Comparison of spectral FRET microscopy approaches for single-cell analysis

Joshua Deal; Naga Annamdevula; Donald John Pleshinger; John Robert Griswold; Aliyah Odom; Alia Tayara; Malvika Lall; Craig Browning; Marina Parker; Thomas C. Rich; Silas J. Leavesley

doi:10.1117/12.2546308

17 February 2020 Comparison of spectral FRET microscopy approaches for single-cell analysis

Joshua Deal, Naga Annamdevula, Donald John Pleshinger, John Robert Griswold, Aliyah Odom, Alia Tayara, Malvika Lall, Craig Browning, Marina Parker, Thomas C. Rich, Silas J. Leavesley

Author Affiliations +

Proceedings Volume 11243, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVIII; 112430Y (2020) https://doi.org/10.1117/12.2546308
Event: SPIE BiOS, 2020, San Francisco, California, United States

Abstract

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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Joshua Deal, Naga Annamdevula, Donald John Pleshinger, John Robert Griswold, Aliyah Odom, Alia Tayara, Malvika Lall, Craig Browning, Marina Parker, Thomas C. Rich, and Silas J. Leavesley "Comparison of spectral FRET microscopy approaches for single-cell analysis", Proc. SPIE 11243, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVIII, 112430Y (17 February 2020); https://doi.org/10.1117/12.2546308

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