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Beyond the H&E: Advanced Technologies for in situ Tissue Biomarker Imaging
摘要： For decades, histopathology with routine hematoxylin and eosin staining has been and remains the gold standard for reaching a morphologic diagnosis in tissue samples from humans and veterinary species. However, within the past decade, there has been exponential growth in advanced techniques for in situ tissue biomarker imaging that bridge the divide between anatomic and molecular pathology. It is now possible to simultaneously observe localization and expression magnitude of multiple protein, nucleic acid, and molecular targets in tissue sections and apply machine learning to synthesize vast, image-derived datasets. As these technologies become more sophisticated and widely available, a team-science approach involving subspecialists with medical, engineering, and physics backgrounds is critical to upholding quality and validity in studies generating these data. The purpose of this manuscript is to detail the scientific premise, tools and training, quality control, and data collection and analysis considerations needed for the most prominent advanced imaging technologies currently applied in tissue sections: immunofluorescence, in situ hybridization, laser capture microdissection, matrix-assisted laser desorption ionization imaging mass spectrometry, and spectroscopic/optical methods. We conclude with a brief overview of future directions for ex vivo and in vivo imaging techniques.
关键词： laser capture microdissection,immunohistochemistry,quality control,in situ hybridization,biomarkers,molecular pathology,fluorescence microscopy,MALDI
更新于2025-09-10 09:29:36
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Automated high-throughput light-sheet fluorescence microscopy of larval zebrafish
摘要： Light sheet fluorescence microscopy enables fast, minimally phototoxic, three-dimensional imaging of live specimens, but is currently limited by low throughput and tedious sample preparation. Here, we describe an automated high-throughput light sheet fluorescence microscope in which specimens are positioned by and imaged within a fluidic system integrated with the sheet excitation and detection optics. We demonstrate the ability of the instrument to rapidly examine live specimens with minimal manual intervention by imaging fluorescent neutrophils over a nearly 0.3 mm3 volume in dozens of larval zebrafish. In addition to revealing considerable inter-individual variability in neutrophil number, known previously from labor-intensive methods, three-dimensional imaging allows assessment of the correlation between the bulk measure of total cellular fluorescence and the spatially resolved measure of actual neutrophil number per animal. We suggest that our simple experimental design should considerably expand the scope and impact of light sheet imaging in the life sciences.
关键词： neutrophils,light sheet fluorescence microscopy,automated imaging,high-throughput imaging,larval zebrafish
更新于2025-09-10 09:29:36
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Red-Shifted Aminated Derivatives of GFP Chromophore for Live-Cell Protein Labeling with Lipocalins
摘要： Fluorogens are an attractive type of dye for imaging applications, eliminating time-consuming washout steps from staining protocols. With just a handful of reported fluorogen-protein pairs, mostly in the green region of spectra, there is a need for the expansion of their spectral range. Still, the origins of solvatochromic and fluorogenic properties of the chromophores suitable for live-cell imaging are poorly understood. Here we report on the synthesis and labeling applications of novel red-shifted fluorogenic cell-permeable green fluorescent protein (GFP) chromophore analogs.
关键词： fluorogenic dyes,fluorescence microscopy,protein-PAINT,fluorescent labeling,arylmethane dyes,GFP chromophore
更新于2025-09-10 09:29:36
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Intraoperative Probe-Based Confocal Endomicroscopy to Histologically Differentiate Thyroid From Parathyroid Tissue Before Resection
摘要： Background. Frozen section is the standard method to histologically distinguish parathyroid tissue from thyroid tissue during endocrine neck surgery. Frozen section can be time-consuming and costly. Its drawback is that it is to be performed only after the removal of a suspected pathological tissue. This study demonstrates the use of probe-based confocal laser endomicroscopy (pCLE) to confirm histology prior to tissue resection. Design. A prospective, single-institution, nonrandomized study was conducted. No sample size calculation was performed for this observational trial. The primary objective was the description of histological rendering of normal and pathological tissues through pCLE. Real-time in vivo fluorescence microscopy imaging was performed with the CystoFlex UHD probe after intravenous injection of 2.5 mL of 10% fluorescein sodium. Results. Eleven patients with hyperparathyroidism and thyroid conditions were included. A total of 104 videos showing thyroid, parathyroid, adipose tissue, muscle, laryngeal nerve, and lymph nodes were recorded. Videos were compared with visual information and pathological samples (when sampling was indicated). Thyroid tissue could be identified based on the presence of colloid follicles (intensely fluorescent area surrounded by a small ridge of low-fluorescence epithelial cells) including the pathognomonic aspect of resorption vacuole. Parathyroid tissue could be identified based on a regular, “diamond-shaped” capillary network encompassing parathyroid chief cells. Blinded reinterpretation of pCLE videos demonstrated an 89.3% sensitivity and a 90% specificity as compared with histology in tissue recognition. Conclusion. This pilot study describes representative renderings of intraoperative pCLE to nontraumatically differentiate thyroid, parathyroid, and lymph nodes before surgical removal.
关键词： parathyroidectomy,thyroid surgery,pCLE,in vivo fluorescence microscopy,probe-based confocal laser endomicroscopy
更新于2025-09-10 09:29:36
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VIPER is a genetically encoded peptide tag for fluorescence and electron microscopy
摘要： Many discoveries in cell biology rely on making specific proteins visible within their native cellular environment. There are various genetically encoded tags, such as fluorescent proteins, developed for fluorescence microscopy (FM). However, there are almost no genetically encoded tags that enable cellular proteins to be observed by both FM and electron microscopy (EM). Herein, we describe a technology for labeling proteins with diverse chemical reporters, including bright organic fluorophores for FM and electron-dense nanoparticles for EM. Our technology uses versatile interacting peptide (VIP) tags, a class of genetically encoded tag. We present VIPER, which consists of a coiled-coil heterodimer formed between the genetic tag, CoilE, and a probe-labeled peptide, CoilR. Using confocal FM, we demonstrate that VIPER can be used to highlight subcellular structures or to image receptor-mediated iron uptake. Additionally, we used VIPER to image the iron uptake machinery by correlative light and EM (CLEM). VIPER compared favorably with immunolabeling for imaging proteins by CLEM, and is an enabling technology for protein targets that cannot be immunolabeled. VIPER is a versatile peptide tag that can be used to label and track proteins with diverse chemical reporters observable by both FM and EM instrumentation.
关键词： biochemistry,fluorescence microscopy,coiled coil,chemical biology,electron microscopy
更新于2025-09-09 09:28:46
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Self-healing dyes for super-resolution fluorescence microscopy
摘要： In recent years, optical microscopy techniques have emerged that allow optical imaging at unprecedented resolution beyond the diffraction limit. These techniques exploit photostabilizing buffers to enable photoswitching and/or the enhancement of fluorophore brightness and stability. A major drawback with the use of photostabilizing buffers, however, is that they cannot be used in live cell imaging. In this paper, we tested the performance of self-healing organic fluorophores, which undergo intramolecular photostabilization, in super-resolution microscopy examining both targeted (stimulated emission depletion (STED) microscopy) and stochastic readout (stochastic optical reconstruction microscopy (STORM)). The overall goal of the study was to identify dyes and conditions that lead to improved spatial and temporal resolution of both techniques without the need for mixtures of photostabilizing agents in the imaging buffer. As a result of previously shown superior performance, we identified an ATTO647N-photostabilizer conjugate as a potential candidate for STED microscopy. We have here characterized the photostability and resulting performance of this nitrophenylalanine (NPA) conjugate of ATTO647N on oligonucleotides in STED microscopy. We found that the superior photophysical performance resulted in optimal STED imaging and demonstrated that single-molecule fluorescent transients of individual fluorophores can be obtained with both the excitation- and STED-laser. In similar experiments, we also tested a nitrophenylacetic acid conjugate of STAR635P, another frequently used dye in STED microscopy, and present a characterization of its photophysical properties. Finally, we performed an analysis of the photoswitching kinetics of self-healing Cy5 dyes (containing trolox, cyclooctatetraene and NPA-based stabilizers) in the presence of Tris(2-carboxyethyl) phosphine and cysteamine, which are typically used in STORM microscopy. In line with previous work, we found that intramolecular photostabilization strongly influences photoswitching kinetics and requires careful attention when designing STORM-experiments. In summary, this contribution explores the possibilities and limitations of self-healing dyes in super-resolution microscopy of differing modalities.
关键词： STORM,super-resolution microscopy,fluorescent dyes,STED,fluorescence microscopy
更新于2025-09-09 09:28:46
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Content-aware image restoration: pushing the limits of fluorescence microscopy
摘要： Fluorescence microscopy is a key driver of discoveries in the life sciences, with observable phenomena being limited by the optics of the microscope, the chemistry of the fluorophores, and the maximum photon exposure tolerated by the sample. These limits necessitate trade-offs between imaging speed, spatial resolution, light exposure, and imaging depth. In this work we show how content-aware image restoration based on deep learning extends the range of biological phenomena observable by microscopy. We demonstrate on eight concrete examples how microscopy images can be restored even if 60-fold fewer photons are used during acquisition, how near isotropic resolution can be achieved with up to tenfold under-sampling along the axial direction, and how tubular and granular structures smaller than the diffraction limit can be resolved at 20-times-higher frame rates compared to state-of-the-art methods. All developed image restoration methods are freely available as open source software in Python, FIJI, and KNIME.
关键词： isotropic resolution,image restoration,sub-diffraction structures,deep learning,content-aware,fluorescence microscopy
更新于2025-09-09 09:28:46
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The Maia Detector and Event Mode
摘要： A decade ago, prototypes of the Maia detector successfully demonstrated a fresh approach to X-ray fluorescence microscopy (XFM) imaging that combined a massively parallel detector architecture with dedicated pulse shaping and capture on each channel, asynchronous acquisition of X-rays as an event stream, and real-time processing of the event data [1, 2]. Today, a number of XFM beamlines that raster scan a sample through a focused X-ray beam to construct images of element concentration and chemical state use a 384-detector array version of Maia for high-throughput, high-definition XFM. The beamlines include those at the Australian Synchrotron (AS) in Melbourne [3], the PETRA III synchrotron at DESY, Hamburg [4], the CHESS synchrotron at Cornell University in Ithaca [5], and the NSLS-II at Brookhaven National Laboratory (BNL) in New York.
关键词： event mode,high-definition imaging,Maia detector,X-ray fluorescence microscopy,synchrotron
更新于2025-09-09 09:28:46
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[IEEE 2018 IEEE 15th International Symposium on Biomedical Imaging (ISBI 2018) - Washington, DC (2018.4.4-2018.4.7)] 2018 IEEE 15th International Symposium on Biomedical Imaging (ISBI 2018) - Segmentation of cell nuclei using intensity-based model fitting and sequential convex programming
摘要： We introduce a convex model-based approach for the segmentation of cell nuclei, which exploits both shape and intensity information. The model is directly fitted to the image intensities. Previous shape-based approaches either are not globally optimal or require prior binarization of an image. Our approach relies on a fast second-order optimization scheme to solve a sequence of convex programs and estimate the globally optimal solution based on the image intensities. Model fitting is performed within image regions which are determined by exploiting the local image structure. We evaluated our approach using fluorescence microscopy images of two different cell types and performed a quantitative comparison with previous methods.
关键词： model fitting,Fluorescence microscopy,convex optimization,cell segmentation
更新于2025-09-09 09:28:46
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Study on the Application of Electric Field to Giant Vesicles Comprised of 1,2-Dilauroyl-Sn-Glycero-3-Phosphatidylcholine Using Optical Fluorescence Microscopy
摘要： The influence of alternating electric field (AC) in the structure and dynamics of giant unilamellar vesicles (GUVs) comprised of 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC) is reported. The investigations were conducted by using optical fluorescence microscopy as the method of analysis. The lipid membrane of the DLPC GUVs at the fluid phase can be deformed and they migrate towards the electrodes under AC electric field. Nevertheless, membrane disruption or vesicle fusion was never noticed. The addition of concentrated glucose solution influences the osmotic pressure of the system leading to the formation of filaments at the outer region of the GUVs. These long flexible cylinders do not retract spontaneously. However, the application of AC electric field (20 V/mm, 20 Hz) enables the filaments to be retracted back to the GUVs membrane at a calculated speed of 0.18 μm.s-1
关键词： Electric field,GUVs,Fluorescence microscopy,DLPC
更新于2025-09-09 09:28:46