1. Live-cell biosensors based on the fluorescence lifetime of environment-sensing dyes.
- Author
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Mehl, Brian, Vairaprakash, Pothiappan, Li, Li, Hinde, Elizabeth, MacNevin, Christopher, Hsu, Chia-Wen, Liu, Bei, Hahn, Klaus, and Gratton, Enrico
- Subjects
CP: Imaging ,Cdc42 ,FLIM ,biosensor ,cell ,dye ,fluorescence ,merocyanine ,phasor ,solvatochromic ,Microscopy ,Fluorescence ,Fluorescent Dyes ,Biosensing Techniques - Abstract
In this work, we examine the use of environment-sensitive fluorescent dyes in fluorescence lifetime imaging microscopy (FLIM) biosensors. We screened merocyanine dyes to find an optimal combination of environment-induced lifetime changes, photostability, and brightness at wavelengths suitable for live-cell imaging. FLIM was used to monitor a biosensor reporting conformational changes of endogenous Cdc42 in living cells. The ability to quantify activity using phasor analysis of a single fluorophore (e.g., rather than ratio imaging) eliminated potential artifacts. We leveraged these properties to determine specific concentrations of activated Cdc42 across the cell.
- Published
- 2024