Super-resolution Microscopy Techniques Research Articles

Universal and High-Fidelity Resolution Extending for Fluorescence Microscopy Using a Single-Training Physics-Informed Sparse Neural Network

As a supplement to optical super-resolution microscopy techniques, computational super-resolution methods have demonstrated remarkable results in alleviating the spatiotemporal imaging trade-off. However, they commonly suffer from low structural fidelity and universality. Therefore, we herein propose a deep-physics-informed sparsity framework designed holistically to synergize the strengths of physical imaging models (image blurring processes), prior knowledge (continuity and sparsity constraints), a back-end optimization algorithm (image deblurring), and deep learning (an unsupervised neural network). Owing to the utilization of a multipronged learning strategy, the trained network can be applied to a variety of imaging modalities and samples to enhance the physical resolution by a factor of at least 1.67 without requiring additional training or parameter tuning. Given the advantages of high accessibility and universality, the proposed deep-physics-informed sparsity method will considerably enhance existing optical and computational imaging techniques and have a wide range of applications in biomedical research.

Spatial frequency shift super-resolution imaging based on quasiperiodic grating and deep learning.

In this study, we propose a pioneering spatially frequency-shifted super-resolution microscopy technique that utilizes the synergy of quasiperiodic gratings and deep learning. First, a quasiperiodic grating capable of converting evanescent waves into propagating waves is designed. The grating is positioned between the object under investigation and the objective lens, and the high-frequency information carried by the evanescent waves in the near-field region of the object is shifted into the detection window and becomes accessible in the far field for imaging. Subsequently, we provide two deep learning models for image and video reconstructions to achieve the reconstruction of static and dynamic samples respectively. Simulation results demonstrate the high feasibility of the proposed method, and both static and dynamic objects with sub-wavelength features can be resolved. The developed method paves the way to the realization of super-resolution imaging by using a traditional bright-field microscope without the need for an extensive optical system design.

Synergistic Pharmacological Therapy to Modulate Glial Cells in Spinal Cord Injury.

Current treatments for modulating the glial-mediated inflammatory response after spinal cord injury (SCI) have limited ability to improve recovery. This is quite likely due to the lack of a selective therapeutic approach acting on microgliosis and astrocytosis, the glia components most involved after trauma, while maximizing efficacy and minimizing side effects. A new nanogel that can selectively release active compounds in microglial cells and astrocytes is developed and characterized. The degree of selectivity and subcellular distribution of the nanogel is evaluated by applying an innovative super-resolution microscopy technique, expansion microscopy. Two different administration schemes are then tested in a SCI mouse model: in an early phase, the nanogel loaded with Rolipram, an anti-inflammatory drug, achieves significant improvement in the animal's motor performance due to the increased recruitment of microglia and macrophages that are able to localize the lesion. Treatment in the late phase, however, gives opposite results, with worse motor recovery because of the widespread degeneration. These findings demonstrate that the nanovector can be selective and functional in the treatment of the glial component in different phases of SCI. They also open a new therapeutic scenario for tackling glia-mediated inflammation after neurodegenerative events in the central nervous system.

A non-iterative frame-reduced structured illumination microscopy using checkerboard modulation

Structured illumination microscopy (SIM) is a tremendously potential super-resolution microscopy technique requiring nine raw images to enhance two-fold lateral resolution. For SIM, it is a challenging dilemma to achieve super-resolution imaging with a reduced number of images and fast data processing speed at the same time. Here, we demonstrate a new SIM reconstruction framework that uses only four raw images without requiring extensive iterative computation. The four-frame method is achieved by applying checkerboard pattern illumination modulation to supersede the sinusoidal fringe illumination. It is worth noting that the proposed scheme prominently shortens image acquisition time, combined with a remarkably higher image reconstruction rate compared to current frame-reduced methods. Meanwhile, the reconstruction process is non-iterative and unlimited by the field of view size. Consequently, our work is expected to bring about breakthroughs in facilitating the improvement of real-time live cell observation.

HIV-1 Gag targeting to the plasma membrane reorganizes sphingomyelin-rich and cholesterol-rich lipid domains

Although the human immunodeficiency virus type 1 lipid envelope has been reported to be enriched with host cell sphingomyelin and cholesterol, the molecular mechanism of the enrichment is not well understood. Viral Gag protein plays a central role in virus budding. Here, we report the interaction between Gag and host cell lipids using different quantitative and super-resolution microscopy techniques in combination with specific probes that bind endogenous sphingomyelin and cholesterol. Our results indicate that Gag in the inner leaflet of the plasma membrane colocalizes with the outer leaflet sphingomyelin-rich domains and cholesterol-rich domains, enlarges sphingomyelin-rich domains, and strongly restricts the mobility of sphingomyelin-rich domains. Moreover, Gag multimerization induces sphingomyelin-rich and cholesterol-rich lipid domains to be in close proximity in a curvature-dependent manner. Our study suggests that Gag binds, coalesces, and reorganizes pre-existing lipid domains during assembly.

Unprecedented enantio-selective live-cell mitochondrial DNA super-resolution imaging and photo-sensitizing by the chiral ruthenium polypyridyl DNA "light-switch".

Mitochondrial DNA (mtDNA) is known to play a critical role in cellular functions. However, the fluorescent probe enantio-selectively targeting live-cell mtDNA is rare. We recently found that the well-known DNA 'light-switch' [Ru(phen)2dppz]Cl2 can image nuclear DNA in live-cells with chlorophenolic counter-anions via forming lipophilic ion-pairing complex. Interestingly, after washing with fresh-medium, [Ru(phen)2dppz]Cl2 was found to re-localize from nucleus to mitochondria via ABC transporter proteins. Intriguingly, the two enantiomers of [Ru(phen)2dppz]Cl2 were found to bind enantio-selectively with mtDNA in live-cells not only by super-resolution optical microscopy techniques (SIM, STED), but also by biochemical methods (mitochondrial membrane staining with Tomo20-dronpa). Using [Ru(phen)2dppz]Cl2 as the new mtDNA probe, we further found that each mitochondrion containing 1-8 mtDNA molecules are distributed throughout the entire mitochondrial matrix, and there are more nucleoids near nucleus. More interestingly, we found enantio-selective apoptotic cell death was induced by the two enantiomers by prolonged visible light irradiation, and in-situ self-monitoring apoptosis process can be achieved by using the unique 'photo-triggered nuclear translocation' property of the Ru complex. This is the first report on enantio-selective targeting and super-resolution imaging of live-cell mtDNA by a chiral Ru complex via formation and dissociation of ion-pairing complex with suitable counter-anions.

Enhanced synaptic protein visualization by multicolor super-resolution expansion microscopy.

Understanding the organization of biomolecules into complexes and their dynamics is crucial for comprehending cellular functions and dysfunctions, particularly in neuronal networks connected by synapses. In the last two decades, various powerful super-resolution (SR) microscopy techniques have been developed that produced stunning images of synapses and their molecular organization. However, current SR microscopy methods do not permit multicolor fluorescence imaging with 20 to 30nm spatial resolution. We developed a method that enables 4-color fluorescence imaging of synaptic proteins in neurons with 20 to 30nm lateral resolution. We used post-expansion immunolabeling of eightfold expanded hippocampal neurons in combination with Airyscan and structured illumination microscopy (SIM). We demonstrate that post-expansion immunolabeling of approximately eightfold expanded hippocampal neurons enables efficient labeling of synaptic proteins in crowded compartments with minimal linkage error and enables in combination with Airyscan and SIM four-color three-dimensional fluorescence imaging with 20 to 30nm lateral resolution. Using immunolabeling of Synaptobrevin 2 as an efficient marker of the vesicle pool allowed us to identify individual synaptic vesicles colocalized with Rab3-interacting molecule 1 and 2 (RIM1/2), a marker of pre-synaptic fusion sites. Our optimized expansion microscopy approach improves the visualization and location of pre- and post-synaptic proteins and can thus provide invaluable insights into the spatial organization of proteins at synapses.

Characterizing the blood-brain barrier and gut barrier with super-resolution imaging: opportunities and challenges.

Brain and gut barriers have been receiving increasing attention in health and diseases including in psychiatry. Recent studies have highlighted changes in the blood-brain barrier and gut barrier structural properties, notably a loss of tight junctions, leading to hyperpermeability, passage of inflammatory mediators, stress vulnerability, and the development of depressive behaviors. To decipher the cellular processes actively contributing to brain and gut barrier function in health and disease, scientists can take advantage of neurophotonic tools and recent advances in super-resolution microscopy techniques to complement traditional imaging approaches like confocal and electron microscopy. Here, we summarize the challenges, pros, and cons of these innovative approaches, hoping that a growing number of scientists will integrate them in their study design exploring barrier-related properties and mechanisms.

Pre-organized landscape of T cell surface.

T cell activation is initiated by the recognition of specific antigenic peptides and subsequently accomplished by complex signaling cascades. These aspects have been extensively studied for decades as pivotal factors in the establishment of adaptive immunity. However, how receptors or signaling molecules are organized in the resting state prior to encountering antigens has received less attention. Recent advancements in super-resolution microscopy techniques have revealed topographically controlled pre-formed organization of key molecules involved in antigen recognition and signal transduction on microvillar projections of T cells before activation and substantial effort has been dedicated to characterizing the topological structure of resting T cells over the past decade. This review will summarize our current understanding of how key surface receptors are pre-organized on the T-cell plasma membrane and discuss the potential role of these receptors, which are preassembled prior to ligand binding in the early activation events of T cells.

Synaptic expression of TAR-DNA-binding protein 43 in the mouse spinal cord determined using super-resolution microscopy.

Amyotrophic Lateral Sclerosis (ALS) is characterised by a loss of motor neurons in the brain and spinal cord that is preceded by early-stage changes in synapses that may be associated with TAR-DNA-Binding Protein 43 (TDP-43) pathology. Cellular inclusions of hyperphosphorylated TDP-43 (pTDP-43) are a key hallmark of neurodegenerative diseases such ALS. However, there has been little characterisation of the synaptic expression of TDP-43 inside subpopulations of spinal cord synapses. This study utilises a range of high-resolution and super-resolution microscopy techniques with immunolabelling, as well as an aptamer-based TDP-43 labelling strategy visualised with single-molecule localisation microscopy, to characterise and quantify the presence of pTDP-43 in populations of excitatory synapses near where motor neurons reside in the lateral ventral horn of the mouse lumbar spinal cord. We observe that TDP-43 is expressed in approximately half of spinal cord synapses as nanoscale clusters. Synaptic TDP-43 clusters are found most abundantly at synapses associated with VGLUT1-positive presynaptic terminals, compared to VGLUT2-associated synapses. Our nanoscopy techniques showed no difference in the subsynaptic expression of pTDP-43 in the ALS mouse model, SOD1G93a, compared to healthy controls, despite prominent structural deficits in VGLUT1-associated synapses in SOD1G93a mice. This research characterises the basic synaptic expression of TDP-43 with nanoscale precision and provides a framework with which to investigate the potential relationship between TDP-43 pathology and synaptic pathology in neurodegenerative diseases.

TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation

TIGIT is an inhibitory receptor expressed on lymphocytes and can inhibit T cells by preventing CD226 co-stimulation through interactions in cis or through competition of shared ligands. Whether TIGIT directly delivers cell-intrinsic inhibitory signals in T cells remains unclear. Here we show, by analysing lymphocytes from matched human tumour and peripheral blood samples, that TIGIT and CD226 co-expression is rare on tumour-infiltrating lymphocytes. Using super-resolution microscopy and other techniques, we demonstrate that ligation with CD155 causes TIGIT to reorganise into dense nanoclusters, which coalesce with T cell receptor (TCR)-rich clusters at immune synapses. Functionally, this reduces cytokine secretion in a manner dependent on TIGIT’s intracellular ITT-like signalling motif. Thus, we provide evidence that TIGIT directly inhibits lymphocyte activation, acting independently of CD226, requiring intracellular signalling that is proximal to the TCR. Within the subset of tumours where TIGIT-expressing cells do not commonly co-express CD226, this will likely be the dominant mechanism of action.

Exploring Lignification Complexity in Plant Cell Walls with Airyscan Super-resolution Microscopy and Bioorthogonal Chemistry.

In this paper, we present the use of multiplex click/bioorthogonal chemistry combined with super-resolution Airyscan microscopy to track biomolecules in living systems with a focus on studying lignin formation in plant cell walls. While laser scanning confocal microscopy (LSCM) provided insights into the tissue-scale dynamics of lignin formation and distribution in our previous reports, its limited resolution precluded an in-depth analysis of lignin composition at the unique cell wall or substructure level. To overcome this limitation, we explored the use of Airyscan microscopy, which, among the super-resolution techniques available, offers an optimal balance between performance, cost, accessibility, and ease of implementation. Our study demonstrates that a triple labeling strategy using copper-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), and inverse electronic-demand Diels-Alder cycloaddition (IEDDA) to label modified lignin metabolic precursors can be combined with Airyscan microscopy to reveal the zones of active lignification at the single cell level with improved sensitivity and resolution. This approach enables insights into the lignin composition in wall substructures, such as pits or in wall layers that are otherwise not distinguishable by classical LSCM. Our work emphasizes the importance of studying lignin formation in plant cell walls and demonstrates the potential of combining bioorthogonal chemistry and super-resolution microscopy techniques for studying biomolecules in living systems.

Switchable and Functional Fluorophores for Multidimensional Single-Molecule Localization Microscopy.

Multidimensional single-molecule localization microscopy (mSMLM) represents a paradigm shift in the realm of super-resolution microscopy techniques. It affords the simultaneous detection of single-molecule spatial locations at the nanoscale and functional information by interrogating the emission properties of switchable fluorophores. The latter is finely tuned to report its local environment through carefully manipulated laser illumination and single-molecule detection strategies. This Perspective highlights recent strides in mSMLM with a focus on fluorophore designs and their integration into mSMLM imaging systems. Particular interests are the accomplishments in simultaneous multiplexed super-resolution imaging, nanoscale polarity and hydrophobicity mapping, and single-molecule orientational imaging. Challenges and prospects in mSMLM are also discussed, which include the development of more vibrant and functional fluorescent probes, the optimization of optical implementation to judiciously utilize the photon budget, and the advancement of imaging analysis and machine learning techniques.

Super-resolution computational saturated absorption microscopy.

Imaging beyond the diffraction limit barrier has attracted wide attention due to the ability to resolve previously hidden image features. Of the various super-resolution microscopy techniques available, a particularly simple method called saturated excitation microscopy (SAX) requires only simple modification of a laser scanning microscope: The illumination beam power is sinusoidally modulated and driven into saturation. SAX images are extracted from the harmonics of the modulation frequency and exhibit improved spatial resolution. Unfortunately, this elegant strategy is hindered by the incursion of shot noise that prevents high-resolution imaging in many realistic scenarios. Here, we demonstrate a technique for super-resolution imaging that we call computational saturated absorption (CSA) in which a joint deconvolution is applied to a set of images with diversity in spatial frequency support among the point spread functions (PSFs) used in the image formation with saturated laser scanning fluorescence microscopy. CSA microscopy allows access to the high spatial frequency diversity in a set of saturated effective PSFs, while avoiding image degradation from shot noise.

Plasmon-Enhanced Expansion Microscopy.

Expansion microscopy (ExM) is a rapidly emerging super-resolution microscopy technique that involves isotropic expansion of biological samples to improve spatial resolution. However, fluorescence signal dilution due to volumetric expansion is a hindrance to the widespread application of ExM. Here, we introduce plasmon-enhanced expansion microscopy (p-ExM) by harnessing an ultrabright fluorescent nanoconstruct, called plasmonic-fluor (PF), as a nanolabel. The unique structure of PFs renders nearly 15000-fold brighter fluorescence signal intensity and higher fluorescence retention following the ExM protocol (nearly 76%) compared to their conventional counterparts (<16% for IR-650). Individual PFs can be easily imaged using conventional fluorescence microscopes, making them excellent "digital" labels for ExM. We demonstrate that p-ExM enables improved tracing and decrypting of neural networks labeled with PFs, as evidenced by improved quantification of morphological markers (nearly a 2.5-fold increase in number of neurite terminal points). Overall, p-ExM complements the existing ExM techniques for probing structure-function relationships of various biological systems.

Super-Resolution Ultrasound Localization Microscopy Using High-Frequency Ultrasound to Measure Ocular Perfusion Velocity in the Rat Eye.

Imaging of the ocular vasculature can provide new insights into the pathophysiology of ocular diseases. This study proposes a novel high-frequency super-resolution ultrasound localization microscopy (SRULM) technique and evaluates its ability to measure in vivo perfusion changes in the rat eye at elevated intraocular pressure (IOP). A 38.4 MHz center frequency linear array transducer on a VisualSonics Vevo F2 imaging platform was used to collect high frame rate (1 kHz) radiofrequency data of the posterior rat eye following systemic microbubble contrast injection. Following clutter and spatiotemporal non-local means filtering, individual microbubbles were localized and tracked. The microbubble tracks were accumulated over 10,000 frames to generate vascular images quantifying perfusion velocity and direction. Experiments were performed using physiologic relevant controlled flow states for algorithm validation and subsequently performed in vivo on the rat eye at 10 mm Hg IOP increments from 10 to 60 mm Hg. The posterior vasculature of the rat eye, including the ophthalmic artery, long posterior ciliary arteries and their branches, central retinal artery and retinal arterioles and venules were successfully visualized, and velocities quantified at each IOP level. Significant reductions in arterial flow were measured as IOP was elevated. High-frequency SRULM can be used to visualize and quantify the perfusion velocity of the rat eye in both the retrobulbar and intraocular vasculature simultaneously. The ability to detect ocular perfusion changes throughout the depth of the eye may help elucidate the role ischemia has in the pathophysiology of ocular diseases such as glaucoma.

RegiSTORM: channel registration for multi-color stochastic optical reconstruction microscopy

BackgroundStochastic optical reconstruction microscopy (STORM), a super-resolution microscopy technique based on single-molecule localizations, has become popular to characterize sub-diffraction limit targets. However, due to lengthy image acquisition, STORM recordings are prone to sample drift. Existing cross-correlation or fiducial marker-based algorithms allow correcting the drift within each channel, but misalignment between channels remains due to interchannel drift accumulating during sequential channel acquisition. This is a major drawback in multi-color STORM, a technique of utmost importance for the characterization of various biological interactions.ResultsWe developed RegiSTORM, a software for reducing channel misalignment by accurately registering STORM channels utilizing fiducial markers in the sample. RegiSTORM identifies fiducials from the STORM localization data based on their non-blinking nature and uses them as landmarks for channel registration. We first demonstrated accurate registration on recordings of fiducials only, as evidenced by significantly reduced target registration error with all the tested channel combinations. Next, we validated the performance in a more practically relevant setup on cells multi-stained for tubulin. Finally, we showed that RegiSTORM successfully registers two-color STORM recordings of cargo-loaded lipid nanoparticles without fiducials, demonstrating the broader applicability of this software.ConclusionsThe developed RegiSTORM software was demonstrated to be able to accurately register multiple STORM channels and is freely available as open-source (MIT license) at https://github.com/oystein676/RegiSTORM.git and https://doi.org/10.5281/zenodo.5509861 (archived), and runs as a standalone executable (Windows) or via Python (Mac OS, Linux).

Quantitative Single-Molecule Localization Microscopy.

Super-resolution fluorescence microscopy allows the investigation of cellular structures at nanoscale resolution using light. Current developments in super-resolution microscopy have focused on reliable quantification of the underlying biological data. In this review, we first describe the basic principles of super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM), and then give a broad overview of methodological developments to quantify super-resolution data, particularly those geared toward SMLM data. We cover commonly used techniques such as spatial point pattern analysis, colocalization, and protein copy number quantification but also describe more advanced techniques such as structural modeling, single-particle tracking, and biosensing. Finally, we provide an outlook on exciting new research directions to which quantitative super-resolution microscopy might be applied.

Titelbild: Reversible Markierung in lebenden Zellen mit retro‐konstruierten HaloTags ermöglicht hoch‐ und superauflösende Langzeitbildgebung (Angew. Chem. 18/2023)

Reversible posttranslational fluorescence labeling opens exciting possibilities for advanced and superresolution microscopy techniques. In their Research Article (e202219050) Michael Holtmannspötter, Jacob Piehler et al. have engineered HaloTag variants with restored dehalogenase activity, which enzymatically exchange fluorogenic substrates at the second-to-minutes time scale. Thus, long-term time-lapse imaging with utmost spatial and temporal resolution is achieved with particularly striking performance in single molecule tracking and localization microscopy.

Cover Picture: Reversible Live‐Cell Labeling with Retro‐engineered HaloTags Enables Long‐Term High‐ and Super‐Resolution Imaging (Angew. Chem. Int. Ed. 18/2023)

Reversible posttranslational fluorescence labeling opens exciting possibilities for advanced and superresolution microscopy techniques. In their Research Article (e202219050) Michael Holtmannspötter, Jacob Piehler et al. have engineered HaloTag variants with restored dehalogenase activity, which enzymatically exchange fluorogenic substrates at the second-to-minutes time scale. Thus, long-term time-lapse imaging with utmost spatial and temporal resolution is achieved with particularly striking performance in single molecule tracking and localization microscopy. Reversible posttranslational fluorescence labeling opens exciting possibilities for advanced and superresolution microscopy techniques. In their Research Article (e202219050) Michael Holtmannspötter, Jacob Piehler et al. have engineered HaloTag variants with restored dehalogenase activity, which enzymatically exchange fluorogenic substrates at the second-to-minutes time scale. Thus, long-term time-lapse imaging with utmost spatial and temporal resolution is achieved with particularly striking performance in single molecule tracking and localization microscopy. Sandwich Compounds DNA Nanotechnology Anticancer Agents