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Super-resolution microscopy (SRM) has transformed our understanding of proteins' subcellular organization and revealed cellular details down to nanometers, far beyond conventional microscopy. While localization precision is independent of the number of fluorophores attached to a biomolecule, labeling density is a decisive factor for resolving complex biological structures. The average distance between adjacent fluorophores should be less than half the desired spatial resolution for optimal clarity. While this was not a major limitation in recent decades, the success of modern microscopy approaching molecular resolution down to the single-digit nanometer range will depend heavily on advancements in fluorescence labeling. This review highlights recent advances and challenges in labeling strategies for SRM, focusing on site-specific labeling technologies. These advancements are crucial for improving SRM precision and expanding our understanding of molecular interactions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The most widely used fluorophore in glioma-resection surgery, 5-aminolevulinic acid (5-ALA), is thought to cause the selective accumulation of fluorescent protoporphyrin IX (PpIX) in tumour cells. Here we show that the clinical detection of PpIX can be improved via a microscope that performs paired stimulated Raman histology and two-photon excitation fluorescence microscopy (TPEF). We validated the technique in fresh tumour specimens from 115 patients with high-grade gliomas across four medical institutions. We found a weak negative correlation between tissue cellularity and the fluorescence intensity of PpIX across all imaged specimens. Semi-supervised clustering of the TPEF images revealed five distinct patterns of PpIX fluorescence, and spatial transcriptomic analyses of the imaged tissue showed that myeloid cells predominate in areas where PpIX accumulates in the intracellular space. Further analysis of external spatially resolved metabolomics, transcriptomics and RNA-sequencing datasets from glioblastoma specimens confirmed that myeloid cells preferentially accumulate and metabolize PpIX. Our findings question 5-ALA-induced fluorescence in glioma cells and show how 5-ALA and TPEF imaging can provide a window into the immune microenvironment of gliomas., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Objective To explore a simple and feasible method for whole-mount immunofluorescence staining of lymphatic vessels in the ApoE -/- mouse model of atherosclerosis. Methods Aortic specimens were carefully excised from the ApoE -/- mouse model. Following immunostaining with specific antibodies against smooth muscle actin (SMA) and lymphatic vessel endothelial receptor 1 (LYVE1), the aortas, including the aortic root, were subjected to a 30-minute treatment with 5 g/L Sudan Black B solution. This step was instrumental in minimizing the autofluorescent background of the tissue. Thereafter, the aortas were processed through a clearing protocol and imaged within a purpose-built chamber under a fluorescence microscope. Results The pretreatment with 5 g/L Sudan Black B effectively suppressed the autofluorescent signals emanating from the vascular structures, thereby enhancing the contrast and clarity of the specific fluorescence signals associated with the lymphatic vessels. This enhancement in signal quality did not compromise the integrity or specificity of the immunofluorescent markers. Conclusion A facile, highly specific, and effective approach for the visualization of lymphatic vessels in whole-mount aortic preparations from ApoE -/- mice is established.

Competing Interests: Declaration of interests No interests are declared.

Fluorescent probes are a powerful tool for imaging amyloid β (Aβ) plaques, the hallmark of Alzheimer's disease (AD). Herein, we report the synthesis and comprehensive characterization of 21 novel probes as well as their optical properties and binding affinities to Aβ fibrils. One of these dyes, 1Ae, exhibited several improvements over FDDNP, an established biomarker for Aβ- and Tau-aggregates. First, 1Ae had large Stokes shifts (138-213 nm) in various solvents, thereby reducing self-absorption. With a high quantum yield ratio (φ(dichloromethane/methanol) = 104), 1Ae also ensures minimal background emission in aqueous environments and high sensitivity. In addition, compound 1Ae exhibited low micromolar binding affinity to Aβ fibrils in vitro (K d = 1.603 µM), while increasing fluorescence emission (106-fold) compared to emission in buffer alone. Importantly, the selective binding of 1Ae to Aβ 1-42 fibrils was confirmed by an in cellulo assay, supported by ex vivo fluorescence microscopy of 1Ae on postmortem AD brain sections, allowing unequivocal identification of Aβ plaques. The intermolecular interactions of fluorophores with Aβ were elucidated by docking studies and molecular dynamics simulations. Density functional theory calculations revealed the unique photophysics of these rod-shaped fluorophores, with a twisted intramolecular charge transfer (TICT) excited state. These results provide valuable insights into the future application of such probes as potential diagnostic tools for AD in vitro and ex vivo such as determination of Aβ 1-42 in cerebrospinal fluid or blood., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Significance: Label-free quantitative phase imaging can potentially measure cellular dynamics with minimal perturbation, motivating efforts to develop faster and more sensitive instrumentation. We characterize fast, single-shot quantitative phase gradient microscopy (ss-QPGM) that simultaneously acquires multiple polarization components required to reconstruct phase images. We integrate a computationally efficient least squares algorithm to provide real-time, video-rate imaging (up to 75   frames / s ). The developed instrument was used to observe changes in cellular morphology and correlate these to molecular measures commonly obtained by staining., Aim: We aim to characterize a fast approach to ss-QPGM and record morphological changes in single-cell phase images. We also correlate these with biochemical changes indicating cell death using concurrently acquired fluorescence images., Approach: Here, we examine nutrient deprivation and anticancer drug-induced cell death in two different breast cell lines, viz. , M2 and MCF7. Our approach involves in-line measurements of ss-QPGM and fluorescence imaging of the cells biochemically labeled for viability., Results: We validate the accuracy of the phase measurement using a USAF1951 pattern phase target. The ss-QPGM system resolves 912.3    lp / mm , and our analysis scheme accurately retrieves the phase with a high correlation coefficient ( ∼ 0.99 ), as measured by calibrated sample thicknesses. Analyzing the contrast in phase, we estimate the spatial resolution achievable to be 0.55    μ m for this microscope. ss-QPGM time-lapse live-cell imaging reveals multiple intracellular and morphological changes during biochemically induced cell death. Inferences from co-registered images of quantitative phase and fluorescence suggest the possibility of necrosis, which agrees with previous findings., Conclusions: Label-free ss-QPGM with high-temporal resolution and high spatial fidelity is demonstrated. Its application for monitoring dynamic changes in live cells offers promising prospects., (© 2024 The Authors.)

G protein-coupled receptors (GPCRs) are membrane proteins that transmit specific external stimuli into cells by changing their conformation. This conformational change allows them to couple and activate G-proteins to initiate signal transduction. A critical challenge in studying and inferring these structural dynamics arises from the complexity of the cellular environment, including the presence of various endogenous factors. Due to the recent advances in cell-expression systems, membrane-protein purification techniques, and labeling approaches, it is now possible to study the structural dynamics of GPCRs at a single-molecule level both in vitro and in live cells. In this review, we discuss state-of-the-art techniques and strategies for expressing, purifying, and labeling GPCRs in the context of single-molecule research. We also highlight four recent studies that demonstrate the applications of single-molecule microscopy in revealing the dynamics of GPCRs. These techniques are also useful as complementary methods to verify the results obtained from other structural biology tools like cryo-electron microscopy and x-ray crystallography., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The point spread function (PSF) of a microscope describes the image of a point emitter. Knowing the accurate PSF model is essential for various imaging tasks, including single-molecule localization, aberration correction and deconvolution. Here we present universal inverse modeling of point spread functions (uiPSF), a toolbox to infer accurate PSF models from microscopy data, using either image stacks of fluorescent beads or directly images of blinking fluorophores, the raw data in single-molecule localization microscopy (SMLM). Our modular framework is applicable to a variety of microscope modalities and the PSF model incorporates system- or sample-specific characteristics, for example, the bead size, field- and depth- dependent aberrations, and transformations among channels. We demonstrate its application in single or multiple channels or large field-of-view SMLM systems, 4Pi-SMLM, and lattice light-sheet microscopes using either bead data or single-molecule blinking data., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

By superlocalizing the positions of millions of single molecules over many camera frames, a class of super-resolution fluorescence microscopy methods known as single-molecule localization microscopy (SMLM) has revolutionized how we understand subcellular structures over the past decade. In this review, we highlight emerging studies that transcend the outstanding structural (shape) information offered by SMLM to extract and map physicochemical parameters in living mammalian cells at single-molecule and super-resolution levels. By encoding/decoding high-dimensional information-such as emission and excitation spectra, motion, polarization, fluorescence lifetime, and beyond-for every molecule, and mass accumulating these measurements for millions of molecules, such multidimensional and multifunctional super-resolution approaches open new windows into intracellular architectures and dynamics, as well as their underlying biophysical rules, far beyond the diffraction limit.

The proliferation of microscopy methods for live-cell imaging offers many new possibilities for users but can also be challenging to navigate. The prevailing challenge in live-cell fluorescence microscopy is capturing intra-cellular dynamics while preserving cell viability. Computational methods can help to address this challenge and are now shifting the boundaries of what is possible to capture in living systems. In this Review, we discuss these computational methods focusing on artificial intelligence-based approaches that can be layered on top of commonly used existing microscopies as well as hybrid methods that integrate computation and microscope hardware. We specifically discuss how computational approaches can improve the signal-to-noise ratio, spatial resolution, temporal resolution and multi-colour capacity of live-cell imaging., (© 2024. Springer Nature Limited.)

A growing consensus that the brain is a mechanosensitive organ is driving the need for tools that mechanically stimulate and simultaneously record the electrophysiological response of neurons within neuronal networks. Here we introduce a synchronized combination of atomic force microscopy, high-density microelectrode array and fluorescence microscopy to monitor neuronal networks and to mechanically characterize and stimulate individual neurons at piconewton force sensitivity and nanometre precision while monitoring their electrophysiological activity at subcellular spatial and millisecond temporal resolution. No correlation is found between mechanical stiffness and electrophysiological activity of neuronal compartments. Furthermore, spontaneously active neurons show exceptional functional resilience to static mechanical compression of their soma. However, application of fast transient (∼500 ms) mechanical stimuli to the neuronal soma can evoke action potentials, which depend on the anchoring of neuronal membrane and actin cytoskeleton. Neurons show higher responsivity, including bursts of action potentials, to slower transient mechanical stimuli (∼60 s). Moreover, transient and repetitive application of the same compression modulates the neuronal firing rate. Seemingly, neuronal networks can differentiate and respond to specific characteristics of mechanical stimulation. Ultimately, the developed multiparametric tool opens the door to explore manifold nanomechanobiological responses of neuronal systems and new ways of mechanical control., (© 2024. The Author(s).)

Three-dimensional (3D) imaging has advanced basic research and clinical medicine. However, limited resolution and imperfections of real-world 3D image material often preclude algorithmic image analysis. Here, we present a methodologic framework for such imaging and analysis for functional and spatial relations in experimental nephritis. First, optical tissue-clearing protocols were optimized to preserve fluorescence signals for light sheet fluorescence microscopy and compensated attenuation effects using adjustable 3D correction fields. Next, we adapted the fast marching algorithm to conduct backtracking in 3D environments and developed a tool to determine local concentrations of extractable objects. As a proof-of-concept application, we used this framework to determine in a glomerulonephritis model the individual proteinuria and periglomerular immune cell infiltration for all glomeruli of half a mouse kidney. A correlation between these parameters surprisingly did not support the intuitional assumption that the most inflamed glomeruli are the most proteinuric. Instead, the spatial density of adjacent glomeruli positively correlated with the proteinuria of a given glomerulus. Because proteinuric glomeruli appear clustered, this suggests that the exact location of a kidney biopsy may affect the observed severity of glomerular damage. Thus, our algorithmic pipeline described here allows analysis of various parameters of various organs composed of functional subunits, such as the kidney, and can theoretically be adapted to processing other image modalities., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Here we present a method to reduce the photobleaching of fluorescent proteins and the associated phototoxicity. It exploits a photophysical process known as reverse intersystem crossing, which we induce by near-infrared co-illumination during fluorophore excitation. This dual illumination method reduces photobleaching effects 1.5-9.2-fold, can be easily implemented on commercial microscopes and is effective in eukaryotic and prokaryotic cells with a wide range of fluorescent proteins., (© 2023. The Author(s).)

Optical tissue clearing and three-dimensional (3D) immunofluorescence (IF) microscopy is transforming imaging of the complex tumor microenvironment (TME). However, current 3D IF microscopy has restricted multiplexity; only 3 or 4 cellular and noncellular TME components can be localized in cleared tumor tissue. Here we report a light-emitting diode (LED) photobleaching method and its application for 3D multiplexed optical mapping of the TME. We built a high-power LED light irradiation device and temperature-controlled chamber for completely bleaching fluorescent signals throughout optically cleared tumor tissues without compromise of tissue and protein antigen integrity. With newly developed tissue mounting and selected region-tracking methods, we established a cyclic workflow involving IF staining, tissue clearing, 3D confocal microscopy, and LED photobleaching. By registering microscope channel images generated through 3 work cycles, we produced 8-plex image data from individual 400 μm-thick tumor macrosections that visualize various vascular, immune, and cancer cells in the same TME at tissue-wide and cellular levels in 3D. Our method was also validated for quantitative 3D spatial analysis of cellular remodeling in the TME after immunotherapy. These results demonstrate that our LED photobleaching system and its workflow offer a novel approach to increase the multiplexing power of 3D IF microscopy for studying tumor heterogeneity and response to therapy., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Fluorescence microscopy allows for the high-throughput imaging of cellular activity across brain areas in mammals. However, capturing rapid cellular dynamics across the curved cortical surface is challenging, owing to trade-offs in image resolution, speed, field of view and depth of field. Here we report a technique for wide-field fluorescence imaging that leverages selective illumination and the integration of focal areas at different depths via a spinning disc with varying thickness to enable video-rate imaging of previously reconstructed centimetre-scale arbitrarily shaped surfaces at micrometre-scale resolution and at a depth of field of millimetres. By implementing the technique in a microscope capable of acquiring images at 1.68 billion pixels per second and resolving 16.8 billion voxels per second, we recorded neural activities and the trajectories of neutrophils in real time on curved cortical surfaces in live mice. The technique can be integrated into many microscopes and macroscopes, in both reflective and fluorescence modes, for the study of multiscale cellular interactions on arbitrarily shaped surfaces., (© 2023. The Author(s).)

The interactions between proteins in membranes govern many cellular functions. Our ability to probe for such interactions has greatly evolved in recent years due to the introduction of new fluorescence techniques. As a result, we currently have a choice of methods that can be used to assess the spatial distribution of a membrane protein, its association state, and the thermodynamic stability of the oligomers in the native milieu. These biophysical measurements have revealed new insights into important biological processes in cellular membranes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Time-resolved fluorescence imaging techniques, like confocal fluorescence lifetime imaging microscopy, are powerful photonic instrumentation tools of modern science with diverse applications, including: biology, medicine, and chemistry. However, complexities of the systems, both at specimen and device levels, cause difficulties in quantifying soft biomarkers. To address the problems, we first aim to understand and model the underlying photophysics of fluorescence decay curves. For this purpose, we provide a set of mathematical functions, called "life models", fittable with the real temporal recordings of histogram of photon counts. For each model, an equivalent electrical circuit, called a "life circuit", is derived for explaining the whole process. In confocal endomicroscopy, the components of excitation laser, specimen, and fluorescence-emission signal as the histogram of photon counts are modelled by a power source, network of resistor-inductor-capacitor circuitry, and multimetre, respectively. We then design a novel pixel-level temporal classification algorithm, called a "fit-flexible approach", where qualities of "intensity", "fall-time", and "life profile" are identified for each point. A model selection mechanism is used at each pixel to flexibly choose the best representative life model based on a proposed Misfit-percent metric. A two-dimensional arrangement of the quantified information detects some kind of structural information. This approach showed a potential of separating microbeads from lung tissue, distinguishing the tri-sensing from conventional methods. We alleviated by 7% the error of the Misfit-percent for recovering the histograms on real samples than the best state-of-the-art competitor. Codes are available online.

Significance: Spectroscopic single-molecule localization microscopy (sSMLM) takes advantage of nanoscopy and spectroscopy, enabling sub-10 nm resolution as well as simultaneous multicolor imaging of multi-labeled samples. Reconstruction of raw sSMLM data using deep learning is a promising approach for visualizing the subcellular structures at the nanoscale., Aim: Develop a novel computational approach leveraging deep learning to reconstruct both label-free and fluorescence-labeled sSMLM imaging data., Approach: We developed a two-network-model based deep learning algorithm, termed DsSMLM, to reconstruct sSMLM data. The effectiveness of DsSMLM was assessed by conducting imaging experiments on diverse samples, including label-free single-stranded DNA (ssDNA) fiber, fluorescence-labeled histone markers on COS-7 and U2OS cells, and simultaneous multicolor imaging of synthetic DNA origami nanoruler., Results: For label-free imaging, a spatial resolution of 6.22 nm was achieved on ssDNA fiber; for fluorescence-labeled imaging, DsSMLM revealed the distribution of chromatin-rich and chromatin-poor regions defined by histone markers on the cell nucleus and also offered simultaneous multicolor imaging of nanoruler samples, distinguishing two dyes labeled in three emitting points with a separation distance of 40 nm. With DsSMLM, we observed enhanced spectral profiles with 8.8% higher localization detection for single-color imaging and up to 5.05% higher localization detection for simultaneous two-color imaging., Conclusions: We demonstrate the feasibility of deep learning-based reconstruction for sSMLM imaging applicable to label-free and fluorescence-labeled sSMLM imaging data. We anticipate our technique will be a valuable tool for high-quality super-resolution imaging for a deeper understanding of DNA molecules' photophysics and will facilitate the investigation of multiple nanoscopic cellular structures and their interactions., (© 2024 The Authors.)

Fluorescence microscopy has undergone rapid advancements, offering unprecedented visualization of biological events and shedding light on the intricate mechanisms governing living organisms. However, the exploration of rapid biological dynamics still poses a significant challenge due to the limitations of current digital camera architectures and the inherent compromise between imaging speed and other capabilities. Here, we introduce sHAPR, a high-speed acquisition technique that leverages the operating principles of sCMOS cameras to capture fast cellular and subcellular processes. sHAPR harnesses custom fiber optics to convert microscopy images into one-dimensional recordings, enabling acquisition at the maximum camera readout rate, typically between 25 and 250 kHz. We have demonstrated the utility of sHAPR with a variety of phantom and dynamic systems, including high-throughput flow cytometry, cardiomyocyte contraction, and neuronal calcium waves, using a standard epi-fluorescence microscope. sHAPR is highly adaptable and can be integrated into existing microscopy systems without requiring extensive platform modifications. This method pushes the boundaries of current fluorescence imaging capabilities, opening up new avenues for investigating high-speed biological phenomena., (© 2024. The Author(s).)

Nanopore sensing is a popular biosensing strategy that is being explored for the quantitative analysis of biomarkers. With low concentrations of analytes, nanopore sensors face challenges related to slow response times and selectivity. Here, we demonstrate an approach to rapidly detect species at ultralow concentrations using an optical nanopore blockade sensor for quantitative detection of the protein vascular endothelial growth factor (VEGF). This sensor relies on monitoring fluorescent polystyrene nanoparticles blocking nanopores in a nanopore array of 676 nanopores. The fluorescent signal is read out using a wide-field fluorescence microscope. Nonspecific blockade events are then distinguished from specific blockade events based on the ability to pull the particles out of the pore using an applied electric field. This allows the detection of VEGF at sub-picomolar concentration in less than 15 min.

High-speed wide-field fluorescence microscopy has the potential to capture biological processes with exceptional spatiotemporal resolution. However, conventional cameras suffer from low signal-to-noise ratio at high frame rates, limiting their ability to detect faint fluorescent events. Here, we introduce an image sensor where each pixel has individually programmable sampling speed and phase, so that pixels can be arranged to simultaneously sample at high speed with a high signal-to-noise ratio. In high-speed voltage imaging experiments, our image sensor significantly increases the output signal-to-noise ratio compared to a low-noise scientific CMOS camera (~2-3 folds). This signal-to-noise ratio gain enables the detection of weak neuronal action potentials and subthreshold activities missed by the standard scientific CMOS cameras. Our camera with flexible pixel exposure configurations offers versatile sampling strategies to improve signal quality in various experimental conditions., (© 2024. The Author(s).)

Selective Plane Illumination Microscopy (SPIM) has become an emerging technology since its first application for 3D in-vivo imaging of the development of a living organism. An extensive number of works have been published, improving both the speed of acquisition and the resolution of the systems. Furthermore, multispectral imaging allows the effective separation of overlapping signals associated with different fluorophores from the spectrum over the whole field-of-view of the analyzed sample. To eliminate the need of using fluorescent dyes, this technique can also be applied to autofluorescence imaging. However, the effective separation of the overlapped spectra in autofluorescence imaging necessitates the use of mathematical tools. In this work, we explore the application of a method based on Principal Component Analysis (PCA) that enables tissue characterization upon spectral autofluorescence data without the use of fluorophores. Thus, enabling the separation of different tissue types in fixed and living samples with no need of staining techniques. Two procedures are described for acquiring spectral data, including a single excitation based method and a multi-excitation scanning approach. In both cases, we demonstrate the effective separation of various tissue types based on their unique autofluorescence spectra., (© 2024. The Author(s).)

Molecular probes with the ability to differentiate between subcellular variations in acidity levels remain important for the investigation of dynamic cellular processes and functions. In this context, a series of cyclic peptide and PEG bio-conjugated dual near-infrared emissive BF 2 -azadipyrromethene fluorophores with maxima emissions at 720 nm (at pH > 6) and 790 nm (at pH < 5) have been developed and their aqueous solution photophysical properties determined. Their inter-converting emissions and fluorescence lifetime characteristics were exploited to track their spatial and temporal progression from first contact with the plasma membrane to subcellular locales to their release within extracellular vesicles. A pH-dependent reversible phenolate/phenol interconversion on the fluorophore controlled the dynamic changes in dual emission responses and corresponding lifetime changes. Live-cell confocal microscopy experiments in the metastatic breast cancer cell line MDA-MB-231 confirmed the usability of the dual emissive properties for imaging over prolonged periods. All three derivatives performed as probes capable of real-time continuous imaging of fundamental cellular processes such as plasma membrane interaction, tracking endocytosis, lysosomal/large acidic vesicle accumulation, and efflux within extracellular vesicles without perturbing cellular function. Furthermore, fluorescence lifetime imaging microscopy provided valuable insights regarding fluorophore progression through intracellular microenvironments over time. Overall, the unique photophysical properties of these fluorophores show excellent potential for their use as information-rich probes.

Fluorescent labels have strongly contributed to many advancements in bioanalysis, molecular biology, molecular imaging, and medical diagnostics. Despite a large toolbox of molecular and nanoscale fluorophores to choose from, there is still a need for brighter labels, e.g., for flow cytometry and fluorescence microscopy, that are preferably of molecular nature. This requires versatile concepts for fluorophore multimerization, which involves the shielding of dyes from other chromophores and possible quenchers in their neighborhood. In addition, to increase the number of readout parameters for fluorescence microscopy and eventually also flow cytometry, control and tuning of the labels' fluorescence lifetimes is desired. Searching for bright multi-chromophoric or multimeric labels, we developed PEGylated dyes bearing functional groups for their bioconjugation and explored their spectroscopic properties and photostability in comparison to those of the respective monomeric dyes for two exemplarily chosen fluorophores excitable at 488 nm. Subsequently, these dyes were conjugated with anti-CD4 and anti-CD8 immunoglobulins to obtain fluorescent conjugates suitable for the labeling of cells and beads. Finally, the suitability of these novel labels for fluorescence lifetime imaging and target discrimination based upon lifetime measurements was assessed. Based upon the results of our spectroscopic studies including measurements of fluorescence quantum yields (QY) and fluorescence decay kinetics we could demonstrate the absence of significant dye-dye interactions and self-quenching in these multimeric labels. Moreover, in a first fluorescence lifetime imaging (FLIM) study, we could show the future potential of this multimerization concept for lifetime discrimination and multiplexing., (© 2024. The Author(s).)

Gamma-prefoldin (γPFD), a unique chaperone found in the extremely thermophilic methanogen Methanocaldococcus jannaschii , self-assembles into filaments in vitro , which so far have been observed using transmission electron microscopy and cryo-electron microscopy. Utilizing three-dimensional stochastic optical reconstruction microscopy (3D-STORM), here we achieve ∼20 nm resolution by precisely locating individual fluorescent molecules, hence resolving γPFD ultrastructure both in vitro and in vivo . Through CF647 NHS ester labeling, we first demonstrate the accurate visualization of filaments and bundles with purified γPFD. Next, by implementing immunofluorescence labeling after creating a 3xFLAG-tagged γPFD strain, we successfully visualize γPFD in M . jannaschii cells. Through 3D-STORM and two-color STORM imaging with DNA, we show the widespread distribution of filamentous γPFD structures within the cell. These findings provide valuable insights into the structure and localization of γPFD, opening up possibilities for studying intriguing nanoscale components not only in archaea but also in other microorganisms.

We present methods for making and testing the membrane biophysics of model lipid droplets (LDs). Methods are described for imaging LDs ranging in size from 0.1 to 40 μm in diameter with high-resolution microscopy and spectroscopy. With known LD compositions, membrane binding, sorting, diffusion, and tension were measured via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), and imaging flow cytometry. Additionally, a custom, small-volume pendant droplet tensiometer is described and used to measure the association of phospholipids to the LD surface. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes on lipid droplet monolayers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Computational super-resolution methods, including conventional analytical algorithms and deep learning models, have substantially improved optical microscopy. Among them, supervised deep neural networks have demonstrated outstanding performance, however, demanding abundant high-quality training data, which are laborious and even impractical to acquire due to the high dynamics of living cells. Here, we develop zero-shot deconvolution networks (ZS-DeconvNet) that instantly enhance the resolution of microscope images by more than 1.5-fold over the diffraction limit with 10-fold lower fluorescence than ordinary super-resolution imaging conditions, in an unsupervised manner without the need for either ground truths or additional data acquisition. We demonstrate the versatile applicability of ZS-DeconvNet on multiple imaging modalities, including total internal reflection fluorescence microscopy, three-dimensional wide-field microscopy, confocal microscopy, two-photon microscopy, lattice light-sheet microscopy, and multimodal structured illumination microscopy, which enables multi-color, long-term, super-resolution 2D/3D imaging of subcellular bioprocesses from mitotic single cells to multicellular embryos of mouse and C. elegans., (© 2024. The Author(s).)

Fluorescence fluctuation super-resolution microscopy (FF-SRM) has emerged as a promising method for the fast, low-cost, and uncomplicated imaging of biological specimens beyond the diffraction limit. Among FF-SRM techniques, super-resolution radial fluctuation (SRRF) microscopy is a popular technique but is prone to artifacts, resulting in low fidelity, especially under conditions of high-density fluorophores. In this Letter, we developed a novel, to the best of our knowledge, combinatory computational super-resolution microscopy method, namely VeSRRF, that demonstrated superior performance in SRRF microscopy. VeSRRF combined intensity and gradient variance reweighted radial fluctuations (VRRF) and enhanced-SRRF (eSRRF) algorithms, leveraging the enhanced resolution achieved through intensity and gradient variance analysis in VRRF and the improved fidelity obtained from the radial gradient convergence transform in eSRRF. Our method was validated using microtubules in mammalian cells as a standard biological model system. Our results demonstrated that VeSRRF consistently achieved the highest resolution and exceptional fidelity compared to those obtained from other algorithms in both single-molecule localization microscopy (SMLM) and FF-SRM. Moreover, we developed the VeSRRF software package that is freely available on the open-source ImageJ/Fiji software platform to facilitate the use of VeSRRF in the broader community of biomedical researchers. VeSRRF is an exemplary method in which complementary microscopy techniques are integrated holistically, creating superior imaging performance and capabilities.

Tracking stem cell fate transition is crucial for understanding their development and optimizing biomanufacturing. Destructive single-cell methods provide a pseudotemporal landscape of stem cell differentiation but cannot monitor stem cell fate in real time. We established a metabolic optical metric using label-free fluorescence lifetime imaging microscopy (FLIM), feature extraction and machine learning-assisted analysis, for real-time cell fate tracking. From a library of 205 metabolic optical biomarker (MOB) features, we identified 56 associated with hematopoietic stem cell (HSC) differentiation. These features collectively describe HSC fate transition and detect its bifurcate lineage choice. We further derived a MOB score measuring the "metabolic stemness" of single cells and distinguishing their division patterns. This score reveals a distinct role of asymmetric division in rescuing stem cells with compromised metabolic stemness and a unique mechanism of PI3K inhibition in promoting ex vivo HSC maintenance. MOB profiling is a powerful tool for tracking stem cell fate transition and improving their biomanufacturing from a single-cell perspective.

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and intercellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we develop Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes., (© 2024. The Author(s).)

Extracellular matrix diseases like fibrosis are elusive to diagnose early on, to avoid complete loss of organ function or even cancer progression, making early diagnosis crucial. Imaging the matrix densities of proteins like collagen in fixed tissue sections with suitable stains and labels is a standard for diagnosis and staging. However, fine changes in matrix density are difficult to realize by conventional histological staining and microscopy as the matrix fibrils are finer than the resolving capacity of these microscopes. The dyes further blur the outline of the matrix and add a background that bottlenecks high-precision early diagnosis of matrix diseases. Here we demonstrate the multiple signal classification method-MUSICAL-otherwise a computational super-resolution microscopy technique to precisely estimate matrix density in fixed tissue sections using fibril autofluorescence with image stacks acquired on a conventional epifluorescence microscope. We validated the diagnostic and staging performance of the method in extracted collagen fibrils, mouse skin during repair, and pre-cancers in human oral mucosa. The method enables early high-precision label-free diagnosis of matrix-associated fibrotic diseases without needing additional infrastructure or rigorous clinical training., (© 2024. The Author(s).)

Fluorescence labeling of chemically fixed specimens, especially immunolabeling, plays a vital role in super-resolution imaging as it offers a convenient way to visualize cellular structures like mitochondria or the distribution of biomolecules with high detail. Despite the development of various distinct probes that enable super-resolved stimulated emission depletion (STED) imaging of mitochondria in live cells, most of these membrane-potential-dependent fluorophores cannot be retained well in mitochondria after chemical fixation. This lack of suitable mitochondrial probes has limited STED imaging of mitochondria to live cell samples. In this study, we introduce a mitochondria-specific probe, PK Mito Orange FX (PKMO FX), which features a fixation-driven cross-linking motif and accumulates in the mitochondrial inner membrane. It exhibits high fluorescence retention after chemical fixation and efficient depletion at 775 nm, enabling nanoscopic imaging both before and after aldehyde fixation. We demonstrate the compatibility of this probe with conventional immunolabeling and other strategies commonly used for fluorescence labeling of fixed samples. Moreover, we show that PKMO FX facilitates correlative super-resolution light and electron microscopy, enabling the correlation of multicolor fluorescence images and transmission EM images via the characteristic mitochondrial pattern. Our probe further expands the mitochondrial toolkit for multimodal microscopy at nanometer resolutions., Competing Interests: Competing interests statement:P.C. is a full-time employee of Genvivo Biotech. Z.C. is the founder of Genvivo Biotech. J.C. and Z.C. have submitted a patent application based on the Cy3.5 conjugates described in this work.

Fluorescence lifetime imaging microscopy (FLIM) has proven to be a useful method for analyzing various aspects of material science and biology, like the supramolecular organization of (slightly) fluorescent compounds or the metabolic activity in non-labeled cells; in particular, FLIM phasor analysis (phasor-FLIM) has the potential for an intuitive representation of complex fluorescence decays and therefore of the analyzed properties. Here we present and make available tools to fully exploit this potential, in particular by coding via hue, saturation, and intensity the phasor positions and their weights both in the phasor plot and in the microscope image. We apply these tools to analyze FLIM data acquired via two-photon microscopy to visualize: (i) different phases of the drug pioglitazone (PGZ) in solutions and/or crystals, (ii) the position in the phasor plot of non-labelled poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), and (iii) the effect of PGZ or PGZ-containing NPs on the metabolism of insulinoma (INS-1 E) model cells. PGZ is recognized for its efficacy in addressing insulin resistance and hyperglycemia in type 2 diabetes mellitus, and polymeric nanoparticles offer versatile platforms for drug delivery due to their biocompatibility and controlled release kinetics. This study lays the foundation for a better understanding via phasor-FLIM of the organization and effects of drugs, in particular, PGZ, within NPs, aiming at better control of encapsulation and pharmacokinetics, and potentially at novel anti-diabetics theragnostic nanotools.

Motivation: Accurate segmentation and recognition of C.elegans cells are critical for various biological studies, including gene expression, cell lineages, and cell fates analysis at single-cell level. However, the highly dense distribution, similar shapes, and inhomogeneous intensity profiles of whole-body cells in 3D fluorescence microscopy images make automatic cell segmentation and recognition a challenging task. Existing methods either rely on additional fiducial markers or only handle a subset of cells. Given the difficulty or expense associated with generating fiducial features in many experimental settings, a marker-free approach capable of reliably segmenting and recognizing C.elegans whole-body cells is highly desirable., Results: We report a new pipeline, called automated segmentation and recognition (ASR) of cells, and applied it to 3D fluorescent microscopy images of L1-stage C.elegans with 558 whole-body cells. A novel displacement vector field based deep learning model is proposed to address the problem of reliable segmentation of highly crowded cells with blurred boundary. We then realize the cell recognition by encoding and exploiting statistical priors on cell positions and structural similarities of neighboring cells. To the best of our knowledge, this is the first method successfully applied to the segmentation and recognition of C.elegans whole-body cells. The ASR-segmentation module achieves an F1-score of 0.8956 on a dataset of 116 C.elegans image stacks with 64 728 cells (accuracy 0.9880, AJI 0.7813). Based on the segmentation results, the ASR recognition module achieved an average accuracy of 0.8879. We also show ASR's applicability to other cell types, e.g. platynereis and rat kidney cells., Availability and Implementation: The code is available at https://github.com/reaneyli/ASR., (© The Author(s) 2024. Published by Oxford University Press.)

Fluorescent imaging of cellular membranes is challenged by the size of lipid bilayers, which are smaller than the diffraction limit of light. Recently, expansion microscopy (ExM) has emerged as an approachable super-resolution method that requires only widely accessible confocal microscopes. In this method, biomolecules of interest are anchored to hydrogel-based, polymeric networks that are expanded through osmosis to physically separate and resolve features smaller than the diffraction limit of light. Whereas ExM has been employed for super-resolution imaging of proteins, DNA, RNA, and glycans, the application of this method to the study of lipids is challenged by the requirement of permeabilization procedures that remove lipids and compromise the integrity of the membrane. Here, we describe our recently developed protocols for lipid expansion microscopy (LExM), a method that enables ExM of membranes without permeabilization. These detailed protocols and accompanying commentary sections aim to make LExM accessible to any experimentalist interested in imaging membranes with super-resolution. © 2024 Wiley Periodicals LLC. Basic Protocol 1: LExM of alkyne-choline lipids Basic Protocol 2: LExM of IMPACT-labeled lipids Basic Protocol 3: LExM of clickable cholesterol Basic Protocol 4: Determining the expansion factor., (© 2024 Wiley Periodicals LLC.)

Both Ca 2+ and protein kinase A (PKA) are multifaceted and ubiquitous signaling molecules, essential for regulating the intricate network of signaling pathways. However, their dynamics within specialized membrane regions are still not well characterized. By using genetically encoded fluorescent indicators specifically targeted to distinct plasma membrane microdomains, we have established a protocol that permits observing Ca 2+ /PKA dynamics in discrete neuronal microdomains with high spatial and temporal resolution. The approach employs a fluorescence microscope with a sensitive camera and a dedicated CFP/YFP/mCherry filter set, enabling the simultaneous detection of donor-acceptor emission and red fluorescence signal. In this detailed step-by-step guide, we outline the experimental procedure, including isolation of rat primary neurons and their transfection with biosensors targeted to lipid rafts or non-raft regions of plasma membrane. We provide information on the necessary equipment and imaging setup required for recording, along with highlighting critical parameters and troubleshooting guidelines for real-time measurements. Finally, we provide examples of the observed Ca 2+ and PKA changes in specific cellular compartments. The application of this technique may have significant implications for studying cross-talk between second messengers and their alterations in various pathological conditions. © 2024 Wiley Periodicals LLC., (© 2024 Wiley Periodicals LLC.)

Herein, we report the development of a macromolecular multifunctional imaging tool for biological investigations, which is comprised of an N-(2-hydroxypropyl)methacrylamide backbone, iridium-based luminescent probe, glutamate carboxypeptidase II (GCPII) targeting ligand, and biotin affinity tag. The iridium luminophore is a tris-cyclometalated complex based on [Ir(ppy) 3 ] with one of its 2-phenylpyridine ligands functionalized to allow conjugation. Synthesized macromolecular probes differed in the structure of the polymer and content of the iridium complex. The applicability of the developed imaging tools has been tested in flow cytometry (FACS) based assay, laser confocal microscopy, and fluorescence lifetime imaging microscopy (FLIM). The FACS analysis has shown that the targeted iBodies containing the iridium luminophore exhibit selective labelling of GCPII expressing cells. This observation was also confirmed in the imaging experiments with laser confocal microscopy. The FLIM experiment has shown that the iBodies with the iridium label exhibit a lifetime greater than 100 ns, which distinguishes them from typically used systems labelled with organic fluorophores exhibiting short fluorescence lifetimes. The results of this investigation indicate that the system exhibits interesting properties, which supports the development of additional biological tools utilizing the key components (iridium complexes, iBody concept), primarily focusing on the longer lifetime of the iridium emitter., (© 2024 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

High-resolution spatio-temporal monitoring of the cell membrane lipid order provides visual insights into the complex and sophisticated systems that control cellular physiological functions. Solvatochromic fluorescent probes are highly promising noninvasive visualization tools for identifying the ordering of the microenvironment of plasma membrane microdomains. However, conventional probes, although capable of structural analysis, lack the necessary long-term photostability required for live imaging at the cellular level. Here, an ultra-high-light-resistant solvatochromic fluorescence probe, 2-N,N-diethylamino-7-(4-methoxycarbonylphenyl)-9,9-dimethylfluorene (FπCM) is reported, which enables live lipid order imaging of cell division. This probe and its derivatives exhibit sufficient fluorescence wavelengths, brightness, polarity responsiveness, low phototoxicity, and remarkable photostability under physiological conditions compared to conventional solvatochromic probes. Therefore, these probes have the potential to overcome the limitations of fluorescence microscopy, particularly those associated with photobleaching. FπCM probes can serve as valuable tools for elucidating mechanisms of cellular processes at the bio-membrane level., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Light-sheet fluorescence microscopy is an invaluable tool for four-dimensional biological imaging of multicellular systems due to the rapid volumetric imaging and minimal illumination dosage. However, it is challenging to retrieve fine subcellular information, especially in living cells, due to the width of the sheet of light (>1 μm). Here, using reversibly switchable fluorescent proteins (RSFPs) and a periodic light pattern for photoswitching, we demonstrate a super-resolution imaging method for rapid volumetric imaging of subcellular structures called multi-sheet RESOLFT. Multiple emission-sheets with a width that is far below the diffraction limit are created in parallel increasing recording speed (1-2 Hz) to provide super-sectioning ability (<100 nm). Our technology is compatible with various RSFPs due to its minimal requirement in the number of switching cycles and can be used to study a plethora of cellular structures. We track cellular processes such as cell division, actin motion and the dynamics of virus-like particles in three dimensions., (© 2024. The Author(s).)

Multicellular systems grow over the course of weeks from single cells to tissues or even full organisms, making live imaging challenging. To bridge spatiotemporal scales, we present an open-top dual-view and dual-illumination light-sheet microscope dedicated to live imaging of large specimens at single-cell resolution. The configuration of objectives together with a customizable multiwell mounting system combines dual view with high-throughput multiposition imaging. We use this microscope to image a wide variety of samples and highlight its capabilities to gain quantitative single-cell information in large specimens such as mature intestinal organoids and gastruloids., (© 2024. The Author(s).)

Fluorescence microscopy is a powerful tool used in scientific and medical research, but it is inextricably linked to phototoxicity. Neglecting phototoxicity can lead to erroneous or inconclusive results. Recently, several reports have addressed this issue, but it is still underestimated by many researchers, even though it can lead to cell death. Phototoxicity can be reduced by appropriate microscopic techniques and carefully designed experiments. This review focuses on recent strategies to reduce phototoxicity in microscopic imaging of living cells and tissues. We describe digital image processing and new hardware solutions. We point out new modifications of microscopy methods and hope that this review will interest microscopy hardware engineers. Our aim is to underscore the challenges and potential solutions integral to the design of microscopy systems. Simultaneously, we intend to engage biologists, offering insight into the latest technological advancements in imaging that can enhance their understanding and practice., (© 2024 The Authors. BioEssays published by Wiley Periodicals LLC.)

Background: Fluorescence-based confocal microscopy (FCM) can be used to create virtual H&E sections in real time. So far, FCM has been used in dermato-, uro-, and gynecopathology. FCM allows the creation of a completely digitized frozen section, which could potentially replace conventional frozen sections in the future., Objective: The aim of the current work is to implement FCM technology as a component of fully digitized processes in the pathological workflow. For this purpose, the current use of FCM in liver transplant pathology will be extended to other disciplines such as urology and otorhinolaryngology., Materials and Methods: The FCM technique continues to be used prospectively on native tissue samples from potential donor livers. Conventional frozen sections are used comparatively to virtual FCM scans., Results: The data show a nearly perfect agreement for the detection of cholangitis, fibrosis, and malignancy, and a high level of agreement for, e.g., macrovesicular steatosis, inflammation, steatohepatitis, and necrosis between virtual FCM scans and conventional routine diagnostic frozen sections., Conclusion: Since the availability of time- and cost-intensive frozen section diagnostics in the context of transplant pathology in continuous operation (24/7) is now only established at very few university centers in Germany due to an increasing shortage of specialists, the use of FCM could be an important building block in the current process leading towards a fully digitized pathology workflow and should thus be extended to various disciplines., (© 2024. The Author(s).)

Imaging-based methods are widely used for studying the subcellular localization of proteins in living cells. While routine for individual proteins, global monitoring of protein dynamics following perturbation typically relies on arrayed panels of fluorescently tagged cell lines, limiting throughput and scalability. Here, we describe a strategy that combines high-throughput microscopy, computer vision and machine learning to detect perturbation-induced changes in multicolour tagged visual proteomics cell (vpCell) pools. We use genome-wide and cancer-focused intron-targeting sgRNA libraries to generate vpCell pools and a large, arrayed collection of clones each expressing two different endogenously tagged fluorescent proteins. Individual clones can be identified in vpCell pools by image analysis using the localization patterns and expression level of the tagged proteins as visual barcodes, enabling simultaneous live-cell monitoring of large sets of proteins. To demonstrate broad applicability and scale, we test the effects of antiproliferative compounds on a pool with cancer-related proteins, on which we identify widespread protein localization changes and new inhibitors of the nuclear import/export machinery. The time-resolved characterization of changes in subcellular localization and abundance of proteins upon perturbation in a pooled format highlights the power of the vpCell approach for drug discovery and mechanism-of-action studies., (© 2024. The Author(s).)

Endo-microscopy is crucial for real-time 3D visualization of internal tissues and subcellular structures. Conventional methods rely on axial movement of optical components for precise focus adjustment, limiting miniaturization and complicating procedures. Meta-device, composed of artificial nanostructures, is an emerging optical flat device that can freely manipulate the phase and amplitude of light. Here, an intelligent fluorescence endo-microscope is developed based on varifocal meta-lens and deep learning (DL). The breakthrough enables in vivo 3D imaging of mouse brains, where varifocal meta-lens focal length adjusts through relative rotation angle. The system offers key advantages such as invariant magnification, a large field-of-view, and optical sectioning at a maximum focal length tuning range of ≈2 mm with 3 µm lateral resolution. Using a DL network, image acquisition time and system complexity are significantly reduced, and in vivo high-resolution brain images of detailed vessels and surrounding perivascular space are clearly observed within 0.1 s (≈50 times faster). The approach will benefit various surgical procedures, such as gastrointestinal biopsies, neural imaging, brain surgery, etc., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

The background light from out-of-focus planes hinders resolution enhancement in structured illumination microscopy when observing volumetric samples. Here we used selective plane illumination and reversibly photoswitchable fluorescent proteins to realize structured illumination within the focal plane and eliminate the out-of-focus background. Theoretical investigation of the imaging properties and experimental demonstrations show that selective plane activation is beneficial for imaging dense microstructures in cells and cell spheroids., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Fluorescence microscopy data describe protein localization patterns at single-cell resolution and have the potential to reveal whole-proteome functional information with remarkable precision. Yet, extracting biologically meaningful representations from cell micrographs remains a major challenge. Existing approaches often fail to learn robust and noise-invariant features or rely on supervised labels for accurate annotations. We developed PIFiA (Protein Image-based Functional Annotation), a self-supervised approach for protein functional annotation from single-cell imaging data. We imaged the global yeast ORF-GFP collection and applied PIFiA to generate protein feature profiles from single-cell images of fluorescently tagged proteins. We show that PIFiA outperforms existing approaches for molecular representation learning and describe a range of downstream analysis tasks to explore the information content of the feature profiles. Specifically, we cluster extracted features into a hierarchy of functional organization, study cell population heterogeneity, and develop techniques to distinguish multi-localizing proteins and identify functional modules. Finally, we confirm new PIFiA predictions using a colocalization assay, suggesting previously unappreciated biological roles for several proteins. Paired with a fully interactive website ( https://thecellvision.org/pifia/ ), PIFiA is a resource for the quantitative analysis of protein organization within the cell., (© 2024. The Author(s).)

Recent developments in real-time, in vivo micro-imaging have allowed for the visualization of tissue pathological changes, facilitating rapid diagnosis. However, miniaturization, magnification, the field of view, and in vivo image stabilization remain challenging factors to reconcile. A key issue for this technology is ensuring it is user friendly for surgeons, enabling them to use the device manually and obtain instantaneous information necessary for surgical decision-making. This descriptive study introduces a handheld, actively stabilized, miniaturized epi-fluorescence widefield microscope (MEW-M) for real-time observation in vivo with high resolution. The methodology of MEW-M system includes high resolution microscopy miniaturization technology, thousandfold shaking suppression (actively stabilized), ultra-photosensitivity, and tailored image signal processing cell image capture and processing technology, which support for the excellent real-time imaging performance of MEW-M system in brain, mammary, liver, lung, and kidney tissue imaging of rats in vivo. With a single-objective and high-frame-rate imaging, the MEW-M system facilitates roving image acquisition, enabling contiguous analysis of large tissue areas. RESEARCH HIGHLIGHTS: A handheld, actively stabilized MEW-M system was introduced. Excellent real-time, in vivo imaging with high resolution and active stabilization in brain, mammary, liver, lung, and kidney tissue of rats., (© 2024 Wiley Periodicals LLC.)