Super-resolution microscopy techniques have been widely adopted in biological sciences. Recently, a new super-resolution microscopy technique, called expansion microscopy (ExM) has been developed. In this technique, biomolecules inside specimens are first labeled with fluorophores, followed by in-situ hydrogel synthesis and physical expansion of the specimens. Image quality, including brightness and signal-to-noise ratio, depends on the extent to which fluorophores have bleached during the in-situ hydrogel synthesis process. In this work, we compared the fluorescence signal brightness of more than 20 fluorophores, after expansion, to identify the best fluorophore set for 4-color expansion microscopy imaging. In addition, we achieved 5-color multiplexed expansion microscopy by photo-bleaching one of the four fluorophores and re-staining thereafter.
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