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

Nature Protocols volume 11, pages 2066–2080 (2016)Cite this article

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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.

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Figure 1: Schematic overview of the setup for measuring biosensor activity in a living animal.
Figure 2: Schematic workflow of the procedure and constructs described in Table 1.
Figure 3: 2PE spectral scan of the indicated FPs expressed individually in living HEK-293 cells.
Figure 4: FRET ratio images of HEK-293 cells expressing the indicated FRET standard probes.
Figure 5: Ratiometric FRET measurements from HEK-293 cells expressing the AKAR4.1 biosensor probe.
Figure 6: Use of TPLSM to measure the response of the AKAR4.1 biosensor to glucagon in hepatocytes in the intact mouse liver.

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Acknowledgements

This research was supported by the National Institutes of Health O'Brien Center for Advanced Renal Microscopic Analysis (NIH-NIDDK P30DK079312 to R.N.D. and K.W.D.). Microscopy studies were conducted at the Indiana Center for Biological Microscopy. The authors thank M. Kamocka and S. Winfree for their assistance in microscopy. This article is dedicated to the memory of M.W. Davidson.

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Authors and Affiliations

  1. Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis

    Richard N Day

  2. Indiana, USA

    Richard N Day

  3. Department of Medicine, Division of Nephrology, Indiana University Medical Center, Indianapolis

    Wen Tao & Kenneth W Dunn

  4. Indiana, USA

    Wen Tao & Kenneth W Dunn

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  1. Richard N Day
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R.N.D. and K.W.D. wrote the manuscript. W.T. conducted the experiments, and R.N.D and K.W.D. supervised the research.

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Correspondence to Richard N Day.

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Sequence archive: DNA sequence information for plasmids used in this protocol (PDF 123 kb)

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Day, R., Tao, W. & Dunn, K. A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy. Nat Protoc 11, 2066–2080 (2016). https://doi.org/10.1038/nprot.2016.121

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