A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy

American Journal of Physiology. Cell Physiology
Wen TaoRichard N Day

Abstract

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 m...Continue Reading

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Citations

Jun 13, 2019·Scientific Reports·Christopher A ReissausAmelia K Linnemann
Aug 14, 2020·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Kapil NichaniJessica P Houston
Apr 25, 2021·Cell Chemical Biology·Michael R Post, David Sulzer
Mar 9, 2021·American Journal of Physiology. Renal Physiology·Kenneth W DunnPierre C Dagher
Apr 22, 2021·British Journal of Pharmacology·Robert FeilSusanne Feil
Aug 3, 2021·Journal of Neuroscience Methods·Crystian I MassengillHaining Zhong

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