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The sarcomere is the fundamental contractile unit in skeletal muscle, and the regularity of its structure is critical for function. Emerging data demonstrates that nanoscale changes to the regularity of sarcomere structure can affect the overall function of the protein dense ~2μm sarcomere. Further, sarcomere structure is implicated in many clinical conditions of muscle weakness. However, our understanding of how sarcomere structure changes in disease, especially at the nanoscale, has been limited in part due to the inability to robustly detect and measure at sub-sarcomere resolution. We optimized several methodological steps and developed a robust pipeline to analyze sarcomere structure using structured illumination super-resolution microscopy in conjunction with commercially-available and fluorescently-conjugated Variable Heavy-Chain only fragment secondary antibodies (nanobodies), and achieved a significant increase in resolution of z-disc width (353nm vs. 62nm) compared to confocal microscopy. The combination of these methods provides a unique approach to probe sarcomere protein localization at the nanoscale and may prove advantageous for analysis of other cellular structures., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Douglas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In this work, we apply single-molecule fluorescence microscopy and spectroscopy to probe plasmon-enhanced fluorescence and Förster resonance energy transfer in a nanoscale assemblies. The structure where the interplay between these two processes was present consists of photoactive proteins conjugated with silver nanowires and deposited on a monolayer graphene. By comparing the results of continuous-wave and time-resolved fluorescence microscopy acquired for this structure with those obtained for the reference samples, where proteins were coupled with either a graphene monolayer or silver nanowires, we find clear indications of the interplay between plasmonic enhancement and the energy transfer to graphene. Namely, fluorescence intensities calculated for the structure, where proteins were coupled to graphene only, are less than for the structure playing the central role in this study, containing both silver nanowires and graphene. Conversely, decay times extracted for the latter are shorter compared to a protein-silver nanowire conjugate, pointing towards emergence of the energy transfer. Overall, the results show that monitoring the optical properties of single emitters in a precisely designed hybrid nanostructure provides an elegant way to probe even complex combination of interactions at the nanoscale.

Fluorescence analysis of β-TCP ceramics is often used to describe cells found on said ceramics. However, we found, to our knowledge, so far undescribed artifacts which might sometimes be hard to differentiate from cells due to shape and fluorescence behavior. We tried prolonged ultrasound washing as well as Technovit 9100 fixation to reduce these artifacts. While untreated dowels showed no reduction in artifacts no matter the further treatment, Technovit fixation reduced the artifacts with even further reduction achieved by mechanical cleaning. As a consequence, scientists working with these dowels and likely even other types should try to avoid creating false positive results by considering the existence of these artifacts, checking additional filters for unusual fluorescence and by reducing them by using Technovit fixation when possible., (© 2024. The Author(s).)

The quantitative description of biological structures is a valuable yet difficult task in the life sciences. This is commonly accomplished by imaging samples using fluorescence microscopy and analyzing resulting images using Pearson's correlation or Manders' co-occurrence intensity-based colocalization paradigms. Though conceptually and computationally simple, these approaches are critically flawed due to their reliance on signal overlap, sensitivity to cursory signal qualities, and inability to differentiate true and incidental colocalization. Point pattern analysis provides a framework for quantitative characterization of spatial relationships between spatial patterns using the distances between observations rather than their overlap, thus overcoming these issues. Here we introduce an image analysis tool called Spatial Pattern Analysis using Closest Events (SPACE) that leverages nearest neighbor-based point pattern analysis to characterize the spatial relationship of fluorescence microscopy signals from image data. The utility of SPACE is demonstrated by assessing the spatial association between mRNA and cell nuclei from confocal images of cardiac myocytes. Additionally, we use synthetic and empirical images to characterize the sensitivity of SPACE to image segmentation parameters and cursory image qualities such as signal abundance and image resolution. Ultimately, SPACE delivers performance superior to traditional colocalization methods and offers a valuable addition to the microscopist's toolbox., Competing Interests: Conflict of Interest: The authors declare that they have no competing interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Microscopy Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Targeting the supportive tumor microenvironment (TME) is an approach of high interest in cancer drug development. However, assessing TME-targeted drug candidates presents a unique set of challenges. We develop a comprehensive screening platform that allows monitoring, quantifying, and ranking drug-induced effects in self-organizing, vascularized tumor spheroids (VTSs). The confrontation of four human-derived cell populations makes it possible to recreate and study complex changes in TME composition and cell-cell interaction. The platform is modular and adaptable for tumor entity or genetic manipulation. Treatment effects are recorded by light sheet fluorescence microscopy and translated by an advanced image analysis routine in processable multi-parametric datasets. The system proved to be robust, with strong interassay reliability. We demonstrate the platform's utility for evaluating TME-targeted antifibrotic and antiangiogenic drugs side-by-side. The platform's output enabled the differential evaluation of even closely related drug candidates according to projected therapeutic needs., (© 2024. The Author(s).)

Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.

Surpassing the diffraction barrier revolutionized modern fluorescence microscopy. However, intrinsic limitations in statistical sampling, the number of simultaneously analyzable channels, hardware requirements, and sample preparation procedures still represent an obstacle to its widespread diffusion in applicative biomedical research. Here, we present a novel pipeline based on automated multimodal microscopy and super-resolution techniques employing easily available materials and instruments and completed with open-source image-analysis software developed in our laboratory. The results show the potential impact of single-molecule localization microscopy (SMLM) on the study of biomolecules' interactions and the localization of macromolecular complexes. As a demonstrative application, we explored the basis of p53-53BP1 interactions, showing the formation of a putative macromolecular complex between the two proteins and the basal transcription machinery in situ, thus providing visual proof of the direct role of 53BP1 in sustaining p53 transactivation function. Moreover, high-content SMLM provided evidence of the presence of a 53BP1 complex on the cell cytoskeleton and in the mitochondrial space, thus suggesting the existence of novel alternative 53BP1 functions to support p53 activity.

Large-scale imaging of brain activity with high spatio-temporal resolution is crucial for advancing our understanding of brain function. The existing neuroimaging techniques are largely limited by restricted field of view, slow imaging speed, or otherwise do not have the adequate spatial resolution to capture brain activities on a capillary and cellular level. To address these limitations, we introduce fluorescence localization microscopy aided with sparsely-labeled red blood cells for cortex-wide morphological and functional cerebral angiography with 4.9 µm spatial resolution and 1 s temporal resolution. When combined with fluorescence calcium imaging, the proposed method enables extended recordings of stimulus-evoked neuro-vascular changes in the murine brain while providing simultaneous multiparametric readings of intracellular neuronal activity, blood flow velocity/direction/volume, and vessel diameter. Owing to its simplicity and versatility, the proposed approach will become an invaluable tool for deciphering the regulation of cortical microcirculation and neurovascular coupling in health and disease., (© 2024. The Author(s).)

Cell-to-cell communication via tunneling nanotubes (TNTs) is a challenging topic with a growing interest. In this work, we proposed several innovative tools that use red/near-infrared dye labeling and employ lifetime-based imaging strategies to investigate the dynamics of TNTs in a living mesothelial H28 cell line that exhibits spontaneously TNT1 and TNT2 subtypes. Thanks to a fluorescence lifetime imaging microscopy module being integrated into confocal microscopy and stimulated emission depletion nanoscopy, we applied lifetime imaging, lifetime dye unmixing, and lifetime denoising techniques to perform multiplexing experiments and time-lapses of tens of minutes, revealing therefore structural and functional characteristics of living TNTs that were preserved from light exposure. In these conditions, vesicle-like structures, and tubular- and round-shaped mitochondria were identified within living TNT1. In addition, mitochondrial dynamic studies revealed linear and stepwise mitochondrial migrations, bidirectional movements, transient backtracking, and fission events in TNT1. Transfer of Nile Red-positive puncta via both TNT1 and TNT2 was also detected between living H28 cells., (© 2024 Bénard et al.)

The small ultrared fluorescent protein (smURFP) is a bright near-infrared (NIR) fluorescent protein (FP) that forms a dimer and binds its fluorescence chromophore, biliverdin, at its dimer interface. To engineer a monomeric NIR FP based on smURFP potentially more suitable for bioimaging, we employed protein design to extend the protein backbone with a new segment of two helices that shield the original dimer interface while covering the biliverdin binding pocket in place of the second chain in the original dimer. We experimentally characterized 13 designs and obtained a monomeric protein with a weak fluorescence. We enhanced the fluorescence of this designed protein through two rounds of directed evolution and obtained designed monomeric smURFP (DMsmURFP), a bright, stable, and monomeric NIR FP with a molecular weight of 19.6 kDa. We determined the crystal structures of DMsmURFP both in the apo state and in complex with biliverdin, which confirmed the designed structure. The use of DMsmURFP in in vivo imaging of mammalian systems was demonstrated. The backbone design-based strategy used here can also be applied to monomerize other naturally multimeric proteins with intersubunit functional sites.

STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial for time-lapse imaging. Unlike fluorescent proteins, organic fluorophores cannot be genetically fused to a target protein but require different labeling strategies. To achieve simultaneous imaging of more than one protein in the interior of the cell with organic fluorophores, bioorthogonal labeling techniques and cell-permeable dyes are needed. In addition, the fluorophores should preferentially emit in the red spectral range to reduce the potential phototoxic effects that can be induced by the STED light, which further restricts the choice of suitable markers. In this work, we selected five different cell-permeable organic dyes that fulfill all of the above requirements and applied them for SPIEDAC click labeling inside living cells. By combining click-chemistry-based protein labeling with other orthogonal and highly specific labeling methods, we demonstrate two-color STED imaging of different target structures in living specimens using different dye pairs. The excellent photostability of the dyes enables STED imaging for up to 60 frames, allowing the observation of dynamic processes in living cells over extended time periods at super-resolution.

Mitochondria are essential organelles in cellular energy metabolism and other cellular functions. Mitochondrial dysfunction is closely linked to cellular damage and can potentially contribute to the aging process. The purpose of this study was to investigate the subcellular structure of mitochondria and their activities in various cellular environments using super-resolution stimulated emission depletion (STED) nanoscopy. We examined the morphological dispersion of mitochondria below the diffraction limit in sub-cultured human primary skin fibroblasts and mouse skin tissues. Confocal microscopy provides only the overall morphology of the mitochondrial membrane and an indiscerptible location of nucleoids within the diffraction limit. Conversely, super-resolution STED nanoscopy allowed us to resolve the nanoscale distribution of translocase clusters on the mitochondrial outer membrane and accurately quantify the number of nucleoids per cell in each sample. Comparable results were obtained by analyzing the translocase distribution in the mouse tissues. Furthermore, we precisely and quantitatively analyzed biomolecular distribution in nucleoids, such as the mitochondrial transcription factor A (TFAM), using STED nanoscopy. Our findings highlight the efficacy of super-resolution fluorescence imaging in quantifying aging-related changes on the mitochondrial sub-structure in cells and tissues., (© 2024. The Author(s).)

Carbon monoxide (CO) as an endogenous gas signaling molecule possesses important physiological functions and is of great significance in the treatment of various diseases. Real-time tracking of CO in living organisms has become a research hotspot in recent years. This article presents a lysosomal targeted near-infrared ratio fluorescence probe (TBM-CO) for selective detection of CO based on the dicyanoisophorone skeleton and morpholine fragment. The probe TBM-CO with weak ICT effect can be transformed to precursor TBM-NH 2 with strong ICT effect by the traditional Tsuji-Trost reaction procession in the presence of Pd 2+ ions. The mechanism was proved by DFT calculation or the MS and HPLC results respectively. In the near-infrared region an obvious ratio fluorescence intensity change (F 686 / F 616 ) is observed in vitro spectral experiments. The concentration titration experiments indicate that there is a good liner relationship between the ratio fluorescence intensity and the concentration in the range of 0 to 50 μM (R 2  = 0.996) and the detection limit is calculated as 0.38 μM. The cell fluorescence imaging and co-localization experiments further demonstrate that TBM-CO is able to detect the exogenous and endogenous CO in lysosomal subcellular organelle. Finally, it was used to detect the changes of CO concentration in living mice successfully. In short, a probe with three advantages of near-infrared emission, ratiometric fluorescence and organelle targeting was reported and used to detect CO successfully in cells and in living mice., 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 B.V. All rights reserved.)

Abnormal physiological processes and diseases can lead to content or activity fluctuations of biocomponents in organelles and whole blood. However, precise monitoring of these abnormalities remains extremely challenging due to the insufficient sensitivity and accuracy of available fluorescence probes, which can be attributed to the background fluorescence arising from two sources, 1) biocomponent autofluorescence (BCAF) and 2) probe intrinsic fluorescence (PIF). To overcome these obstacles, we have re-engineered far-red to NIR II rhodol derivatives that possess weak BCAF interference. And a series of "zero" PIF sensing-platforms were created by systematically regulating the open-loop/spirocyclic forms. Leveraging these advancements, we devised various ultra-sensitive NIR indicators, achieving substantial fluorescence boosts (190 to 1300-fold). Among these indicators, 8-LAP demonstrated accurate tracking and quantifying of leucine aminopeptidase (LAP) in whole blood at various stages of tumor metastasis. Furthermore, coupling 8-LAP with an endoplasmic reticulum-targeting element enabled the detection of ERAP1 activity in HCT116 cells with p53 abnormalities. This delicate design of eliminating PIF provides insights into enhancing the sensitivity and accuracy of existing fluorescence probes toward the detection and imaging of biocomponents in abnormal physiological processes and diseases., (© 2024 Wiley‐VCH GmbH.)

Advances in fluorescence microscopy and tissue-clearing have revolutionised 3D imaging of fluorescently labelled tissues, organs and embryos. However, the complexity and high cost of existing software and computing solutions limit their widespread adoption, especially by researchers with limited resources. Here, we present Acto3D, an open-source software, designed to streamline the generation and analysis of high-resolution 3D images of targets labelled with multiple fluorescent probes. Acto3D provides an intuitive interface for easy 3D data import and visualisation. Although Acto3D offers straightforward 3D viewing, it performs all computations explicitly, giving users detailed control over the displayed images. Leveraging an integrated graphics processing unit, Acto3D deploys all pixel data to system memory, reducing visualisation latency. This approach facilitates accurate image reconstruction and efficient data processing in 3D, eliminating the need for expensive high-performance computers and dedicated graphics processing units. We have also introduced a method for efficiently extracting lumen structures in 3D. We have validated Acto3D by imaging mouse embryonic structures and by performing 3D reconstruction of pharyngeal arch arteries while preserving fluorescence information. Acto3D is a cost-effective and efficient platform for biological research., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The development of near-infrared (NIR) fluorophores that have both excellent chemical stability and photostability, as well as efficient cell permeability, is highly demanded. In this study, we present phospha-rhodamine (POR) dyes which display significantly improved performance for protein labeling. This is achieved by incorporating a 2-carboxy-3-benzothiophenyl group at the 9-position of the xanthene scaffold. The resulting cis and trans isomers were successfully isolated and structurally characterized using X-ray diffraction. The HaloTag ligand conjugates of the two isomers exhibited different staining abilities in live cells. While the cis isomer showed non-specific accumulation on the organelle membranes, the trans isomer selectively labeled the HaloTag-fused proteins, enabling the long-term imaging of cell division and the 5-color imaging of cell organelles. Molecular dynamics simulations of the HaloTag ligand conjugates within the lipid membrane suggested that the cis isomer is more prone to forming oligomers in the membrane. In contrast, the oligomerization of the trans isomer is effectively suppressed by its interaction with the lipid molecules. By taking advantage of the superior labeling performance of the trans isomer and its NIR-emissive properties, multi-color time-lapse super-resolution 3D imaging, namely super-resolution 5D-imaging, of the interconnected network between the endoplasmic reticulum and microtubules was achieved in living cells., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Mitochondria are important organelles that provide energy for cellular physiological activities. Changes in their structures may indicate the occurrence of diseases, and the super-resolution imaging of mitochondria is of great significance. However, developing fluorescent probes for mitochondrial super-resolution visualization still remains challenging due to insufficient fluorescence brightness and poor stability. Herein, we rationally synthesized an ultrabright xanthene fluorescence probe Me-hNR for mitochondria-specific super-resolution imaging using structured illumination microscopy (SIM). The rigid structure of Me-hNR provided its ultrahigh fluorescence quantum yield of up to 0.92 and ultrahigh brightness of up to 16,000. Occupying the para -position of the O atom in the xanthene skeleton by utilizing the smallest methyl group ensured its excellent stability. The study of the photophysical process indicated that Me-hNR mainly emitted fluorescence via radiative decay, and nonradiative decay and inter-system crossing were rare due to the slow nonradiative decay rate and large energy gap (Δ E s t = 0.55 eV). Owing to these excellent merits, Me-hNR can specifically light up mitochondria at ultralow concentrations down to 5 nM. The unprecedented spatial resolution for mitochondria with an fwhm of 174 nm was also achieved. Therefore, this ultrabright xanthene fluorescence probe has great potential in visualizing the structural changes of mitochondria and revealing the pathogenesis of related diseases using SIM.

Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes. Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes but also measure values of membrane potentials. This study discloses a fluorescent indicator which can address both. We describe the synthesis of a sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence. Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials (APs) with greater signal-to-noise than state-of-the-art BeRST 1 (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of APs in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages., Competing Interests: Competing interests statement:The authors declare no competing interest.

Significance: Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful and versatile imaging technique renowned for its remarkable features, including high-speed 3D tomography, minimal photobleaching, and low phototoxicity. The interference light-sheet fluorescence microscope, with its larger field of view (FOV) and more uniform axial resolution, possesses significant potential for a wide range of applications in biology and medicine., Aim: The aim of this study is to investigate the interference behavior among multiple light sheets (LSs) in LSFM and optimize the FOV and resolution of the light-sheet fluorescence microscope., Approach: We conducted a detailed investigation of the interference effects among LSs through theoretical derivation and numerical simulations, aiming to find optimal parameters. Subsequently, we constructed a customized system of multi-LSFM that incorporates both interference light sheets (ILS) and noninterference light-sheet configurations. We performed beam imaging and microsphere imaging tests to evaluate the FOV and axial resolution of these systems., Results: Using our custom-designed light-sheet fluorescence microscope, we captured the intensity distribution profiles of both interference and noninterference light sheets (NILS). Additionally, we conducted imaging tests on microspheres to assess their imaging outcomes. The ILS not only exhibits a larger FOV compared to the NILS but also demonstrates a more uniform axial resolution., Conclusions: By effectively modulating the interference among multiple LSs, it is possible to optimize the intensity distribution of the LSs, expand the FOV, and achieve a more uniform axial resolution., (© 2024 The Authors.)

Fluorescent dyes synergize with advanced microscopy for researchers to investigate the location and dynamic processes of biomacromolecules with high spatial and temporal resolution. However, the instability of fluorescent dyes, including photobleaching and photoconversion, represent fundamental limits for super-resolution and time-lapse imaging. In this review, we discuss the latest advances in improving the photostability of fluorescent dyes. We summarize the primary photobleaching processes of cyanine and rhodamine dyes and highlight a range of strategies developed in recent years to strengthen these fluorophores. Additionally, we discuss the influence of protein microenvironments and labeling methods on the photostability of fluorophores. We aim to inspire next-generation robust and bright fluorophores that ultimately enable the routine practice of time-lapse super-resolution imaging of live cells., Competing Interests: Declaration of competing interest Z.C. is an inventor of a patent on mitochondria dyes described in this review (CN 202010492298.8). Z.C. and Y.Z. are the inventors of a patent on the fluorophores with S-arylation-type conjugation chemistry in this review (CN202210228995.1, patent pending)., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The pathogenesis of cancer and cardiovascular diseases is subjected to spatiotemporal regulation by the tissue microenvironment. Multiplex visualization of the microenvironmental components, including immune cells, vasculature and tissue hypoxia, provides critical information underlying the disease progression and therapy resistance, which is often limited by imaging depth and resolution in large-volume tissues. To this end, light sheet fluorescence microscopy, following tissue clarification and immunostaining, may generate three-dimensional high-resolution images at a whole-organ level. Here we provide a detailed description of light sheet fluorescence microscopy imaging analysis of immune cell composition, vascularization, tissue perfusion and hypoxia in mouse normal brains and hearts, as well as brain tumors. We describe a procedure for visualizing tissue vascularization, perfusion and hypoxia with a transgenic vascular labeling system. We provide the procedures for tissue collection, tissue semi-clearing and immunostaining. We further describe standard methods for analyzing tissue immunity and vascularity. We anticipate that this method will facilitate the spatial illustration of structure and function of the tissue microenvironmental components in cancer and cardiovascular diseases. The procedure requires 1-2 weeks and can be performed by users with expertise in general molecular biology., (© 2024. Springer Nature Limited.)

Transcription factors (TFs) regulate gene expression by recognizing specific target enhancers in the genome. The DNA-binding and regulatory activity of TFs depend on the presence of additional protein partners, leading to the formation of versatile and dynamic multimeric protein complexes. Visualizing these protein-protein interactions (PPIs) in the nucleus is key for decrypting the molecular cues underlying TF specificity in vivo. Over the last few years, Bimolecular Fluorescence Complementation (BiFC) has been developed in several model systems and applied in the analysis of different types of PPIs. In particular, BiFC has been applied when analyzing PPIs with hundreds of TFs in the nucleus of live Drosophila embryos. However, the visualization of PPIs at the level of specific target enhancers or genomic regions of interest awaits the advent of DNA-labelling methods that can be coupled with BiFC. Here, we present a novel experimental strategy that we have called BiFOR and that is based on the coupling of BiFC with the bacterial ANCHOR DNA-labelling system. We demonstrate that BiFOR enables the precise quantification of the enrichment of specific dimeric protein complexes on target enhancers in Drosophila salivary gland nuclei. Given its versatility and sensitivity, BiFOR could be applied more widely to other tissues during Drosophila development. Our work sets up the experimental basis for future applications of this strategy.

The utility of fluorescence lifetime imaging microscopy (FLIM) for identifying bacteria in complex mineral matrices was investigated. Baseline signals from unlabelled Bacillus subtilis and Euglena gracilis, and Bacillus subtilis labelled with SYTO 9 were obtained using two-photon excitation at 730, 750 and 800 nm, identifying characteristic lifetimes of photosynthetic pigments, unpigmented cellular autofluorescence, and SYTO 9. Labelled and unlabelled B. subtilis were seeded onto marble and gypsum samples containing endolithic photosynthetic cyanobacteria and the ability to distinguish cells from mineral autofluorescence and nonspecific dye staining was examined in parallel with ordinary multichannel confocal imaging. It was found that FLIM enabled discrimination of SYTO 9 labelled cells from background, but that the lifetime of SYTO 9 was shorter in cells on minerals than in pure culture under our conditions. Photosynthetic microorganisms were easily observed using both FLIM and confocal. Unlabelled, nonpigmented bacteria showed weak signals that were difficult to distinguish from background when minerals were present, though cellular autofluorescence consistent with NAD(P)H could be seen in pure cultures, and phasor analysis permitted detection on rocks. Gypsum and marble samples showed similar autofluorescence profiles, with little autofluorescence in the yellow-to-red range. Lifetime or time-gated imaging may prove a useful tool for environmental microbiology. LAY DESCRIPTION: The standard method of bacterial enumeration is to label the cells with a fluorescent dye and count them under high-power fluorescence microscopy. However, this can be difficult when the cells are embedded in soil and rock due to fluorescence from the surrounding minerals and dye binding to ambiguous features of the substrate. The use of fluorescence lifetime imaging (FLIM) can disambiguate these signals and allow for improved detection of bacteria in environmental samples., (© 2024 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

Expansion microscopy (ExM) is a revolutionary novel approach to increase resolution in light microscopy. In contrast to super-resolution microscopy methods that rely on sophisticated technological advances, including novel instrumentation, ExM instead is entirely based on sample preparation. In ExM, labeled target molecules in fixed cells are anchored in a hydrogel, which is then physically enlarged by osmotic swelling. The isotropic swelling of the hydrogel pulls the labels apart from one another, and their relative organization can thus be resolved using conventional microscopes even if it was below the diffraction limit of light beforehand. As ExM can additionally benefit from the technical resolution enhancements achieved by super-resolution microscopy, it can reach into the nanometer range of resolution with an astoundingly low degree of error induced by distortion during the physical expansion process. Because the underlying chemistry is well understood and the technique is based on a relatively simple procedure, ExM is easily reproducible in non-expert laboratories and has quickly been adopted to address an ever-expanding spectrum of problems across the life sciences. In this Review, we provide an overview of this rapidly expanding new field, summarize the most important insights gained so far and attempt to offer an outlook on future developments., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Super-resolution fluorescence microscopy technologies developed over the past two decades have pushed the resolution limit for fluorescently labeled molecules into the nanometer range. These technologies have the potential to study bacterial structures, for example, macromolecular assemblies such as secretion systems, with single-molecule resolution on a millisecond time scale. Here we review recent applications of super-resolution fluorescence microscopy with a focus on bacterial secretion systems. We also describe MINFLUX fluorescence nanoscopy, a relatively new technique that promises to one day produce molecular movies of molecular machines in action., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

During starvation in the yeast Saccharomyces cerevisiae vacuolar vesicles fuse and lipid droplets (LDs) can become internalized into the vacuole in an autophagic process named lipophagy. There is a lack of tools to quantitatively assess starvation-induced vacuole fusion and lipophagy in intact cells with high resolution and throughput. Here, we combine soft X-ray tomography (SXT) with fluorescence microscopy and use a deep-learning computational approach to visualize and quantify these processes in yeast. We focus on yeast homologs of mammalian NPC1 (NPC intracellular cholesterol transporter 1; Ncr1 in yeast) and NPC2 proteins, whose dysfunction leads to Niemann Pick type C (NPC) disease in humans. We developed a convolutional neural network (CNN) model which classifies fully fused versus partially fused vacuoles based on fluorescence images of stained cells. This CNN, named Deep Yeast Fusion Network (DYFNet), revealed that cells lacking Ncr1 ( ncr1∆ cells) or Npc2 ( npc2∆ cells) have a reduced capacity for vacuole fusion. Using a second CNN model, we implemented a pipeline named LipoSeg to perform automated instance segmentation of LDs and vacuoles from high-resolution reconstructions of X-ray tomograms. From that, we obtained 3D renderings of LDs inside and outside of the vacuole in a fully automated manner and additionally measured droplet volume, number, and distribution. We find that ncr1∆ and npc2∆ cells could ingest LDs into vacuoles normally but showed compromised degradation of LDs and accumulation of lipid vesicles inside vacuoles. Our new method is versatile and allows for analysis of vacuole fusion, droplet size and lipophagy in intact cells. Abbreviations: BODIPY493/503: 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza- s -Indacene; BPS: bathophenanthrolinedisulfonic acid disodium salt hydrate; CNN: convolutional neural network; DHE; dehydroergosterol; npc2∆ , yeast deficient in Npc2; DSC, Dice similarity coefficient; EM, electron microscopy; EVs, extracellular vesicles; FIB-SEM, focused ion beam milling-scanning electron microscopy; FM 4-64, N -(3-triethylammoniumpropyl)-4-(6-[4-{diethylamino} phenyl] hexatrienyl)-pyridinium dibromide; LDs, lipid droplets; Ncr1, yeast homolog of human NPC1 protein; ncr1∆ , yeast deficient in Ncr1; NPC, Niemann Pick type C; NPC2, Niemann Pick type C homolog; OD 600 , optical density at 600 nm; ReLU, rectifier linear unit; PPV, positive predictive value; NPV, negative predictive value; MCC, Matthews correlation coefficient; SXT, soft X-ray tomography; UV, ultraviolet; YPD, yeast extract peptone dextrose.

Projection imaging accelerates volumetric interrogation in fluorescence microscopy, but for multi-cellular samples, the resulting images may lack contrast, as many structures and haze are summed up. Here, we demonstrate rapid projective light-sheet imaging with parameter selection (props) of imaging depth, position and viewing angle. This allows us to selectively image different sub-volumes of a sample, rapidly switch between them and exclude background fluorescence. Here we demonstrate the power of props by functional imaging within distinct regions of the zebrafish brain, monitoring calcium firing inside muscle cells of moving Drosophila larvae, super-resolution imaging of selected cell layers, and by optically unwrapping the curved surface of a Drosophila embryo. We anticipate that props will accelerate volumetric interrogation, ranging from subcellular to mesoscopic scales., (© 2024. The Author(s).)

Mapping the intricate spatial relationships between the many different molecules inside a cell is essential to understanding cellular functions in all their complexity. Super-resolution fluorescence microscopy offers the required spatial resolution but struggles to reveal more than four different targets simultaneously. Exchanging labels in subsequent imaging rounds for multiplexed imaging extends this number but is limited by its low throughput. Here, we present a method for rapid multiplexed super-resolution microscopy that can, in principle, be applied to a nearly unlimited number of molecular targets by leveraging fluorogenic labeling in conjunction with transient adapter-mediated switching for high-throughput DNA-PAINT (FLASH-PAINT). We demonstrate the versatility of FLASH-PAINT with four applications: mapping nine proteins in a single mammalian cell, elucidating the functional organization of primary cilia by nine-target imaging, revealing the changes in proximity of thirteen different targets in unperturbed and dissociated Golgi stacks, and investigating and quantifying inter-organelle contacts at 3D super-resolution., Competing Interests: Declaration of interests F.S. and J.B. filed patent applications with the U.S. patent office covering the conceptional ideas of this study. J.B. has licensed IP to Bruker Corp. and Hamamatsu Photonics. J.B. is a consultant for Bruker Corp. J.B. is a founder of panluminate, Inc., (Copyright © 2024 Elsevier Inc. All rights reserved.)

To understand biological processes, it is necessary to reveal the molecular heterogeneity of cells by gaining access to the location and interaction of all biomolecules. Significant advances were achieved by super-resolution microscopy, but such methods are still far from reaching the multiplexing capacity of proteomics. Here, we introduce secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a high-throughput imaging method that is capable of achieving virtually unlimited multiplexing at better than 15 nm resolution. Using SUM-PAINT, we generated 30-plex single-molecule resolved datasets in neurons and adapted omics-inspired analysis for data exploration. This allowed us to reveal the complexity of synaptic heterogeneity, leading to the discovery of a distinct synapse type. We not only provide a resource for researchers, but also an integrated acquisition and analysis workflow for comprehensive spatial proteomics at single-protein resolution., Competing Interests: Declaration of interests E.M.U., M.G. and R.J. have filed a patent on the method published in this paper. F.O. is a shareholder of NanoTag Biotechnologies GmbH. The other authors declare no competing financial interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

A wide-field microscope with epi-fluorescence and selective plane illumination was combined with a single-photon avalanche diode (SPAD) array camera to enable live-cell fluorescence lifetime imaging (FLIM) using time-correlated single-photon counting (TCSPC). The camera sensor comprised of 192 × 128 pixels, each integrating a single SPAD and a time-to-digital converter. Jointly, they produced a stream of single-photon images of photon arrival times with ≈ 38 ps accuracy. The photon arrival times were subject to systematic delays and nonlinearities, which were corrected by a Monte-Carlo algorithm. The SPAD camera was then applied to FLIM where histogramming the resulting photon arrival times in each pixel resulted in decays compatible with common data processing pipelines for fluorescence lifetime analysis. The capabilities of the TCSPC camera-based FLIM microscope were demonstrated by imaging living unicellular photosynthetic algae and artificial lipid vesicles. Epi-fluorescence illumination enabled rapid fluorescence lifetime imaging of living cells and selective-plane illumination enabled 3-dimensional FLIM of stationary samples., (© 2024. The Author(s).)

Computational analysis of fluorescent timelapse microscopy images at the single-cell level is a powerful approach to study cellular changes that dictate important cell fate decisions. Core to this approach is the need to generate reliable cell segmentations and classifications necessary for accurate quantitative analysis. Deep learning-based convolutional neural networks (CNNs) have emerged as a promising solution to these challenges. However, current CNNs are prone to produce noisy cell segmentations and classifications, which is a significant barrier to constructing accurate single-cell lineages. To address this, we developed a novel algorithm called Single Cell Track (SC-Track), which employs a hierarchical probabilistic cache cascade model based on biological observations of cell division and movement dynamics. Our results show that SC-Track performs better than a panel of publicly available cell trackers on a diverse set of cell segmentation types. This cell-tracking performance was achieved without any parameter adjustments, making SC-Track an excellent generalized algorithm that can maintain robust cell-tracking performance in varying cell segmentation qualities, cell morphological appearances and imaging conditions. Furthermore, SC-Track is equipped with a cell class correction function to improve the accuracy of cell classifications in multiclass cell segmentation time series. These features together make SC-Track a robust cell-tracking algorithm that works well with noisy cell instance segmentation and classification predictions from CNNs to generate accurate single-cell lineages and classifications., (© The Author(s) 2024. Published by Oxford University Press.)

For generations researchers have been observing the dynamic processes of life through the lens of a microscope. This has offered tremendous insights into biological phenomena that span multiple orders of time- and length-scales ranging from the pure magic of molecular reorganization at the membrane of immune cells, to cell migration and differentiation during development or wound healing. Standard fluorescence microscopy techniques offer glimpses at such processes in vitro, however, when applied in intact systems, they are challenged by reduced signal strengths and signal-to-noise ratios that result from deeper imaging. As a remedy, two-photon excitation (TPE) microscopy takes a special place, because it allows us to investigate processes in vivo, in their natural environment, even in a living animal. Here, we review the fundamental principles underlying TPE aimed at basic and advanced microscopy users interested in adopting TPE for intravital imaging. We focus on applications in neurobiology, present current trends towards faster, wider and deeper imaging, discuss the combination with photon counting technologies for metabolic imaging and spectroscopy, as well as highlight outstanding issues and drawbacks in development and application of these methodologies., (© 2024. The Author(s).)

In this work, we examine the use of environment-sensitive fluorescent dyes in fluorescence lifetime imaging microscopy (FLIM) biosensors. We screened merocyanine dyes to find an optimal combination of environment-induced lifetime changes, photostability, and brightness at wavelengths suitable for live-cell imaging. FLIM was used to monitor a biosensor reporting conformational changes of endogenous Cdc42 in living cells. The ability to quantify activity using phasor analysis of a single fluorophore (e.g., rather than ratio imaging) eliminated potential artifacts. We leveraged these properties to determine specific concentrations of activated Cdc42 across the cell., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Hypoxia, a significant feature of the tumor microenvironment, is closely associated with tumor growth, metastasis, and drug resistance. In the field of tumor microenvironment analysis, accurately imaging and quantifying hypoxia - a critical factor associated with tumor progression, metastasis, and resistance to therapy - remains a significant challenge. Herein, a hypoxia-activated red-emission fluorescent probe, ODP, for in vivo imaging of hypoxia in the tumor microenvironment is presented. Among various imaging methods, optical imaging is particularly convenient due to its rapid response and high sensitivity. The ODP probe specifically targets nitroreductase (AzoR), an enzyme highly expressed in hypoxic cells, playing a vital role by catalyzing the cleavage of azo bonds. The optical properties of ODP exhibited excellent performance in terms of fluorescence enhancement, fluorescence lifetime (0.81 ns), and detection limit (0.86 µM) in response to SDT. Cell imaging experiments showed that ODP could effectively detect and image intracellular hypoxia and the imaging capability of ODP was studied under various conditions including cell migration, antioxidant treatment, and different incubation times. Through comprehensive in vitro and in vivo experiments, including cellular imaging and mouse tumor models, this work demonstrates the efficacy of ODP in accurately detecting and imaging hypoxia. Moreover, ODP's potential in inducing apoptosis in cancer cells offers a promising avenue for integrating diagnostic and therapeutic strategies in cancer treatment. This innovative approach not only contributes to the understanding and assessment of tumor hypoxia but also opens new possibilities for targeted cancer therapy., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

In the emerging field of whole-brain imaging at single-cell resolution, which represents one of the new frontiers to investigate the link between brain activity and behavior, the nematode Caenorhabditis elegans offers one of the most characterized models for systems neuroscience. Whole-brain recordings consist of 3D time series of volumes that need to be processed to obtain neuronal traces. Current solutions for this task are either computationally demanding or limited to specific acquisition setups. Here, we propose See Elegans, a direct programming algorithm that combines different techniques for automatic neuron segmentation and tracking without the need for the RFP channel, and we compare it with other available algorithms. While outperforming them in most cases, our solution offers a novel method to guide the identification of a subset of head neurons based on position and activity. The built-in interface allows the user to follow and manually curate each of the processing steps. See Elegans is thus a simple-to-use interface aimed at speeding up the post-processing of volumetric calcium imaging recordings while maintaining a high level of accuracy and low computational demands. (Contact: enrico.lanza@iit.it)., Competing Interests: Viola Folli is an employee and scientific advisor of D-tails s.r.l. Valeria Lucente and Ilaria Cavallo are employees of D-tails s.r.l." However, this does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Lanza et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The varied functions of lipid droplets, which encompass the regulation of lipid and energy homeostasis, as well as their association with the occurrence of various metabolic diseases, are intricately linked to their dynamic properties. Super-resolution imaging techniques have emerged to decipher physiological processes and molecular mechanisms on the nanoscale. However, achieving long-term dynamic super-resolution imaging faces challenges due to the need for fluorescent probes with high photostability. This paper introduces LD-CF , a "buffering probe" for imaging lipid droplet dynamics using structured illumination microscopy (SIM). The polarity-sensitive LD-CF eliminates background fluorescence with a "cyan filter" strategy, enabling wash-free imaging of lipid droplets. In the fluorescent "off" state outside droplets, the probes act as a "buffering pool", replacing photobleached probes inside droplets and enabling photostable long-term SIM imaging. With this probe, three modes of lipid droplet fusion were observed, including the discovery of fusion from large to small lipid droplets. Fluorescence intensity tracking also revealed the direction of lipid transport during the lipid droplet fusion.

Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes., (© 2024. The Author(s).)

Ferroptosis, as a new form of regulated cell death, is implicated in various physiological and pathological processes. Developing a single probe for an independent analysis of multiple analytes related to ferroptosis can provide more accurate information and simplify the detection procedures, but it faces great challenges. In this work, we develop a fluorescent probe for the simultaneous detection of GSH through ratiometric fluorescence response and microviscosity via a fluorescence lifetime model. Based on the reversible Michael addition reaction between GSH and unsaturated C═C bond, the probe responds reversibly to GSH with a ratiometric fluorescence variation and a fast response time ( t 1/2 = 4.7 s). At the same time, the probe is sensitive to environmental viscosity by changing its fluorescence lifetimes. The probe was applied to monitor the drug-induced ferroptosis process through both the classical X c - /GSH/GPX4- and DHODH-mediated defense mechanisms. We hope that the probe will provide a useful molecular tool for the real-time live-cell imaging of GSH dynamics, which is benefit to unveiling related physiological and pathological processes.

Here, we present a protocol for estimating nuclear transport parameters in single cells. We describe steps for performing four consecutive fluorescence recovery after photobleaching experiments, fitting the obtained data to an ordinary differential equations model, and statistical analysis of the fittings using a specialized R package. This protocol permits the estimation of import and export rates, nuclear or cytosolic fixed fractions, and total number of molecules. For complete details on the use and execution of this protocol, please refer to Durrieu et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Insulin secretion from pancreatic β-cells is integral in maintaining the delicate equilibrium of blood glucose levels. Calcium is known to be a key regulator and triggers the release of insulin. This sub-cellular process can be monitored and tracked through live-cell imaging and subsequent cell segmentation, registration, tracking, and analysis of the calcium level in each cell. Current methods of analysis typically require the manual outlining of β-cells, involve multiple software packages, and necessitate multiple researchers-all of which tend to introduce biases. Utilizing deep learning algorithms, we have therefore created a pipeline to automatically segment and track thousands of cells, which greatly reduces the time required to gather and analyze a large number of sub-cellular images and improve accuracy. Tracking cells over a time-series image stack also allows researchers to isolate specific calcium spiking patterns and spatially identify those of interest, creating an efficient and user-friendly analysis tool. Using our automated pipeline, a previous dataset used to evaluate changes in calcium spiking activity in β-cells post-electric field stimulation was reanalyzed. Changes in spiking activity were found to be underestimated previously with manual segmentation. Moreover, the machine learning pipeline provides a powerful and rapid computational approach to examine, for example, how calcium signaling is regulated by intracellular interactions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Alsup et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rheumatoid arthritis (RA) is a destructive autoimmune disease, where nitric oxide (NO) is closely implicated in the inflammatory processes of RA. Therefore, direct visualization of NO is essential to assess the pathological changes in RA. Herein, a mitochondrial-targeted near-infrared ratiometric fluorescent probe (NFL-NH 2 ), based on the intramolecular charge transfer effect, was synthesized and applied to monitor the changes of NO content in early RA. Specially, probe NFL-NH 2 showed a 44-fold fluorescent intensity ratio ( I 705 / I 780 ) response toward NO with a detection limit of 0.536 nM, enabling qualitative and quantitative analysis of NO. Additionally, NFL-NH 2 can accurately target mitochondria and sensitively detect exogenous and endogenous NO in RAW 264.7 cells. Notably, in vivo RA monitoring assays demonstrated that NFL-NH 2 can rapidly detect NO levels associated with the inflammatory damage degree in RA mice models by ratiometric fluorescence imaging. These results validate that NFL-NH 2 holds significant potential for diagnosing NO-mediated RA diseases.

Superior photostability, minimal phototoxicity, red-shifted absorption/emission wavelengths, high brightness, and an enlarged Stokes shift are essential characteristics of top-tier organic fluorophores, particularly for long-lasting super-resolution imaging in live cells (e.g., via stimulated emission depletion (STED) nanoscopy). However, few existing fluorophores possess all of these properties. In this study, we demonstrate a general approach for simultaneously enhancing these parameters through the introduction of 9,9-dimethyl-9,10-dihydroacridine (DMA) as an electron-donating auxochrome. DMA not only induces red shifts in emission wavelengths but also suppresses photooxidative reactions and prevents the formation of triplet states in DMA-based fluorophores, greatly improving photostability and remarkably minimizing phototoxicity. Moreover, the DMA group enhances the fluorophores' brightness and enlarges the Stokes shift. Importantly, the "universal" benefits of attaching the DMA auxochrome have been exemplified in various fluorophores including rhodamines, difluoride-boron complexes, and coumarin derivatives. The resulting fluorophores successfully enabled the STED imaging of organelles and HaloTag-labeled membrane proteins.

Long-term continuous imaging of endogenous HClO burst is of great importance for the elucidation of various physiological or pathological processes. However, most of the currently reported HClO probes have failed to achieve this goal due to their insufficient photobleaching resistance under a laser source. Herein, a highly stable ratiometric probe, HFTC-HClO 1, which is capable of continuously monitoring endogenous HClO burst over a long period of time, has been judiciously developed. Briefly, the de novo development of HFTC-HClO 1 mainly involved three main steps: (1) novel coumarins (HFTC 1-5) were designed and synthesized; (2) the most stable scaffold, HFTC 3, was selected through dye screening and cell imaging validation; and (3) based on HFTC 3, three candidate HClO probes were constructed, and HFTC-HClO 1 was finally selected due to its superior sensing properties toward HClO. Furthermore, HFTC-HClO 1 can quantitatively measure HClO levels in various real samples with excellent recovery (>90.4%), and the use of HFTC-HClO 1-coated test strips for qualitative analysis of HClO in real samples was also achieved. In addition, the application of HFTC-HClO 1 for long-term continuous monitoring of intracellular HClO burst was successfully demonstrated. Significantly, HFTC-HClO 1 was able to visualize HClO generated in the rheumatoid arthritis mouse model.

Fluorogenic dyes with high brightness, large turn-on ratios, excellent photostability, favorable specificity, low cytotoxicity, and high membrane permeability are essential for high-resolution fluorescence imaging in live cells. In this study, we endowed these desirable properties to a rhodamine derivative by simply replacing the N, N-diethyl group with a pyrrole substituent. The resulting dye, Rh-NH, exhibited doubled Stokes shifts (54 nm) and a red-shift of more than 50 nm in fluorescence spectra compared to Rhodamine B. Rh-NH preferentially exists in a non-emissive but highly permeable spirolactone form. Upon binding to lysosomes, the collective effects of low pH, low polarity, and high viscosity endow Rh-NH with significant fluorescence turn-on, making it a suitable candidate for wash-free, high-contrast lysosome tracking. Consequently, Rh-NH enabled us to successfully explore stimulated emission depletion (STED) super-resolution imaging of lysosome dynamics, as well as fluorescence lifetime imaging of lysosomes in live cells., (© 2024 Wiley-VCH GmbH.)

Understanding the influence of oxygen tension on cellular functions and behaviors is crucial for investigating various physiological and pathological conditions. In vitro cell culture models, particularly those based on hydrogel extracellular matrices, have been developed to study cellular responses in specific oxygen microenvironments. However, accurately characterizing oxygen tension variations with great spatiotemporal resolutions, especially in three dimensions, remains challenging. This paper presents an approach for rapid time-lapse 3D oxygen tension measurements in hydrogels using a widely available inverted fluorescence microscope. Oxygen-sensitive fluorescent microbeads and widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) are utilized for oxygen tension estimation. To incorporate the third dimension, a motorized sample stage is implanted that enables automated image acquisition in the vertical direction. A machine learning algorithm based on K-means clustering is employed for microbead position identification. Using an upside-down microfluidic device, 3D oxygen gradients are generated within a hydrogel sample, and z -stack images are acquired using the FD-FLIM system. Analyses of the acquired images, involving microbead position identification, lifetime calculation, and oxygen tension conversion, are then performed offline. The results demonstrate the functionality of the developed approach for rapid time-lapse 3D oxygen tension measurements in hydrogels. Furthermore, the 3D oxygen tension adjacent to a tumor spheroid within a hydrogel during media exchange is characterized. The results further confirm that the 3D spatiotemporal oxygen tension profiles can be successfully measured quantitatively using the established setup and analysis process and that the approach may have great potential for investigating cellular activities within oxygen microenvironments.

Correlative microscopy is a sophisticated imaging technique that combines optical and electron microscopes, with the most common approach being the integration of light microscopy and electron microscopy, known as correlative light and electron microscopy (CLEM). While CLEM provides a comprehensive view of biological samples, it presents a significant challenge in sample preparation due to the distinct processes involved in each technique. Striking a balance between these methods is crucial. Despite numerous approaches, achieving seamless imaging with CLEM remains a complex task. Exosomes, nanovesicles ranging from 30 to 150 nm in size, are enclosed by a lipid bilayer and released by various cell types. Visualizing exosomes poses difficulties due to their small size and minimal electric charge. However, imaging exosomes at high resolution offers a direct method to understand their morphology and functions. In this study, we evaluated exosome imaging with CLEM using a combination of confocal, transmission electron microscope, and scanning electron microscope (SEM). In addition, we conducted a comparative analysis of these two techniques, evaluating their suitability and efficiency in imaging nanoscale structures. In this study, we found that confocal-SEM correlation is more applicable for imaging exosomes. Moreover, we observed that exosomes were found in clusters in confocal-SEM correlation., Competing Interests: Competing InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ageing of paper is a complex process of great relevance for application purposes because of its widespread use as support for information storage in books and documents, and as common low-cost and green packaging material, to name a few. A key factor in paper ageing is the oxidation of cellulose, a macromolecule of natural origin that constitutes the main chemical component of paper. Such a complex process results in changes in the cellulose polymeric chains in chemical and structural properties. The scope of this work is to explore the effects of oxidation of cellulose as one of the principal mechanisms of ageing of paper using a fluorescence-based approach. To this aim, fluorescence-lifetime imaging microscopy (FLIM) measurements on pure cellulose samples stained using Carbotrace 680 dye were performed, and data were analyzed by phasor approach. The comparison with results from conventional techniques allowed to map paper microstructure as a function of the sample oxidation degree correlating the fluorescence-lifetime changes to cellulose oxidation. A two-step oxidation kinetics that produced specific modification in paper organization was highlighted indicating that FLIM measurements using Carbotrace 680 dye may provide a simple tool to obtain information on the oxidation process also adding spatial information at sub-micrometric scale., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

We report a simple, inexpensive design of a fluorescence microscope with light-emitting diode (LED) excitation for detection of labeled and unlabeled microorganisms in mineral substrates. The use of deep UV (DUV) excitation with visible emission requires no specialized optics or slides and can be implemented easily and inexpensively using an oblique illumination geometry. DUV excitation (<280 nm) is preferable to near UV (365 nm) for avoidance of mineral autofluorescence. When excited with DUV, unpigmented bacteria show two emission peaks: one in the near UV ∼320 nm, corresponding to proteins, and another peak in the blue to green range, corresponding to flavins and/or reduced nicotinamide adenine dinucleotide (NADH). Many commonly used dyes also show secondary excitation peaks in the DUV, with identical emission spectra and quantum yields as their primary peak. However, DUV fails to excite key biosignature molecules, especially chlorophyll in cyanobacteria. Visible excitation (violet to blue) also results in less mineral autofluorescence than near UV, and most autofluorescence in the minerals seen here is green, so that red dyes and red autofluorescence of chlorophyll and porphyrins are readily distinguished. The pairing of DUV and near UV or visible excitation, with emission across the visible, represents the most thorough approach to detection of labeled and unlabeled bacteria in soil and rock.

Single-molecule localization microscopy (SMLM) is a powerful technique to achieve super-resolution imaging beyond the diffraction limit. Although various types of blinking fluorophores are currently considered for SMLM, intrinsic blinking fluorophores remain rare at the single-molecule level. Here, we report the synthesis of nanographene-based intrinsic burst-blinking fluorophores for highly versatile SMLM. We image amyloid fibrils in air and in various pH solutions without any additive and lysosome dynamics in live mammalian cells under physiological conditions. In addition, the single-molecule labeling of nascent proteins in primary sensory neurons was achieved with azide-functionalized nanographenes via click chemistry. SMLM imaging reveals higher local translation at axonal branching with unprecedented detail, while the size of translation foci remained similar throughout the entire network. These various results demonstrate the potential of nanographene-based fluorophores to drastically expand the applicability of super-resolution imaging.

Currently, in situ monitoring of the adenosine triphosphate (ATP) level in lysosomes is critical to understand their involvement in various biological processes, but it remains difficult due to the interferences of limited targeting and low resolution of fluorescent probes. Herein, we report a classic Mn(II) probe ( FX2-MnCl 2 ) with near-infrared (NIR) nonlinear (NLO) properties, accompanied by three-four photon transition and fivefold fluorescence enhancement in the presence of ATP. FX2-MnCl 2 combines with ATP through dual recognition sites of diethoxy and manganese ions to reflect slightly fluorescence lifetime change. Through the synergy of multiphoton fluorescence imaging (MP-FI) and multiphoton fluorescence lifetime imaging microscopy (MP-FLIM), it is further demonstrated that FX2-MnCl 2 displays lysosome-specific targeting behavior, which can monitor lysosome-related ATP migration under NIR laser light. This work provides a novel multiphoton transformation fluorescence complex, which might be a potential candidate as a simple and straightforward biomarker of lysosome ATP in vitro for clinical diagnosis.