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Indian Journal of Pathology &... 2021The last two decades have seen considerable progress in the use of digital technology in histopathology. Digital photography of microscopic slides and the use of static... (Review)
Review
The last two decades have seen considerable progress in the use of digital technology in histopathology. Digital photography of microscopic slides and the use of static images gave way to robotic microscopes. These technologies had their own limitations that precluded their widespread use. Creation of whole slide scanners that can produce digitized whole slide images (WSI) and the "comparable to conventional microscope" experience opened multiple avenues for their utilization not only in specific applications such as expert consults, quality assessment programs, education and archiving, but also for routine day-to-day reporting. Industry pressures driven by consumer requirements have led to great development in image quality, speed of scanning, size of stored files, and capital cost of scanners. User-friendly software and analytical algorithms have further enhanced user experience. Challenges that need to be either accepted or overcome would include capital expense not significantly yielding a return on investment, and management of storage space. This review attempts to take the reader through the evolution of WSI scanners and to share the author's experience with WSI for routine histopathology reporting, education, and external quality assessment along with a review of available literature.
Topics: Humans; Image Processing, Computer-Assisted; Microscopy
PubMed: 33433403
DOI: 10.4103/IJPM.IJPM_356_20 -
Quarterly Reviews of Biophysics Jun 2021The application of cryo-correlative light and cryo-electron microscopy (cryo-CLEM) gives us a way to locate structures of interest in the electron microscope. In brief,...
The application of cryo-correlative light and cryo-electron microscopy (cryo-CLEM) gives us a way to locate structures of interest in the electron microscope. In brief, the structures of interest are fluorescently tagged, and images from the cryo-fluorescent microscope (cryo-FM) maps are superimposed on those from the cryo-electron microscope (cryo-EM). By enhancing cryo-FM to include single-molecule localization microscopy (SMLM), we can achieve much better localization. The introduction of cryo-SMLM increased the yield of photons from fluorophores, which can benefit localization efforts. Dahlberg and Moerner (2021, Annual Review of Physical Chemistry, 72, 253-278) have a recent broad and elegant review of super-resolution cryo-CLEM. This paper focuses on cryo(F)PALM/STORM for the cryo-electron tomography community. I explore the current challenges to increase the accuracy of localization by SMLM and the mapping of those positions onto cryo-EM images and maps. There is much to consider: we need to know if the excitation of fluorophores damages the structures we seek to visualize. We need to determine if higher numerical aperture (NA) objectives, which add complexity to image analysis but increase resolution and the efficiency of photon collection, are better than lower NA objectives, which pose fewer problems. We need to figure out the best way to determine the axial position of fluorophores. We need to have better ways of aligning maps determined by FM with those determined by EM. We need to improve the instrumentation to be easier to use, more accurate, and ice-contamination free. The bottom line is that we have more work to do.
Topics: Cryoelectron Microscopy; Electron Microscope Tomography; Fluorescent Dyes; Microscopy, Fluorescence; Single Molecule Imaging
PubMed: 34165063
DOI: 10.1017/S003358352100007X -
Advanced Biology Jan 2021Stimulated Raman scattering (SRS) microscopy is a nonlinear optical imaging method for visualizing chemical content based on molecular vibrational bonds. Featuring high... (Review)
Review
Stimulated Raman scattering (SRS) microscopy is a nonlinear optical imaging method for visualizing chemical content based on molecular vibrational bonds. Featuring high speed, high resolution, high sensitivity, high accuracy, and 3D sectioning, SRS microscopy has made tremendous progress toward biochemical information acquisition, cellular function investigation, and label-free medical diagnosis in the biosciences. In this review, the principle of SRS, system design, and data analysis are introduced, and the current innovations of the SRS system are reviewed. In particular, combined with various bio-orthogonal Raman tags, the applications of SRS microscopy in cell metabolism, tumor diagnosis, neuroscience, drug tracking, and microbial detection are briefly examined. The future prospects for SRS microscopy are also shared.
Topics: Microscopy; Nonlinear Optical Microscopy; Spectrum Analysis, Raman; Vibration
PubMed: 33724734
DOI: 10.1002/adbi.202000184 -
Current Opinion in Structural Biology Oct 2020Recent advances in single-particle cryogenic-electron microscopy have facilitated an exponential growth in the number of membrane protein structures determined to close... (Review)
Review
Recent advances in single-particle cryogenic-electron microscopy have facilitated an exponential growth in the number of membrane protein structures determined to close to atomic resolution. Nevertheless, despite improvements in microscope hardware, cryo-EM software and sample preparation techniques, challenges remain for structural analysis of small-sized membrane proteins (i.e.<150 kilodalton). Here we discuss recent examples of structures of macromolecules from this category determined by cryo-EM. We analyze the underlying difficulties, the enabling technologies such as the use of antibody fragments to gain size and provide fiducials for particle alignment, and the unresolved issues like dislocation of complexes at the air-water interface. Finally, we briefly highlight the biological relevance of some of these success stories, and our predictions for the future.
Topics: Cryoelectron Microscopy; Macromolecular Substances; Membrane Proteins; Single Molecule Imaging; Software
PubMed: 32603877
DOI: 10.1016/j.sbi.2020.05.009 -
Nature Communications Mar 2022Scanning probe microscopy techniques, such as atomic force microscopy and scanning tunnelling microscopy, are harnessed to image nanoscale structures with an exquisite...
Scanning probe microscopy techniques, such as atomic force microscopy and scanning tunnelling microscopy, are harnessed to image nanoscale structures with an exquisite resolution, which has been of significant value in a variety of areas of nanotechnology. These scanning probe techniques, however, are not generally suitable for high-throughput imaging, which has, from the outset, been a primary challenge. Traditional approaches to increasing the scalability have involved developing multiple probes for imaging, but complex probe design and electronics are required to carry out the detection method. Here, we report a probe-based imaging method that utilizes scalable cantilever-free elastomeric probe design and hierarchical measurement architecture, which readily reconstructs high-resolution and high-throughput topography images. In a single scan, we demonstrate imaging with a 100-tip array to obtain 100 images over a 1-mm area with 10 pixels in less than 10 min. The potential for large-scale tip integration and the advantage of a simple probe array suggest substantial promise for our approach to high-throughput imaging far beyond what is currently possible.
Topics: Microscopy, Atomic Force; Microscopy, Scanning Probe; Microscopy, Scanning Tunneling; Nanotechnology; Proteins
PubMed: 35301324
DOI: 10.1038/s41467-022-29181-z -
Methods in Molecular Biology (Clifton,... 2022Optical tweezers and fluorescence microscopy are powerful methods for investigating the mechanical and structural properties of biomolecules and for studying the...
Optical tweezers and fluorescence microscopy are powerful methods for investigating the mechanical and structural properties of biomolecules and for studying the dynamics of the biomolecular processes that these molecules are involved in. Here we provide an outline of the concurrent use of optical tweezers and fluorescence microscopy for analyzing biomolecular processes. In particular, we focus on the use of super-resolution microscopy in optical tweezers, which allows visualization of molecules at the higher molecular densities that are typically encountered in living systems. We provide specific details on the alignment procedures of the optical pathways for confocal fluorescence microscopy and 1D-STED microscopy and elaborate on how to diagnose and correct optical aberrations and STED phase plate misalignments.
Topics: Microscopy, Confocal; Microscopy, Fluorescence; Optical Tweezers
PubMed: 36063320
DOI: 10.1007/978-1-0716-2229-2_6 -
Annual Review of Biophysics May 2023Recent advances in cryo-electron microscopy have marked only the beginning of the potential of this technique. To bring structure into cell biology, the modality of... (Review)
Review
Recent advances in cryo-electron microscopy have marked only the beginning of the potential of this technique. To bring structure into cell biology, the modality of cryo-electron tomography has fast developed into a bona fide in situ structural biology technique where structures are determined in their native environment, the cell. Nearly every step of the cryo-focused ion beam-assisted electron tomography (cryo-FIB-ET) workflow has been improved upon in the past decade, since the first windows were carved into cells, unveiling macromolecular networks in near-native conditions. By bridging structural and cell biology, cryo-FIB-ET is advancing our understanding of structure-function relationships in their native environment and becoming a tool for discovering new biology.
Topics: Electron Microscope Tomography; Cryoelectron Microscopy
PubMed: 37159298
DOI: 10.1146/annurev-biophys-111622-091327 -
Current Opinion in Structural Biology Dec 2022All steps of cryogenic electron-microscopy (cryo-EM) workflows have rapidly evolved over the last decade. Advances in both single-particle analysis (SPA) cryo-EM and... (Review)
Review
All steps of cryogenic electron-microscopy (cryo-EM) workflows have rapidly evolved over the last decade. Advances in both single-particle analysis (SPA) cryo-EM and cryo-electron tomography (cryo-ET) have facilitated the determination of high-resolution biomolecular structures that are not tractable with other methods. However, challenges remain. For SPA, these include improved resolution in an additional dimension: time. For cryo-ET, these include accessing difficult-to-image areas of a cell and finding rare molecules. Finally, there is a need for automated and faster workflows, as many projects are limited by throughput. Here, we review current developments in SPA cryo-EM and cryo-ET that push these boundaries. Collectively, these advances are poised to propel our spatial and temporal understanding of macromolecular processes.
Topics: Cryoelectron Microscopy; Electron Microscope Tomography; Macromolecular Substances; Single Molecule Imaging
PubMed: 36323134
DOI: 10.1016/j.sbi.2022.102484 -
Histochemistry and Cell Biology Jun 2022Fluorescence lifetime imaging microscopy (FLIM) allows the characterization of cellular metabolism by quantifying the rate of free and unbound nicotinamide adenine...
Fluorescence lifetime imaging microscopy (FLIM) allows the characterization of cellular metabolism by quantifying the rate of free and unbound nicotinamide adenine dinucleotide hydrogen (NADH). This study delineates the correlative imaging of cells with FLIM and electron microscopy (EM). Human fibroblasts were cultivated in a microscopy slide bearing a coordinate system and FLIM measurement was conducted. Following chemical fixation, embedding in Epon and cutting with an ultramicrotome, tomograms of selected cells were acquired with a scanning transmission electron microscope (STEM). Correlative imaging of antimycin A-treated fibroblasts shows a decrease in fluorescence lifetime as well as swollen mitochondria with large cavities in STEM tomography. To our knowledge, this is the first correlative FLIM and EM workflow. Combining the high sensitivity of FLIM with the high spatial resolution of EM could boost the research of pathophysiological processes involving cell metabolism, such as cancer, neurodegenerative disorders, and viral infection.
Topics: Electron Microscope Tomography; Humans; Microscopy, Electron; Microscopy, Fluorescence; Optical Imaging; Workflow
PubMed: 35267057
DOI: 10.1007/s00418-022-02094-0 -
Nature Communications Mar 2024Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread...
Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce biological throughput and applicability by requiring lower labeling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMLFM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We demonstrate this optical robustness experimentally with high accuracy ( > 99.2 ± 0.1%, 0.1 locs μm) and sensitivity ( > 86.6 ± 0.9%, 0.1 locs μm) through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high-density volumetric imaging (0.15 locs μm) in dense cytosolic tubulin datasets.
Topics: Microscopy; Imaging, Three-Dimensional; Single Molecule Imaging; Nanotechnology
PubMed: 38431671
DOI: 10.1038/s41467-024-45828-5