Nanoscale insights into hematology: super-resolved imaging on blood cell structure, function, and pathology.

Fluorescence nanoscopy, also known as super-resolution microscopy, has transcended the conventional resolution barriers and enabled visualization of biological samples at nanometric resolutions. A series of super-resolution techniques have been developed and applied to investigate the molecular distribution, organization, and interactions in blood cells, as well as the underlying mechanisms of blood-cell-associated diseases. In this review, we provide an overview of various fluorescence nanoscopy technologies, outlining their current development stage and the challenges they are facing in terms of functionality and practicality. We specifically explore how these innovations have propelled forward the analysis of thrombocytes (platelets), erythrocytes (red blood cells) and leukocytes (white blood cells), shedding light on the nanoscale arrangement of subcellular components and molecular interactions. We spotlight novel biomarkers uncovered by fluorescence nanoscopy for disease diagnosis, such as thrombocytopathies, malignancies, and infectious diseases. Furthermore, we discuss the technological hurdles and chart out prospective avenues for future research directions. This review aims to underscore the significant contributions of fluorescence nanoscopy to the field of blood cell analysis and disease diagnosis, poised to revolutionize our approach to exploring, understanding, and managing disease at the molecular level.

4Pi MINFLUX arrangement maximizes spatio-temporal localization precision of fluorescence emitter.

We introduce MINFLUX localization with interferometric illumination through opposing objective lenses for maximizing the attainable precision in 3D-localization of single inelastic scatterers, such as fluorophores. Our 4Pi optical configuration employs three sequentially tilted counter-propagating beam pairs for illumination, each providing a narrow interference minimum of illumination intensity at the focal point. The localization precision is additionally improved by adding the inelastically scattered or fluorescence photons collected through both objective lenses. Our 4Pi configuration yields the currently highest precision per detected photon among all localization schemes. Tracking gold nanoparticles as non-blinking inelastic scatterers rendered a position uncertainty <0.4 nm3 in volume at a localization frequency of 2.9 kHz. We harnessed the record spatio-temporal precision of our 4Pi MINFLUX approach to examine the diffusion of single fluorophores and fluorescent nanobeads in solutions of sucrose in water, revealing local heterogeneities at the nanoscale. Our results show the applicability of 4Pi MINFLUX to study molecular nano-environments of diffusion and its potential for quantifying rapid movements of molecules in cells and other material composites.

Fluorogenic polymethine dyes by intramolecular cyclization.

Fluorescence imaging plays a pivotal role in the study of biological processes, and cell-permeable fluorogenic dyes are crucial to visualize intracellular structures with high specificity. Polymethine dyes are vitally important fluorophores in single-molecule localization microscopy and in vivo imaging, but their use in live cells has been limited by high background fluorescence and low membrane permeability. In this review, we summarize recent advances in the development of fluorogenic polymethine dyes via intramolecular cyclization. Finally, we offer an outlook on the prospects of fluorogenic polymethine dyes for bioimaging.

Structural changes in perineuronal nets and their perforating GABAergic synapses precede motor coordination recovery post stroke.

BACKGROUND: Stroke remains one of the leading causes of long-term disability worldwide, and the development of effective restorative therapies is hindered by an incomplete understanding of intrinsic brain recovery mechanisms. Growing evidence indicates that the brain extracellular matrix (ECM) has major implications for neuroplasticity. Here we explored how perineuronal nets (PNNs), the facet-like ECM layers surrounding fast-spiking interneurons, contribute to neurological recovery after focal cerebral ischemia in mice with and without induced stroke tolerance. METHODS: We investigated the structural remodeling of PNNs after stroke using 3D superresolution stimulated emission depletion (STED) and structured illumination (SR-SIM) microscopy. Superresolution imaging allowed for the precise reconstruction of PNN morphology using graphs, which are mathematical constructs designed for topological analysis. Focal cerebral ischemia was induced by transient occlusion of the middle cerebral artery (tMCAO). PNN-associated synapses and contacts with microglia/macrophages were quantified using high-resolution confocal microscopy. RESULTS: PNNs undergo transient structural changes after stroke allowing for the dynamic reorganization of GABAergic input to motor cortical L5 interneurons. The coherent remodeling of PNNs and their perforating inhibitory synapses precedes the recovery of motor coordination after stroke and depends on the severity of the ischemic injury. Morphological alterations in PNNs correlate with the increased surface of contact between activated microglia/macrophages and PNN-coated neurons. CONCLUSIONS: Our data indicate a novel mechanism of post stroke neuroplasticity involving the tripartite interaction between PNNs, synapses, and microglia/macrophages. We propose that prolonging PNN loosening during the post-acute period can extend the opening neuroplasticity window into the chronic stroke phase.

Designing chromatic optical retarder stacks for segmented next-generation easySTED phase plates.

Fluorescence nanoscopy methods based on the RESOLFT principle, such as beam-scanning STED nanoscopy, require the co-alignment of optical beams for molecular state (on/off) switching and fluorescence excitation. The complexity and stability of the beam alignment can be drastically simplified and improved by using a single-mode fibre as the sole light source for all required laser beams. This in turn then requires a chromatic optical element for shaping the off-switching beam into a focal-plane donut while simultaneously leaving the focal intensity distributions at other wavelengths shaped as regular focal spots. Here we describe novel designs of such so-called 'easySTED phase plates' and provide a rationale how to find the desired spectral signature for combinations of multiple wavelengths.

Photobleaching reduction in modulated super-resolution microscopy.

Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases in photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.

Superresolution Fluorescence Imaging of Mutant Huntingtin Aggregation in Cells.

Fluorescence-based nanoscopy methods (also known as "superresolution" microscopy) have substantially expanded our options to examine the distributions of molecules inside cells with nanometer-scale resolution and molecular specificity. In the biophysical analysis of aggregation-prone misfolded proteins and peptides, this has enabled the visualization of distinct populations of aggregated species such as fibrillar assemblies within intact neuronal cells, well below previous limits of sensitivity and resolution. With the Huntington's disease protein, polyglutamine-expanded mutant huntingtin, as an example, we provide sample preparation and imaging protocols for superresolution microscopy down to the ~30 nm-level.

Robust nanoscopy of a synaptic protein in living mice by organic-fluorophore labeling.

Extending superresolution fluorescence microscopy to living animals has remained a challenging frontier ever since the first demonstration of STED (stimulated emission depletion) nanoscopy in the mouse visual cortex. The use of fluorescent proteins (FPs) in in vivo STED analyses has been limiting available fluorescence photon budgets and attainable image contrasts, in particular for far-red FPs. This has so far precluded the definition of subtle details in protein arrangements at sufficient signal-to-noise ratio. Furthermore, imaging with longer wavelengths holds promise for reducing photostress. Here, we demonstrate that a strategy based on enzymatic self-labeling of the HaloTag fusion protein by high-performance synthetic fluorophore labels provides a robust avenue to superior in vivo analysis with STED nanoscopy in the far-red spectral range. We illustrate our approach by mapping the nanoscale distributions of the abundant scaffolding protein PSD95 at the postsynaptic membrane of excitatory synapses in living mice. With silicon-rhodamine as the reporter fluorophore, we present imaging with high contrast and low background down to ∼70-nm lateral resolution in the visual cortex at ≤25-µm depth. This approach allowed us to identify and characterize the diversity of PSD95 scaffolds in vivo. Besides small round/ovoid shapes, a substantial fraction of scaffolds exhibited a much more complex spatial organization. This highly inhomogeneous, spatially extended PSD95 distribution within the disk-like postsynaptic density, featuring intricate perforations, has not been highlighted in cell- or tissue-culture experiments. Importantly, covisualization of the corresponding spine morphologies enabled us to contextualize the diverse PSD95 patterns within synapses of different orientations and sizes.

The Actin/Spectrin Membrane-Associated Periodic Skeleton in Neurons.

Neurons are the most asymmetric cell types, with their axons commonly extending over lengths that are thousand times longer than the diameter of the cell soma. Fluorescence nanoscopy has recently unveiled that actin, spectrin and accompanying proteins form a membrane-associated periodic skeleton (MPS) that is ubiquitously present in mature axons from all neuronal types evaluated so far. The MPS is a regular supramolecular protein structure consisting of actin "rings" separated by spectrin tetramer "spacers". Although the MPS is best organized in axons, it is also present in dendrites, dendritic spine necks and thin cellular extensions of non-neuronal cells such as oligodendrocytes and microglia. The unique organization of the actin/spectrin skeleton has raised the hypothesis that it might serve to support the extreme physical and structural conditions that axons must resist during the lifespan of an organism. Another plausible function of the MPS consists of membrane compartmentalization and subsequent organization of protein domains. This review focuses on what we know so far about the structure of the MPS in different neuronal subdomains, its dynamics and the emerging evidence of its impact in axonal biology.

Adaptive-illumination STED nanoscopy.

The concepts called STED/RESOLFT superresolve features by a light-driven transfer of closely packed molecules between two different states, typically a nonfluorescent "off" state and a fluorescent "on" state at well-defined coordinates on subdiffraction scales. For this, the applied light intensity must be sufficient to guarantee the state difference for molecules spaced at the resolution sought. Relatively high intensities have therefore been applied throughout the imaging to obtain the highest resolutions. At regions where features are far enough apart that molecules could be separated with lower intensity, the excess intensity just adds to photobleaching. Here, we introduce DyMIN (standing for Dynamic Intensity Minimum) scanning, generalizing and expanding on earlier concepts of RESCue and MINFIELD to reduce sample exposure. The principle of DyMIN is that it only uses as much on/off-switching light as needed to image at the desired resolution. Fluorescence can be recorded at those positions where fluorophores are found within a subresolution neighborhood. By tuning the intensity (and thus resolution) during the acquisition of each pixel/voxel, we match the size of this neighborhood to the structures being imaged. DyMIN is shown to lower the dose of STED light on the scanned region up to ∼20-fold under common biological imaging conditions, and >100-fold for sparser 2D and 3D samples. The bleaching reduction can be converted into accordingly brighter images at <30-nm resolution.

Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent.

Photobleaching remains a limiting factor in superresolution fluorescence microscopy. This is particularly true for stimulated emission depletion (STED) and reversible saturable/switchable optical fluorescence transitions (RESOLFT) microscopy, where adjacent fluorescent molecules are distinguished by sequentially turning them off (or on) using a pattern of light formed as a doughnut or a standing wave. In sample regions where the pattern intensity reaches or exceeds a certain threshold, the molecules are essentially off (or on), whereas in areas where the intensity is lower, that is, around the intensity minima, the molecules remain in the initial state. Unfortunately, the creation of on/off state differences on subdiffraction scales requires the maxima of the intensity pattern to exceed the threshold intensity by a large factor that scales with the resolution. Hence, when recording an image by scanning the pattern across the sample, each molecule in the sample is repeatedly exposed to the maxima, which exacerbates bleaching. Here, we introduce MINFIELD, a strategy for fundamentally reducing bleaching in STED/RESOLFT nanoscopy through restricting the scanning to subdiffraction-sized regions. By safeguarding the molecules from the intensity of the maxima and exposing them only to the lower intensities (around the minima) needed for the off-switching (on-switching), MINFIELD largely avoids detrimental transitions to higher molecular states. A bleaching reduction by up to 100-fold is demonstrated. Recording nanobody-labeled nuclear pore complexes in Xenopus laevis cells showed that MINFIELD-STED microscopy resolved details separated by <25 nm where conventional scanning failed to acquire sufficient signal.

Nanoscale characterization of vesicle adhesion by normalized total internal reflection fluorescence microscopy.

We recently proposed a straightforward fluorescence microscopy technique to study adhesion of Giant Unilamellar Vesicles. This technique is based on dual observations which combine epi-fluorescence microscopy and total internal reflection fluorescence (TIRF) microscopy: TIRF images are normalized by epi-fluorescence ones. By this way, it is possible to map the membrane/substrate separation distance with a nanometric resolution, typically ~20 nm, with a maximal working range of 300-400 nm. The purpose of this paper is to demonstrate that this technique is useful to quantify vesicle adhesion from ultra-weak to strong membrane-surface interactions. Thus, we have examined unspecific and specific adhesion conditions. Concerning unspecific adhesion, we have controlled the strength of electrostatic forces between negatively charged vesicles and various functionalized surfaces which exhibit a positive or a negative effective charge. Specific adhesion was highlighted with lock-and-key forces mediated by the well defined biotin/streptavidin recognition.

Fluorescence nanoscopy. Methods and applications.

Fluorescence nanoscopy refers to the experimental techniques and analytical methods used for fluorescence imaging at a resolution higher than conventional, diffraction-limited, microscopy. This review explains the concepts behind fluorescence nanoscopy and focuses on the latest and promising developments in acquisition techniques, labelling strategies to obtain highly detailed super-resolved images and in the quantitative methods to extract meaningful information from them.