A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy.
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.
Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana USA.
Richard N
RN
Day
Department of Medicine, Division of Nephrology, Indiana University Medical Center, Indianapolis, Indiana, USA.
Wen
W
Tao
Department of Medicine, Division of Nephrology, Indiana University Medical Center, Indianapolis, Indiana, USA.
Kenneth W
KW
Dunn
D000818:Animals; D015374:Biosensing Techniques; D002470:Cell Survival; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*
D017868:Cyclic AMP-Dependent Protein Kinases
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