperatures, such an approach offers the possibility to use the orientation of the dipole as an additional information for image analysis. In particular, assignment of localizations to individual dye molecules becomes possible with high reliability. We quantitatively characterized the new approach based on the analysis of simulated oligomeric structures. Side lengths can be determined with a relative error of less than 1% for tetramers with a nominal side length of 5 nm, even if the assumed localization precision for single molecules is more than 2 nm.

Rapid 3D Enhanced Resolution Microscopy Reveals Diversity in Dendritic Spinule Dynamics, Regulation, and Function

Dendritic spinules are thin protrusions, formed by neuronal spines, not adequately resolved by diffraction-limited light microscopy, which has limited our understanding of their behavior. Here we performed rapid structured illumination microscopy and enhanced resolution confocal microscopy to study spatiotemporal spinule dynamics in cortical pyramidal neurons. Spinules recurred at the same locations on mushroom spine heads. Most were short-lived, dynamic, exploratory, and originated near simple PSDs, whereas a subset was long-lived, elongated, and associated with complex PSDs. These subtypes were differentially regulated by Ca2+ transients. Furthermore, the postsynaptic Rac1-GEF kalirin-7 regulated spinule formation, elongation, and recurrence. Long-lived spinules often contained PSD fragments, contacted distal presynaptic terminals, and formed secondary synapses. NMDAR activation increased spinule number, length, and contact with distal presynaptic elements. Spinule subsets, dynamics, and recurrence were validated in cortical neurons of acute brain slices. Thus, we identified unique properties, regulatory mechanisms, and functions of spinule subtypes, supporting roles in neuronal connectivity.

Verifying molecular clusters by 2-color localization microscopy and significance testing

While single-molecule localization microscopy (SMLM) offers the invaluable prospect to visualize cellular structures below the diffraction limit of light microscopy, its potential has not yet been fully capitalized due to its inherent susceptibility to blinking artifacts. Particularly, overcounting of single molecule localizations has impeded a reliable and sensitive detection of biomolecular nanoclusters. Here we introduce a 2-Color Localization microscopy And Significance Testing Approach (2-CLASTA), providing a parameter-free statistical framework for the qualitative analysis of two-dimensional SMLM data via significance testing methods. 2-CLASTA yields p-values for the null hypothesis of random biomolecular distributions, independent of the blinking behavior of the chosen fluorescent labels. The method is parameter-free and does not require any additional measurements nor grouping of localizations. We validated the method both by computer simulations as well as experimentally, using protein concatemers as a mimicry of biomolecular clustering. As the new approach is not affected by overcounting artifacts, it is able to detect biomolecular clustering of various shapes at high sensitivity down to a level of dimers.

Light Microscopy of Mitochondria at the Nanoscale.

Mitochondria are essential for eukaryotic life. These double-membrane organelles often form highly dynamic tubular networks interacting with many cellular structures. Their highly convoluted contiguous inner membrane compartmentalizes the organelle, which is crucial for mitochondrial function. Since the diameter of the mitochondrial tubules is generally close to the diffraction limit of light microscopy, it is often challenging, if not impossible, to visualize submitochondrial structures or protein distributions using conventional light microscopy. This renders super-resolution microscopy particularly valuable, and attractive, for studying mitochondria. Super-resolution microscopy encompasses a diverse set of approaches that extend resolution, as well as nanoscopy techniques that can even overcome the diffraction limit. In this review, we provide an overview of recent studies using super-resolution microscopy to investigate mitochondria, discuss the strengths and opportunities of the various methods in addressing specific questions in mitochondrial biology, and highlight potential future developments.

Coupling APEX labeling to imaging mass spectrometry of single organelles reveals heterogeneity in lysosomal protein turnover.

Quantification of stable isotope tracers after metabolic labeling provides a snapshot of the dynamic state of living cells and tissue. A form of imaging mass spectrometry quantifies isotope ratios with a lateral resolution <50 nm, using a methodology that we refer to as multi-isotope imaging mass spectrometry (MIMS). Despite lateral resolution exceeding diffraction-limited light microscopy, lack of contrast has largely limited use of MIMS to large or specialized subcellular structures, such as the nucleus and stereocilia. In this study, we repurpose the engineered peroxidase APEX2 as the first genetically encoded marker for MIMS. Coupling APEX2 labeling of lysosomes and metabolic labeling of protein, we identify that individual lysosomes exhibit substantial heterogeneity in protein age, which is lost in iPSC-derived neurons lacking the lysosomal protein progranulin. This study expands the practical use of MIMS for cell biology by enabling measurements of metabolic function from stable isotope labeling within individual organelles in situ.

Correlative single-molecule localization microscopy and electron tomography reveals endosome nanoscale domains.

Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which, however, cannot be resolved by diffraction‐limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional subdomains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single‐molecule photo‐switching are opposed. Here, we developed a novel superCLEM workflow that combines triple‐color SMLM (dSTORM & PALM) and electron tomography using semi‐thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labeled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nanodomains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub‐compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.

Temporal Filtering to Improve Single Molecule Identification in High Background Samples.

Single molecule localization microscopy is currently revolutionizing the life sciences as it offers, for the first time, insights into the organization of biological samples below the classical diffraction limit of light microscopy. While there have been numerous examples of new biological findings reported in the last decade, the technique could not reach its full potential due to a set of limitations immanent to the samples themselves. Particularly, high background signals impede the proper performance of most single-molecule identification and localization algorithms. One option is to exploit the characteristic blinking of single molecule signals, which differs substantially from the residual brightness fluctuations of the fluorescence background. To pronounce single molecule signals, we used a temporal high-pass filtering in Fourier space on a pixel-by-pixel basis. We evaluated the performance of temporal filtering by assessing statistical parameters such as true positive rate and false discovery rate. For this, ground truth signals were generated by simulations and overlaid onto experimentally derived movies of samples with high background signals. Compared to the nonfiltered case, we found an improvement of the sensitivity by up to a factor 3.5 while no significant change in the localization accuracy was observable.

Astrocytic Atrophy Following Status Epilepticus Parallels Reduced Ca2+ Activity and Impaired Synaptic Plasticity in the Rat Hippocampus.

Epilepsy is a group of neurological disorders commonly associated with the neuronal malfunction leading to generation of seizures. Recent reports point to a possible contribution of astrocytes into this pathology. We used the lithium-pilocarpine model of status epilepticus (SE) in rats to monitor changes in astrocytes. Experiments were performed in acute hippocampal slices 2–4 weeks after SE induction. Nissl staining revealed significant neurodegeneration in the pyramidal cell layers of hippocampal CA1, CA3 areas, and the hilus, but not in the granular cell layer of the dentate gyrus. A significant increase in the density of astrocytes stained with an astrocyte-specific marker, sulforhodamine 101, was observed in CA1 stratum (str.) radiatum. Astrocytes in this area were also whole-cell loaded with a morphological tracer, Alexa Fluor 594, for two-photon excitation imaging. Sholl analyses showed no changes in the size of the astrocytic domain or in the number of primary astrocytic branches, but a significant reduction in the number of distal branches that are resolved with diffraction-limited light microscopy (and are thought to contain Ca2+ stores, such as mitochondria and endoplasmic reticulum). The atrophy of astrocytic branches correlated with the reduced size, but not overall frequency of Ca2+ events. The volume tissue fraction of nanoscopic (beyond the diffraction limit) astrocytic leaflets showed no difference between control and SE animals. The results of spatial entropy-complexity spectrum analysis were also consistent with changes in ratio of astrocytic branches vs. leaflets. In addition, we observed uncoupling of astrocytes through the gap-junctions, which was suggested as a mechanism for reduced K+ buffering. However, no significant difference in time-course of synaptically induced K+ currents in patch-clamped astrocytes argued against possible alterations in K+ clearance by astrocytes. The magnitude of long-term-potentiation (LTP) was reduced after SE. Exogenous D-serine, a co-agonist of NMDA receptors, has rescued the initial phase of LTP. This suggests that the reduced Ca2+-dependent release of D-serine by astrocytes impairs initiation of synaptic plasticity. However, it does not explain the failure of LTP maintenance which may be responsible for cognitive decline associated with epilepsy.

Correlative Stimulated Emission Depletion and Scanning Ion Conductance Microscopy.

Correlation microscopy combining fluorescence and scanning probe or electron microscopy is limited to fixed samples due to the sample preparation and nonphysiological imaging conditions required by most probe or electron microscopy techniques. Among the few scanning probe techniques that allow imaging of living cells under physiological conditions, scanning ion conductance microscopy (SICM) has been shown to be the technique that minimizes the impact on the investigated sample. However, combinations of SICM and fluorescence microscopy suffered from the mismatch in resolution due to the limited resolution of conventional light microscopy. In the last years, the diffraction limit of light microscopy has been circumvented by various techniques, one of which is stimulated emission depletion (STED) microscopy. Here, we aimed at demonstrating the combination of STED and SICM. We show that both methods allow recording a living cellular specimen and provide a SICM and STED image of the same sample, which allowed us to correlate the membrane surface topography and the distribution of the cytoskeletal protein actin. Our proof-of-concept study exemplifies the benefit of correlating SICM with a subdiffraction fluorescence method and might form the basis for the development of a combined instrument that would allow the simultaneous recording of subdiffraction fluorescence and topography information.