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Title: Designing a Compact, Low-Cost FRET Microscopy Platform for the Undergraduate Classroom
ABSTRACT Advances in fluorescent biosensors allow researchers to spatiotemporally monitor a diversity of biochemical reactions and secondary messengers. However, commercial microscopes for the specific application of Förster Resonance Energy Transfer (FRET) are prohibitively expensive to implement in the undergraduate classroom, owing primarily to the dynamic range required and need for ratiometric emission imaging. The purpose of this article is to provide a workflow to design a low-cost, FRET-enabled microscope and to equip the reader with sufficient knowledge to compare commercial light sources, optics, and cameras to modify the device for a specific application. We used this approach to construct a microscope that was assembled by undergraduate students with no prior microscopy experience that is suitable for most single-cell cyan and yellow fluorescent protein FRET applications. The utility of this design was demonstrated by measuring small metabolic oscillations by using a lactate FRET sensor expressed in primary mouse pancreatic islets, highlighting the biologically suitable signal-to-noise ratio and dynamic range of our compact microscope. The instructions in this article provide an effective teaching tool for undergraduate educators and students interested in implementing FRET in a cost-effective manner.
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The Biophysicist
Sponsoring Org:
National Science Foundation
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