Sensing and visualizing RNA molecules in live mammalian cells quantitatively is an emerging field with broad interest across disciplines. The Riboglow biosensor consists of a short RNA that is genetically tagged to a RNA of interest and binds a small fluorogenic molecule for tracking of RNAs of interest using fluorescence lifetime imaging microscopy (FLIM) in live mammalian cells. We established combining research and teaching of robust sample preparation, data collection, and image processing in the undergraduate classroom. We demonstrate workflows of benchmarking image acquisition and quantitative processing of FLIM datasets first in vitro for purified RNA samples and then in live mammalian cells where single cells are analyzed and subcellular resolution is demonstrated. Finally, complex multicellular environments are evaluated by Riboglow-FLIM. The Riboglow technology is complex but of broad interest, necessitating a detailed step-by-step demonstration of the method to ensure accessibility. A special emphasis is to achieve unbiased analysis of microscopy datasets. This includes demonstrating workflows to design experiments for sample and analysis blinding. Finally, principles to design future iterations of the FLIM platform from experimental insights are demonstrated, as implemented in the undergraduate classroom.
Shafik et al. (Sun,) studied this question.
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