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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 -
Current Opinion in Structural Biology Oct 2020Since the days of the first acknowledged microscopist, Antonie van Leeuwenhoek, the 'animalcules', that is, bacteria and other microbes have been subject to increasingly... (Review)
Review
Since the days of the first acknowledged microscopist, Antonie van Leeuwenhoek, the 'animalcules', that is, bacteria and other microbes have been subject to increasingly detailed visualization. With the currently most sophisticated molecular imaging method; cryo electron tomography (Cryo-ET), we are reaching the milestone of being able to image an entire organism in a single dataset at nanometer resolution. Cryo-ET will enable the next revolution in our understanding of bacterial cells, their ultra-structure and intricate molecular nanomachines. Here, we highlight recent research discoveries based on constantly progressing technology developments. We discuss advantages and challenges of using Cryo-ET to visualize spatial structure of microorganisms and macromolecular complexes in their native environment.
Topics: Bacteria; Cryoelectron Microscopy; Electron Microscope Tomography; Macromolecular Substances
PubMed: 32846309
DOI: 10.1016/j.sbi.2020.07.002 -
Annual Review of Analytical Chemistry... Jun 2022Since its first demonstration, stimulated Raman scattering (SRS) microscopy has become a powerful chemical imaging tool that shows promise in numerous biological and... (Review)
Review
Since its first demonstration, stimulated Raman scattering (SRS) microscopy has become a powerful chemical imaging tool that shows promise in numerous biological and biomedical applications. The spectroscopic capability of SRS enables identification and tracking of specific molecules or classes of molecules, often without labeling. SRS microscopy also has the hallmark advantage of signal strength that is directly proportional to molecular concentration, allowing for in situ quantitative analysis of chemical composition of heterogeneous samples with submicron spatial resolution and subminute temporal resolution. However, it is important to recognize that quantification through SRS microscopy requires assumptions regarding both system and sample. Such assumptions are often taken axiomatically, which may lead to erroneous conclusions without proper validation. In this review, we focus on the tacitly accepted, yet complex, quantitative aspect of SRS microscopy. We discuss the various approaches to quantitative analysis, examples of such approaches, challenges in different systems, and potential solutions. Through our examination of published literature, we conclude that a scrupulous approach to experimental design can further expand the powerful and incisive quantitative capabilities of SRS microscopy.
Topics: Microscopy; Nonlinear Optical Microscopy; Spectrum Analysis, Raman
PubMed: 35300525
DOI: 10.1146/annurev-anchem-061020-015110 -
Cytometry. Part a : the Journal of the... Apr 2020Short half-life proteins regulate many essential processes, including cell cycle, transcription, and apoptosis. However, few well-characterized protein-turnover pathways...
Short half-life proteins regulate many essential processes, including cell cycle, transcription, and apoptosis. However, few well-characterized protein-turnover pathways have been identified because traditional methods to measure protein half-life are time and labor intensive. To overcome this barrier, we developed a protein stability probe and high-content screening pipeline for novel regulators of short half-life proteins using automated image analysis. Our pilot probe consists of the short half-life protein c-MYC (MYC) fused to Venus fluorescent protein (MYC-Venus). This probe enables protein half-life to be scored as a function of fluorescence intensity and distribution. Rapid turnover prevents maximal fluorescence of the probe due to the relatively longer maturation time of the fluorescent protein. Cells expressing the MYC-Venus probe were analyzed using a pipeline in which automated confocal microscopy and image analyses were used to score MYC-Venus stability by two strategies: assaying the percentage of cells with Venus fluorescence above background, and phenotypic comparative analysis. To evaluate this high-content screening pipeline and our probe, a kinase inhibitor library was screened by confocal microscopy to identify known and novel kinases that regulate MYC stability. Compounds identified were shown to increase the half-life of both MYC-Venus and endogenous MYC, validating the probe and pipeline. Fusion of another short half-life protein, myeloid cell leukemia 1 (MCL1), with Venus also demonstrated an increase in percent Venus-positive cells after treatment with inhibitors known to stabilize MCL1. Together, the results validate the use of our automated microscopy and image analysis pipeline of stability probe-expressing cells to rapidly and quantitatively identify regulators of short half-life proteins. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Topics: Apoptosis; Humans; Microscopy, Confocal; Microscopy, Fluorescence; Protein Stability; Proteins
PubMed: 31774248
DOI: 10.1002/cyto.a.23928 -
Annual Review of Biophysics May 2023Single particle cryo-electron microscopy (cryo-EM) has matured into a robust method for the determination of biological macromolecule structures in the past decade,... (Review)
Review
Single particle cryo-electron microscopy (cryo-EM) has matured into a robust method for the determination of biological macromolecule structures in the past decade, complementing X-ray crystallography and nuclear magnetic resonance. Constant methodological improvements in both cryo-EM hardware and image processing software continue to contribute to an exponential growth in the number of structures solved annually. In this review, we provide a historical view of the many steps that were required to make cryo-EM a successful method for the determination of high-resolution protein complex structures. We further discuss aspects of cryo-EM methodology that are the greatest pitfalls challenging successful structure determination to date. Lastly, we highlight and propose potential future developments that would improve the method even further in the near future.
Topics: Cryoelectron Microscopy; Electrons; Single Molecule Imaging
PubMed: 37159297
DOI: 10.1146/annurev-biophys-111622-091300 -
Annual Review of Biochemistry Jun 2021The bedrock of drug discovery and a key tool for understanding cellular function and drug mechanisms of action is the structure determination of chemical compounds,... (Review)
Review
The bedrock of drug discovery and a key tool for understanding cellular function and drug mechanisms of action is the structure determination of chemical compounds, peptides, and proteins. The development of new structure characterization tools, particularly those that fill critical gaps in existing methods, presents important steps forward for structural biology and drug discovery. The emergence of microcrystal electron diffraction (MicroED) expands the application of cryo-electron microscopy to include samples ranging from small molecules and membrane proteins to even large protein complexes using crystals that are one-billionth the size of those required for X-ray crystallography. This review outlines the conception, achievements, and exciting future trajectories for MicroED, an important addition to the existing biophysical toolkit.
Topics: Cryoelectron Microscopy; Crystallization; Drug Discovery; Electrons; Microscopy, Electron, Transmission; Nanoparticles; Proteins; Workflow
PubMed: 34153215
DOI: 10.1146/annurev-biochem-081720-020121 -
Recent Developments in Correlative Super-Resolution Fluorescence Microscopy and Electron Microscopy.Molecules and Cells Jan 2022The recently developed correlative super-resolution fluorescence microscopy (SRM) and electron microscopy (EM) is a hybrid technique that simultaneously obtains the... (Review)
Review
The recently developed correlative super-resolution fluorescence microscopy (SRM) and electron microscopy (EM) is a hybrid technique that simultaneously obtains the spatial locations of specific molecules with SRM and the context of the cellular ultrastructure by EM. Although the combination of SRM and EM remains challenging owing to the incompatibility of samples prepared for these techniques, the increasing research attention on these methods has led to drastic improvements in their performances and resulted in wide applications. Here, we review the development of correlative SRM and EM (sCLEM) with a focus on the correlation of EM with different SRM techniques. We discuss the limitations of the integration of these two microscopy techniques and how these challenges can be addressed to improve the quality of correlative images. Finally, we address possible future improvements and advances in the continued development and wide application of sCLEM approaches.
Topics: Microscopy, Electron; Microscopy, Fluorescence
PubMed: 35114646
DOI: 10.14348/molcells.2021.5011 -
Optics Express Apr 2022Image scanning microscopy (ISM) overcomes the trade-off between spatial resolution and signal volume in confocal microscopy by rearranging the signal distribution on a...
Image scanning microscopy (ISM) overcomes the trade-off between spatial resolution and signal volume in confocal microscopy by rearranging the signal distribution on a two-dimensional detector array to achieve a spatial resolution close to the theoretical limit achievable by infinitesimal pinhole detection without sacrificing the detected signal intensity. In this paper, we improved the spatial resolution of ISM in three dimensions by exploiting saturated excitation (SAX) of fluorescence. We theoretically investigated the imaging properties of ISM, when the fluorescence signals are nonlinearly induced by SAX, and show combined SAX-ISM fluorescence imaging to demonstrate the improvement of the spatial resolution in three dimensions. In addition, we confirmed that the SNR of SAX-ISM imaging of fluorescent beads and biological samples, which is one of the challenges in conventional SAX microscopy, was improved.
Topics: Microscopy, Confocal; Microscopy, Fluorescence; Optical Imaging; Radionuclide Imaging
PubMed: 35472987
DOI: 10.1364/OE.455621 -
Nature Communications May 2023Single-molecule localization microscopy (SMLM) can be used to resolve subcellular structures and achieve a tenfold improvement in spatial resolution compared to that...
Single-molecule localization microscopy (SMLM) can be used to resolve subcellular structures and achieve a tenfold improvement in spatial resolution compared to that obtained by conventional fluorescence microscopy. However, the separation of single-molecule fluorescence events that requires thousands of frames dramatically increases the image acquisition time and phototoxicity, impeding the observation of instantaneous intracellular dynamics. Here we develop a deep-learning based single-frame super-resolution microscopy (SFSRM) method which utilizes a subpixel edge map and a multicomponent optimization strategy to guide the neural network to reconstruct a super-resolution image from a single frame of a diffraction-limited image. Under a tolerable signal density and an affordable signal-to-noise ratio, SFSRM enables high-fidelity live-cell imaging with spatiotemporal resolutions of 30 nm and 10 ms, allowing for prolonged monitoring of subcellular dynamics such as interplays between mitochondria and endoplasmic reticulum, the vesicle transport along microtubules, and the endosome fusion and fission. Moreover, its adaptability to different microscopes and spectra makes it a useful tool for various imaging systems.
Topics: Deep Learning; Microscopy, Fluorescence; Single Molecule Imaging; Neural Networks, Computer
PubMed: 37202407
DOI: 10.1038/s41467-023-38452-2 -
Methods in Molecular Biology (Clifton,... 2023Transmission electron microscopy (TEM) is currently the only method that enables the observation of extracellular vesicles (EVs) at a nanometer scale. Direct...
Transmission electron microscopy (TEM) is currently the only method that enables the observation of extracellular vesicles (EVs) at a nanometer scale. Direct visualization of the whole content of EV preparation provides not only crucial insights on the morphology of EVs but also an objective evaluation of the content and purity of the preparation. Coupled to immunogold labeling, TEM allows the detection and association of proteins at the surface of EVs. In these techniques, EVs are deposited on grids and are chemically immobilized and contrasted to withstand a high-voltage electron beam. Under high vacuum, the electron beam hits the sample and the electrons that scatter forward are collected to form an image. Here, we describe the steps needed to observe EVs by classical TEM and the extra steps required to label proteins through immunolabeling electron microscopy (IEM).
Topics: Microscopy, Electron, Transmission; Microscopy, Electron; Extracellular Vesicles; Proteins
PubMed: 37140788
DOI: 10.1007/978-1-0716-3203-1_4