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

Research output: Contribution to journalArticle

12 Citations (Scopus)

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

Fingerprint

Energy Transfer
Biosensing Techniques
Photons
Biosensors
Energy transfer
Microscopy
Microscopic examination
Confocal Microscopy
Scanning
Lasers
Cells
Photobleaching
Cyclic AMP-Dependent Protein Kinases
Liver
Hepatocytes
Phosphotransferases
Cell Culture Techniques
Tissue
Imaging techniques
Monitoring

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy. / Day, Richard; Tao, Wen; Dunn, Kenneth.

In: Nature Protocols, Vol. 11, No. 11, 01.11.2016, p. 2066-2080.

Research output: Contribution to journalArticle

@article{80476b2504364029ab38344c5ba7a959,
title = "A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy",
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 F{\"o}rster 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.",
author = "Richard Day and Wen Tao and Kenneth Dunn",
year = "2016",
month = "11",
day = "1",
doi = "10.1038/nprot.2016.121",
language = "English (US)",
volume = "11",
pages = "2066--2080",
journal = "Nature Protocols",
issn = "1754-2189",
publisher = "Nature Publishing Group",
number = "11",

}

TY - JOUR

T1 - A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy

AU - Day, Richard

AU - Tao, Wen

AU - Dunn, Kenneth

PY - 2016/11/1

Y1 - 2016/11/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84992563858&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84992563858&partnerID=8YFLogxK

U2 - 10.1038/nprot.2016.121

DO - 10.1038/nprot.2016.121

M3 - Article

C2 - 27685098

AN - SCOPUS:84992563858

VL - 11

SP - 2066

EP - 2080

JO - Nature Protocols

JF - Nature Protocols

SN - 1754-2189

IS - 11

ER -