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Förster resonance energy transfer (FRET) is a valuable tool for measuring molecular distances and the effects of biological processes such as cyclic nucleotide messenger signaling and protein localization. Most FRET techniques require two fluorescent proteins with overlapping excitation/emission spectral pairing to maximize detection sensitivity and FRET efficiency. FRET microscopy often utilizes differing peak intensities of the selected fluorophores measured through different optical filter sets to estimate the FRET index or efficiency. Microscopy platforms used to make these measurements include wide-field, laser scanning confocal, and fluorescence lifetime imaging. Each platform has associated advantages and disadvantages, such as speed, sensitivity, specificity, out-of-focus fluorescence, and Zresolution. In this study, we report comparisons among multiple microscopy and spectral filtering platforms such as standard 2-filter FRET, emission-scanning hyperspectral imaging, and excitation-scanning hyperspectral imaging. Samples of human embryonic kidney (HEK293) cells were grown on laminin-coated 28 mm round gridded glass coverslips (10816, Ibidi, Fitchburg, Wisconsin) and transfected with adenovirus encoding a cAMP-sensing FRET probe composed of a FRET donor (Turquoise) and acceptor (Venus). Additionally, 3 FRET “controls” with fixed linker lengths between Turquoise and Venus proteins were used for inter-platform validation. Grid locations were logged, recorded with light micrographs, and used to ensure that whole-cell FRET was compared on a cell-by-cell basis among the different microscopy platforms. FRET efficiencies were also calculated and compared for each method. Preliminary results indicate that hyperspectral methods increase the signal-to-noise ratio compared to a standard 2-filter approach.
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Simplified Instrument Calibration for Wide‐Field Fluorescence Resonance Energy Transfer (FRET) Measured by the Sensitized Emission Method
Abstract Fӧrster (or fluorescence) resonance energy transfer (FRET) is a quantifiable energy transfer in which a donor fluorophore nonradiatively transfers its excitation energy to an acceptor fluorophore. A change in FRET efficiency indicates a change of proximity and environment of these fluorophores, which enables the study of intermolecular interactions. Measurement of FRET efficiency using the sensitized emission method requires a donor–acceptor calibrated system. One of these calibration factors named theGfactor, which depends on instrument parameters related to the donor and acceptor measurement channels and on the fluorophores quantum efficiencies, can be determined in several different ways and allows for conversion of the raw donor and acceptor emission signals to FRET efficiency. However, the calculated value of the G factor from experimental data can fluctuate significantly depending on the chosen experimental method and the size of the sample. In this technical note, we extend the results of Gates et al. (Cytometry Part A 95A (2018) 201–213) by refining the calibration method used for calibration of FRET from image pixel data. Instead of using the pixel histograms of two constructs with high and low FRET efficiency to determine theGfactor, we use pixel histogram data from one construct of known efficiency. We validate this method by determining theGfactor with the same constructs developed and used by Gates et al. and comparing the results from the two approaches. While the two approaches are equivalent theoretically, we demonstrate that the use of a single construct with known efficiency provides a more precise experimental measurement of theGfactor that can be attained by collecting a smaller number of images. © 2020 International Society for Advancement of Cytometry
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- Award ID(s):
- 1825433
- PAR ID:
- 10185470
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Cytometry Part A
- Volume:
- 99
- Issue:
- 4
- ISSN:
- 1552-4922
- Page Range / eLocation ID:
- p. 407-416
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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