A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy.
The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes.
Department of Medicine, Division of Nephrology, Indiana University Medical Center, Indianapolis, Indiana;
Wen
W
Tao
Riley Heart Research Center, Wells Center for Pediatric Research, and Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Indiana;
Michael
M
Rubart
Department of Medicine, Division of Nephrology, Indiana University Medical Center, Indianapolis, Indiana;
Jennifer
J
Ryan
Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and.
Xiao
X
Xiao
Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and.
Chunping
C
Qiao
Department of Medicine, Division of Nephrology, Indiana University Medical Center, Indianapolis, Indiana;
Takashi
T
Hato
National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, Tallahassee, Florida;
Michael W
MW
Davidson
Department of Medicine, Division of Nephrology, Indiana University Medical Center, Indianapolis, Indiana;
Kenneth W
KW
Dunn
Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana rnday@iupui.edu.
Richard N
RN
Day
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D032383:Myocytes, Cardiac / Q000737:chemistry*
D005456:Fluorescent Dyes; D008164:Luminescent Proteins
Förster resonance energy transfer; PKA activity; biosensor probe; fluorescent proteins; intravital microscopy; two-photon excitation