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Monitoring biosensor activity in living cells with fluorescence lifetime imaging microscopy.

Abstract：

:Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing "FRET standard" fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail.

journal_name

Int J Mol Sci

journal_title

International journal of molecular sciences

authors

Hum JM,Siegel AP,Pavalko FM,Day RN

doi

10.3390/ijms131114385

subject

Has Abstract

pub_date

2012-11-07 00:00:00

pages

14385-400

issue

11

issn

1422-0067

pii

ijms131114385

journal_volume

13

pub_type

杂志文章,评审

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