Linear Behavior of the Phase Lifetime in Frequency-Domain Fluorescence Lifetime Imaging of FRET Constructs
| Autor: | Marina M. Cararo-Lopes, James Jiang, Nada N. Boustany, Bonnie L. Firestein, Audrey Menaesse, Marina A. Ayad, Timothy Mahon, Daniel Sumetsky, Mihir V. Patel |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Fluorescence-lifetime imaging microscopy
mTFP1 Microscope QC1-999 Materials Science (miscellaneous) fluorescence resonance energy transfer Phase (waves) Biophysics General Physics and Astronomy frequency domain 01 natural sciences law.invention 010309 optics 03 medical and health sciences law imaging microscopy 0103 physical sciences fluorescence lifetime Physical and Theoretical Chemistry Mathematical Physics 030304 developmental biology Physics 0303 health sciences Fluorescence Acceptor live cell assays Förster resonance energy transfer Modulation Frequency domain Atomic physics |
| Zdroj: | Frontiers in Physics, Vol 9 (2021) |
| ISSN: | 2296-424X |
| DOI: | 10.3389/fphy.2021.648016 |
| Popis: | We utilize a cost-effective frequency-domain fluorescence lifetime imaging microscope to measure the phase lifetime of mTFP1 in mTFP1-mVenus fluorescence resonance energy transfer (FRET) constructs relevant to the VinTS molecular tension probe. Our data were collected at 15 modulation frequencies ω/2π selected between 14 and 70 MHz. The lifetime of mTFP1 was τD = 3.11 ± 0.02 ns in the absence of acceptor. For modulation frequencies, ω, such that (ω · τD) < 1.1, the phase lifetime of mTFP1in the presence of acceptor (mVenus), τϕDA, was directly related to the amplitude-weighted lifetime τaveDA inferred from the known FRET efficiency (EFRETtrue) of the constructs. A linear fit to a plot of (ω·τϕDA) vs. (ω·τaveDA) yielded a slope of 0.79 ± 0.05 and intercept of 0.095 ± 0.029 (R2 = 0.952). Thus, our results suggest that a linear relationship exists between the apparent EFRETapp based on the measured phase lifetime and EFRETtrue for frequencies such that (ω · τD) < 1.1. We had previously reported a similar relationship between EFRETapp and EFRETtrue at 42 MHz. Our current results provide additional evidence in support of this observation, but further investigation is still required to fully characterize these results. A direct relationship between τϕDAand τaveDA has the potential to simplify significantly data acquisition and interpretation in fluorescence lifetime measurements of FRET constructs. |
| Databáze: | OpenAIRE |
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