Widefield Fluorescence Microscopy
Widefield Fluorescence Microscopy - Related Videos
06:42
Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone
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Cited by 32 •
2013
We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.
09:28
Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System
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Cited by 4 •
2022
Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.
09:22
Introduction to Fluorescence Microscopy
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2023
Fluorescence microscopy is a very powerful analytical tool that combines the magnifying properties of light microscopy with visualization of fluorescence. Fluorescence is a phenomenon that involves absorbance and emission of a small range of light wavelengths by a fluorescent molecule known as a fluorophore. Fluorescence microscopy is accomplished in conjunction with the basic light microscope by the addition of a powerful light source, specialized filters, and a means of fluorescently...
09:59
Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy
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Cited by 3 •
2013
Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.
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.
FAQs
Fluorescent molecules absorb light at selected excitation wavelengths and then emit light at longer wavelengths. The microscope uses emission filters to pass this emitted signal while limiting unwanted excitation light in the image. Matching the excitation source, fluorescent label, and filter characteristics is therefore essential for making labeled structures distinguishable from the surrounding specimen.
Because the excitation source illuminates the entire field, fluorescent material above and below the focal plane can also contribute emitted light. This out-of-focus fluorescence overlaps the signal from the region being examined and reduces image contrast, especially in thick specimens. The limitation matters when interpreting molecular localization or cellular organization across three-dimensional biological material.
The entire field receives illumination at once, allowing images to be acquired rapidly across a broad area. This supports observations that require high temporal resolution, including changes in living cells and other dynamic biological events. The approach is especially useful when recording the timing or progression of activity is more important than eliminating every out-of-focus signal.
First, the labeled specimen is positioned for optical observation and illuminated with the selected excitation source. Fluorescent molecules absorb that light and emit longer-wavelength light, which is directed through emission filters before image formation. The resulting image can then be examined for cellular organization, protein distribution, morphology, or changes occurring during the observation period.
It is well suited to rapid screening, broad-area examination, and live-cell studies. Researchers can survey cells, tissues, or microorganisms while tracking labeled structures and morphology across the field. The method is particularly appropriate when experiments prioritize speed, high temporal resolution, or efficient inspection of many biological features rather than maximum contrast in thick samples.
Images can show where fluorescently labeled proteins or other structures are distributed within cells and tissues. They can also reveal cell morphology, overall cellular organization, and features of microorganisms. When images are collected over time, the same approach can document dynamic biological events, linking molecular localization with visible changes in cellular form or activity.
In a thick specimen, fluorescence originating outside the focal region can be superimposed on the signal from the structure of interest. This may make boundaries or localized protein patterns less distinct than they would appear in a specimen with less overlapping out-of-focus fluorescence. Interpretations should therefore account for reduced contrast when assessing spatial molecular distribution.