A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

Genetically encoded fluorescent protein (FP)-based biosensor probes are useful tools for monitoring cellular events in living cells and tissues. Because these probes were developed for one-photon excitation approaches, their broad two-photon excitation (2PE) and poorly understood photobleaching characteristics have made their implementation in studies using two-photon laser-scanning microscopy (TPLSM) challenging. Here we describe a protocol that simplifies the use of Förster resonance energy transfer (FRET)-based biosensors in TPLSM. First, the TPLSM system is evaluated and optimized using FRET standards expressed in living cells, which enables the determination of spectral bleed-Through (SBT) and the confirmation of FRET measurements from the known standards. Next, we describe how to apply the approach experimentally using a modified version of the A kinase activity reporter (AKAR) protein kinase A (PKA) biosensor as an example-first in cells in culture and then in hepatocytes in the liver of living mice. The microscopic imaging can be accomplished in a day in laboratories that routinely use TPLSM.

Original languageEnglish (US)
Pages (from-to)2066-2080
Number of pages15
JournalNature protocols
Volume11
Issue number11
DOIs
StatePublished - Nov 1 2016

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Fingerprint Dive into the research topics of 'A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy'. Together they form a unique fingerprint.

  • Cite this