Confocal Immunofluorescence Microscopy
Confocal Immunofluorescence Microscopy - Related Videos
01:12
Immunofluorescence Microscopy
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2023
A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange. The...
09:56
Immunofluorescence Microscopy: Immunofluorescence Staining of Paraffin-Embedded Tissue Sections
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2023
Source: Thomas Chaffee1, Thomas S. Griffith2,3,4, and Kathryn L. Schwertfeger1,3,4 1 Department of Lab Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455 2 Department of Urology, University of Minnesota, Minneapolis, MN 55455 3 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455 4 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 Pathologic analyses of tissue sections can be used to obtain a better understanding of normal tissue...
10:48
Imaging Biological Samples with Optical and Confocal Microscopy
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2023
Source: Peiman Shahbeigi-Roodposhti and Sina Shahbazmohamadi, Biomedical Engineering Department, University of Connecticut, Storrs, Connecticut Optical microscopes have been around for centuries, and while they reached their theoretical limitation of resolution decades ago, new equipment and techniques, such as confocal and digital image processing, have created new niches within the field of optical imaging. The best optical microscopes will typically have a resolution down to 200 nm in ideal...
03:16
Three-dimensional Imaging of Immunolabeled Astrocytes Using Confocal Microscopy
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2025
Source: Bagheri, M., et. al. Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes. J. Vis. Exp. (2015).This video demonstrates the imaging of fluorescently labeled astrocytes in a mouse brain section using a confocal microscope. The brain section is positioned under the appropriate objective lens, and suitable fluorophores and filters are selected. After optimizing the exposure automatically, the scanning parameters are defined to ensure high-resolution 2D images. Z-stack...
01:16
Confocal Fluorescence Microscopy
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2023
Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
FAQs
Fluorescent antibodies provide molecular specificity, but the pinhole determines how selectively the microscope detects that signal. By rejecting light originating outside the focal plane, it reduces visual interference from other depths and produces sharper optical sections. This separation is especially useful when labeled neuronal or glial markers occupy densely organized brain tissue.
Optical sectioning converts depth into a sequence of individually resolved images. The microscope collects sections at successive depths rather than treating the specimen as one undifferentiated field, then uses those sections to reconstruct three-dimensional structures. In neuroscience, this preserves the relative placement of labeled proteins, receptors, and glial markers within complex tissue.
Different fluorescent antibody labels can identify different molecular targets within the same specimen, allowing their distributions to be examined together. The resulting images can show whether neuronal proteins, neurotransmitter receptors, or glial markers occupy overlapping spatial patterns. This makes colocalization analysis useful for relating molecular identity to cellular organization while retaining tissue context.
After fluorescent antibody labeling, a focused laser excites the selected fluorophores. The microscope uses a pinhole to reject out-of-focus light, records an image from one depth, and repeats acquisition through successive depths. These optical sections can then be combined to reconstruct three-dimensional structures, providing an ordered path from labeling to spatial interpretation.
It is useful when researchers need to map specific molecules within cellular or tissue organization rather than view molecular signals without spatial context. In neuroscience, applications include examining neuronal proteins, neurotransmitter receptors, and glial markers, as well as studying cellular organization and neural connectivity in brain tissue.
By imaging the locations of labeled targets across brain tissue, researchers can compare patterns associated with disease-related changes or treatment responses. The method links those molecular distributions to preserved spatial relationships, helping interpret whether alterations occur within particular cellular arrangements or tissue structures. Its value therefore extends from marker detection to contextualized analysis of neural organization.