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 language | English (US) |
|---|---|
| Pages (from-to) | 2066-2080 |
| Number of pages | 15 |
| Journal | Nature Protocols |
| Volume | 11 |
| Issue number | 11 |
| DOIs | |
| State | Published - Nov 1 2016 |
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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 journal › Article
}
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.
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U2 - 10.1038/nprot.2016.121
DO - 10.1038/nprot.2016.121
M3 - Article
VL - 11
SP - 2066
EP - 2080
JO - Nature Protocols
JF - Nature Protocols
SN - 1754-2189
IS - 11
ER -