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Current Protocols in Mouse Biology Jun 2020The light (or optical) microscope is the icon of science. The aphorism "seeing is believing" is often quoted in scientific papers involving microscopy. Unlike many...
The light (or optical) microscope is the icon of science. The aphorism "seeing is believing" is often quoted in scientific papers involving microscopy. Unlike many scientific instruments, the light microscope will deliver an image however badly it is set up. Fluorescence microscopy is a widely used research tool across all disciplines of biological and biomedical science. Most universities and research institutions have microscopes, including confocal microscopes. This introductory paper in a series detailing advanced light microscopy techniques explains the foundations of both electron and light microscopy for biologists and life scientists working with the mouse. An explanation is given of how an image is formed. A description is given of how to set up a light microscope, whether it be a brightfield light microscope on the laboratory bench, a widefield fluorescence microscope, or a confocal microscope. These explanations are accompanied by operational protocols. A full explanation on how to set up and adjust a microscope according to the principles of Köhler illumination is given. The importance of Nyquist sampling is discussed. Guidelines are given on how to choose the best microscope to image the particular sample or slide preparation that you are working with. These are the basic principles of microscopy that a researcher must have an understanding of when operating core bioimaging facility instruments, in order to collect high-quality images. © 2020 The Authors. Basic Protocol 1: Setting up Köhler illumination for a brightfield microscope Basic Protocol 2: Aligning the fluorescence bulb and setting up Köhler illumination for a widefield fluorescence microscope Basic Protocol 3: Generic protocol for operating a confocal microscope.
Topics: Animals; Humans; Microscopy; Microscopy, Confocal
PubMed: 32497416
DOI: 10.1002/cpmo.76 -
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 Analytical Chemistry... Jun 2022An optical microscope is probably the most intuitive, simple, and commonly used instrument to observe objects and discuss behaviors through images. Although the idea of... (Review)
Review
An optical microscope is probably the most intuitive, simple, and commonly used instrument to observe objects and discuss behaviors through images. Although the idea of imaging electrochemical processes operando by optical microscopy was initiated 40 years ago, it was not until significant progress was made in the last two decades in advanced optical microscopy or plasmonics that it could become a mainstream electroanalytical strategy. This review illustrates the potential of different optical microscopies to visualize and quantify local electrochemical processes with unprecedented temporal and spatial resolution (below the diffraction limit), up to the single object level with subnanoparticle or single-molecule sensitivity. Developed through optically and electrochemically active model systems, optical microscopy is now shifting to materials and configurations focused on real-world electrochemical applications.
Topics: Electrochemistry; Microscopy; Nanotechnology
PubMed: 35216529
DOI: 10.1146/annurev-anchem-061020-015943 -
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 -
Current Protocols in Human Genetics Jul 2017This unit provides an overview of light microscopy, including objectives, light sources, filters, film, and color photography for fluorescence microscopy and... (Review)
Review
This unit provides an overview of light microscopy, including objectives, light sources, filters, film, and color photography for fluorescence microscopy and fluorescence in situ hybridization (FISH). We believe there are excellent opportunities for cytogeneticists, pathologists, and other biomedical readers, to take advantage of specimen optical clearing techniques and expansion microscopy-we briefly point to these new opportunities. © 2017 by John Wiley & Sons, Inc.
Topics: Animals; Color; Cytogenetics; Humans; In Situ Hybridization, Fluorescence; Microscopy; Microscopy, Fluorescence; Pathology; Photography
PubMed: 28696557
DOI: 10.1002/cphg.42 -
The Analyst Feb 2021Many dermatological studies have had limited success in revealing skin function because conventional histological methods are known to affect skin components. Recent... (Review)
Review
Many dermatological studies have had limited success in revealing skin function because conventional histological methods are known to affect skin components. Recent progress in non-invasive optical imaging has enabled non-invasive visualization of the structure of each skin layer. However, it remains difficult to identify individual skin components. Alternatively, it is possible to obtain molecular vibrational signatures using spontaneous Raman scattering microscopy. Spontaneous Raman scattering microscopy requires long acquisition times and is rarely applied to skin imaging, especially because skin components, such as water and transepidermal agents, undergo relatively rapid changes. Consequently, non-linear Raman microscopies, such as coherent anti-Stokes Raman scattering and stimulated Raman scattering, have gradually been applied to acquire molecular imaging of skin tissue. In this review, the applications of Raman microscopies used to evaluate skin and research trends are presented. The applications of spontaneous Raman microscopy to in vivo human skin evaluation are first demonstrated with typical applications. Finally, the latest application of coherent Raman scattering microscopy to visualize 3D intracellular morphologies in the human epidermis during differentiation is described.
Topics: Humans; Microscopy; Optical Imaging; Skin; Spectrum Analysis, Raman; Vibration
PubMed: 33459306
DOI: 10.1039/d0an02039g -
Current Opinion in Microbiology Jun 2018Understanding how microbes utilize their environment is aided by visualizing them in their natural context at high resolution. Correlative imaging enables efficient... (Review)
Review
Understanding how microbes utilize their environment is aided by visualizing them in their natural context at high resolution. Correlative imaging enables efficient targeting and identification of labelled viral and bacterial components by light microscopy combined with high resolution imaging by electron microscopy. Advances in genetic and bioorthogonal labelling, improved workflows for targeting and image correlation, and large-scale data collection are increasing the applicability of correlative imaging methods. Furthermore, developments in mass spectroscopy and soft X-ray imaging are expanding the correlative imaging modalities available. Investigating the structure and organization of microbes within their host by combined imaging methods provides important insights into mechanisms of infection and disease which cannot be obtained by other techniques.
Topics: Bacteria; Communicable Diseases; Host Microbial Interactions; Humans; Mass Spectrometry; Microscopy; Microscopy, Electron; Single Molecule Imaging; Viruses
PubMed: 29414444
DOI: 10.1016/j.mib.2018.01.009 -
Expert Review of Hematology May 2017Platelet granule deficiencies lead to bleeding disorders, but their specific diagnosis typically requires whole mount transmission electron microscopy, which is often... (Review)
Review
Platelet granule deficiencies lead to bleeding disorders, but their specific diagnosis typically requires whole mount transmission electron microscopy, which is often not available and has a number of important limitations. We recently proposed the use of advanced forms of fluorescence microscopy - the so-called 'super-resolution' microscopies - as a possible solution. Areas covered: In this special report, we review the diagnosis of platelet granule deficiencies, and discuss how recent developments in fluorescence microscopy may be useful in improving diagnostic approaches to these and related disorders. In particular, we conclude that super-resolution fluorescence microscopy may have advantages over transmission electron microscopy in this application. Expert commentary: The value of the super-resolution microscopies has been amply demonstrated in research; however, their potential in diagnostic applications is ripe for further exploration. Hematology is a field particularly likely to benefit because of the relative simplicity of sample preparation. We anticipate that the costs of the necessary instrumentation will continue to fall rapidly, making these technologies widely accessible to clinicians.
Topics: Blood Platelets; Gray Platelet Syndrome; Humans; Microscopy, Fluorescence
PubMed: 28374619
DOI: 10.1080/17474086.2017.1315302 -
The Science of the Total Environment Jun 2021Nanoscale contaminants (including engineered nanoparticles and nanoplastics) pose a significant threat to organisms and environment. Rapid and non-destructive detection... (Review)
Review
Nanoscale contaminants (including engineered nanoparticles and nanoplastics) pose a significant threat to organisms and environment. Rapid and non-destructive detection and identification of nanosized materials in cells, tissues and organisms is still challenging, although a number of conventional methods exist. These approaches for nanoparticles imaging and characterisation both inside the cytoplasm and on the cell or tissue outer surfaces, such as electron or scanning probe microscopies, are unquestionably potent tools, having excellent resolution and supplemented with chemical analysis capabilities. However, imaging and detection of nanomaterials in situ, in wet unfixed and even live samples, such as living isolated cells, microorganisms, protozoans and miniature invertebrates using electron microscopy is practically impossible, because of the elaborate sample preparation requiring chemical fixation, contrast staining, matrix embedding and exposure into vacuum. Atomic force microscopy, in several cases, can be used for imaging and mechanical analysis of live cells and organisms under ambient conditions, however this technique allows for investigation of surfaces. Therefore, a different approach allowing for imaging and differentiation of nanoscale particles in wet samples is required. Dark-field microscopy as an optical microscopy technique has been popular among researchers, mostly for imaging relatively large specimens. In recent years, the so-called "enhanced dark field" microscopy based on using higher numerical aperture light condensers and variable numerical aperture objectives has emegred, which allows for imaging of nanoscale particles (starting from 5 nm nanospheres) using almost conventional optical microscopy methodology. Hyperspectral imaging can turn a dark-field optical microscope into a powerful chemical characterisation tool. As a result, this technique is becoming popular in environmental nanotoxicology studies. In this Review Article we introduce the reader into the methodology of enhanced dark-field and dark-field-based hyperspectral microscopy, covering the most important advances in this rapidly-expanding area of environmental nanotoxicology.
Topics: Microscopy, Atomic Force; Microscopy, Electron; Nanoparticles; Nanostructures
PubMed: 33571774
DOI: 10.1016/j.scitotenv.2021.145478 -
Current Opinion in Structural Biology Oct 2021With a strong understanding of how proteins fold in hand, it is now possible to ask how in-cell environments modulate their folding, binding and function. Studies... (Review)
Review
With a strong understanding of how proteins fold in hand, it is now possible to ask how in-cell environments modulate their folding, binding and function. Studies accessing fast (ns to s) in-cell dynamics have accelerated over the past few years through a combination of in-cell NMR spectroscopy and time-resolved fluorescence microscopies. Here, we discuss this recent work and the emerging picture of protein surfaces as not just hydrophilic coats interfacing the solvent to the protein's core and functional regions, but as critical components in cells controlling protein mobility, function and communication with post-translational modifications.
Topics: Hydrophobic and Hydrophilic Interactions; Magnetic Resonance Spectroscopy; Microscopy; Protein Folding; Proteins; Solvents
PubMed: 33662744
DOI: 10.1016/j.sbi.2021.02.001