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Live-cell biosensors based on the fluorescence lifetime of environment-sensing dyes.

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.

SUBMITTER: Mehl BP

PROVIDER: S-EPMC10985238 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Live-cell biosensors based on the fluorescence lifetime of environment-sensing dyes.

Mehl Brian P BP Vairaprakash Pothiappan P Li Li L Hinde Elizabeth E MacNevin Christopher J CJ Hsu Chia-Wen CW Gratton Enrico E Liu Bei B Hahn Klaus M KM

Cell reports methods 20240318 3

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 fluoropho ...[more]

PMID: 38503289

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