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The Journal of Investigative Dermatology Dec 2012
Review
Topics: Dermoscopy; Humans; Microscopy, Confocal; Microscopy, Electron, Scanning Transmission; Skin Diseases
PubMed: 23187113
DOI: 10.1038/jid.2012.429 -
Annual Review of Biophysics May 2023Super-resolution fluorescence microscopy allows the investigation of cellular structures at nanoscale resolution using light. Current developments in super-resolution... (Review)
Review
Super-resolution fluorescence microscopy allows the investigation of cellular structures at nanoscale resolution using light. Current developments in super-resolution microscopy have focused on reliable quantification of the underlying biological data. In this review, we first describe the basic principles of super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM), and then give a broad overview of methodological developments to quantify super-resolution data, particularly those geared toward SMLM data. We cover commonly used techniques such as spatial point pattern analysis, colocalization, and protein copy number quantification but also describe more advanced techniques such as structural modeling, single-particle tracking, and biosensing. Finally, we provide an outlook on exciting new research directions to which quantitative super-resolution microscopy might be applied.
Topics: Single Molecule Imaging; Microscopy, Fluorescence
PubMed: 37159293
DOI: 10.1146/annurev-biophys-111622-091212 -
BMC Biology Sep 2018Array tomography encompasses light and electron microscopy modalities that offer unparalleled opportunities to explore three-dimensional cellular architectures in...
Array tomography encompasses light and electron microscopy modalities that offer unparalleled opportunities to explore three-dimensional cellular architectures in extremely fine structural and molecular detail. Fluorescence array tomography achieves much higher resolution and molecular multiplexing than most other fluorescence microscopy methods, while electron array tomography can capture three-dimensional ultrastructure much more easily and rapidly than traditional serial-section electron microscopy methods. A correlative fluorescence/electron microscopy mode of array tomography furthermore offers a unique capacity to merge the molecular discrimination strengths of multichannel fluorescence microscopy with the ultrastructural imaging strengths of electron microscopy. This essay samples the first decade of array tomography, highlighting applications in neuroscience.
Topics: Cytological Techniques; Electron Microscope Tomography; Imaging, Three-Dimensional; Microscopy, Electron, Scanning; Microscopy, Fluorescence
PubMed: 30189863
DOI: 10.1186/s12915-018-0560-1 -
Microscopy (Oxford, England) Jun 2023Microscopy has been essential to elucidate micro- and nano-scale processes in space and time and has provided insights into cell and organismic functions. It is widely...
Microscopy has been essential to elucidate micro- and nano-scale processes in space and time and has provided insights into cell and organismic functions. It is widely employed in cell biology, microbiology, physiology, clinical sciences and virology. While label-dependent microscopy, such as fluorescence microscopy, provides molecular specificity, it has remained difficult to multiplex in live samples. In contrast, label-free microscopy reports on overall features of the specimen at minimal perturbation. Here, we discuss modalities of label-free imaging at the molecular, cellular and tissue levels, including transmitted light microscopy, quantitative phase imaging, cryogenic electron microscopy or tomography and atomic force microscopy. We highlight how label-free microscopy is used to probe the structural organization and mechanical properties of viruses, including virus particles and infected cells across a wide range of spatial scales. We discuss the working principles of imaging procedures and analyses and showcase how they open new avenues in virology. Finally, we discuss orthogonal approaches that enhance and complement label-free microscopy techniques.
Topics: Humans; Virus Diseases; Viruses; Microscopy, Atomic Force; Microscopy, Electron; Microscopy, Fluorescence; Cryoelectron Microscopy
PubMed: 37079744
DOI: 10.1093/jmicro/dfad024 -
Current Protocols in Cytometry Oct 2014Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with... (Comparative Study)
Comparative Study Review
Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associated with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology has led to rapid improvement in the protocols and has ushered in a new generation of instruments to reach the next level of correlation--integration.
Topics: Animals; Humans; Microscopy, Electron; Microscopy, Fluorescence
PubMed: 25271959
DOI: 10.1002/0471142956.cy1236s70 -
Trends in Biotechnology Feb 2020The spatial distribution of molecules and cells is fundamental to understanding biological systems. Traditionally, microscopies based on electromagnetic waves such as... (Review)
Review
The spatial distribution of molecules and cells is fundamental to understanding biological systems. Traditionally, microscopies based on electromagnetic waves such as visible light have been used to localize cellular components by direct visualization. However, these techniques suffer from limitations of transmissibility and throughput. Complementary to optical approaches, biochemical techniques such as crosslinking can colocalize molecules without suffering the same limitations. However, biochemical approaches are often unable to combine individual colocalizations into a map across entire cells or tissues. Microscopy-by-sequencing techniques aim to biochemically colocalize DNA-barcoded molecules and, by tracking their thus unique identities, reconcile all colocalizations into a global spatial map. Here, we review this new field and discuss its enormous potential to answer a broad spectrum of questions.
Topics: DNA Barcoding, Taxonomic; High-Throughput Nucleotide Sequencing; Microscopy; Molecular Imaging
PubMed: 31416630
DOI: 10.1016/j.tibtech.2019.06.001 -
The International Journal of... 2019In recent years, we have witnessed an unprecedented advancement of light microscopy techniques which has allowed us to better understand biological processes occurring... (Review)
Review
In recent years, we have witnessed an unprecedented advancement of light microscopy techniques which has allowed us to better understand biological processes occurring during oogenesis and early embryonic development in mammals. In short, two modes of cellular imaging are now available: those involving fluorescent labels and those which are fluorophore-free. Fluorescence microscopy, in its various forms, is used predominantly in research, as it provides detailed information about cellular processes; however, it can involove an increased risk of photodamage. Fluorophore-free techniques provide, on the other hand, a smaller amount of biological data but they are safer for cells and therefore can be potentially used in a clinical setting. Here, we review various fluorescence and fluorophore-free visualisation approaches and discuss their applicability in developmental biology and reproductive medicine.
Topics: Blastocyst; Embryonic Development; Microscopy; Microscopy, Fluorescence; Microscopy, Polarization; Oocytes
PubMed: 31058300
DOI: 10.1387/ijdb.180300aa -
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 -
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 -
Annual Review of Physical Chemistry Apr 2021We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and... (Review)
Review
We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications.
Topics: Caulobacter crescentus; Cryoelectron Microscopy; Electron Microscope Tomography; HEK293 Cells; Humans; Microscopy, Electron; Microscopy, Fluorescence; Nanotechnology; Single Molecule Imaging; Subcellular Fractions
PubMed: 33441030
DOI: 10.1146/annurev-physchem-090319-051546