Widefield Optical Microscopy
Widefield Optical Microscopy - Related Videos
08:31
Three-dimensional Optical-resolution Photoacoustic Microscopy
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Cited by 27 •
2011
Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.
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...
09:45
Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes
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Cited by 21 •
2011
This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.
14:09
Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope
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Cited by 11 •
2014
We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.
14:58
Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters
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2010
We demonstrate a dark-field microscopy method based on Gabor-like filtering to measure subcellular dynamics within single living cells. The technique is sensitive to alterations in the structure of organelles, such as mitochondrial fragmentation.
FAQs
The sample receives excitation light, and fluorescent labels emit light at a longer wavelength. Objective lenses collect this emitted signal and project it onto a camera, allowing labeled structures to be visualized. This wavelength change provides the basis for examining protein localization and other cellular features that have been marked with fluorescent labels.
Because illumination covers the field and light is collected from many sample depths, signals outside the focal plane can contribute to the recorded image. In thick specimens, this out-of-focus light can reduce contrast and make structures harder to distinguish. The limitation is especially relevant when interpreting tissue organization or features distributed through different depths.
Fluorescence imaging records longer-wavelength light emitted by labeled structures after excitation, whereas transmitted-light imaging records light that passes through the specimen. The two modes therefore provide different visual information: fluorescence highlights selected labeled components, while transmitted light supports direct examination of specimen appearance and morphology without relying on the same emission signal.
For fluorescence observations, excitation light is directed through the sample, emitted light is collected by objective lenses, and the resulting image is projected onto a camera. In transmitted-light observations, the camera records light passing through the specimen instead. Selecting the appropriate imaging mode connects the optical pathway to the biological feature being examined.
Its rapid image acquisition and broad spatial coverage make it useful for routine imaging, live-cell studies, and observations requiring a large field of view. Researchers can examine cells, tissues, or microorganisms while following dynamic biological events. The method is particularly practical when intuitive images and speed are more important than resolving information from every specimen depth.
Images can reveal cell morphology, protein localization, tissue organization, and dynamic biological events. Fluorescent labeling helps associate signals with selected structures, while transmitted-light observations show light passing through the specimen. These complementary observations support studies ranging from individual cells and microorganisms to broader tissue patterns, provided that reduced contrast from out-of-focus light is considered.