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Abstract Visualizing fluorescence‐tagged molecules is a powerful strategy that can reveal the complex dynamics of the cell. One robust and broadly applicable method is immunofluorescence microscopy, in which a fluorescence‐labeled antibody binds the molecule of interest and then the location of the antibody is determined by fluorescence microscopy. The effective application of this technique includes several considerations, such as the nature of the antigen, specificity of the antibody, permeabilization and fixation of the specimen, and fluorescence imaging of the cell. Although each protocol will require fine‐tuning depending on the cell type, antibody, and antigen, there are steps common to nearly all applications. This article provides protocols for staining the cytoskeleton and organelles in two very different kinds of cells: flat, adherent fibroblasts and thick, free‐swimmingTetrahymenacells. Additional protocols enable visualization with widefield, laser scanning confocal, and eSRRF super‐resolution fluorescence microscopy. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescence staining of adherent cells such as fibroblasts Basic Protocol 2: Immunofluorescence of suspension cells such asTetrahymena Basic Protocol 3: Visualizing samples with a widefield fluorescence microscope Alternate Protocol 1: Staining suspension cells adhered to poly‐l‐lysine‐coated coverslips Alternate Protocol 2: Visualizing samples with a laser scanning confocal microscope Alternate Protocol 3: Generating super‐resolution images with SRRF microscopy
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Imaging of surface acoustic waves on GaAs using 2D confocal Raman microscopy and atomic force microscopy
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Guichard, P.; Hamel, V. (Ed.)This chapter describes two mechanical expansion microscopy methods with accompanying step-by-step protocols. The first method, mechanically resolved expansion microscopy, uses non-uniform expansion of partially digested samples to provide the imaging contrast that resolves local mechanical properties. Examining bacterial cell wall with this method, we are able to distinguish bacterial species in mixed populations based on their distinct cell wall rigidity and detect cell wall damage caused by various physiological and chemical perturbations. The second method is mechanically locked expansion microscopy, in which we use a mechanically stable gel network to prevent the original polyacrylate network from shrinking in ionic buffers. This method allows us to use anti-photobleaching buffers in expansion microscopy, enabling detection of novel ultra-structures under the optical diffraction limit through super-resolution single molecule localization microscopy on bacterial cells and whole-mount immunofluorescence imaging in thick animal tissues. We also discuss potential applications and assess future directions.more » « less
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Finer resolution with greater stability is possible using unique low-power (aW), low-noise (20 dB S/N), microwave harmonics generated within a nanoscale tip-sample junction for feedback control in place of the DC tunneling current. Please see the attached poster to be presented at the Microscopy & Microanalysis-2018 meeting in Baltimore Monday August 6th as Post-deadline poster PDP-18. Applications include true sub-nm resolution in the carrier profiling of semiconductors. This method is especially appropriate for resistive samples where the spreading resistance flattens plots of the tunneling current vs. tip-sample distance with a scanning tunneling microscope.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1063/5.0034572
