FRET Efficiency Measurement with a Low-Cost Frequency Domain Fluorescence Lifetime Imaging Microscope

2020 ◽  
Author(s):  
John-Paul Dumas ◽  
James Y. Jiang ◽  
Evan M. Gates ◽  
Brenton D. Hoffman ◽  
Mark C. Pierce ◽  
...  
Keyword(s):  
Frequency Domain ◽  
Fluorescence Lifetime ◽  
Low Cost ◽  
Efficiency Measurement ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Fret Efficiency

scholarly journals FRET efficiency measurement in a molecular tension probe with a low-cost frequency-domain fluorescence lifetime imaging microscope

2019 ◽  
Vol 24 (12) ◽  
pp. 1 ◽  
Author(s):  
John-Paul Dumas ◽  
James Y. Jiang ◽  
Evan M. Gates ◽  
Brenton D. Hoffman ◽  
Mark C. Pierce ◽  
...  
Keyword(s):  
Frequency Domain ◽  
Fluorescence Lifetime ◽  
Low Cost ◽  
Efficiency Measurement ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Fret Efficiency

scholarly journals Linear Behavior of the Phase Lifetime in Frequency-Domain Fluorescence Lifetime Imaging of FRET Constructs

2021 ◽  
Vol 9 ◽  
Author(s):  
Daniel Sumetsky ◽  
James Y. Jiang ◽  
Marina A. Ayad ◽  
Timothy Mahon ◽  
Audrey Menaesse ◽  
...  
Keyword(s):  
Frequency Domain ◽  
Fluorescence Lifetime ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cost Effective ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Linear Behavior ◽  
Fret Efficiency ◽  
Linear Fit

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.

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

2009 ◽  
Vol 72 (5) ◽  
pp. 371-379 ◽  
Author(s):  
Aymeric Leray ◽  
Franck B. Riquet ◽  
Elodie Richard ◽  
Corentin Spriet ◽  
Dave Trinel ◽  
...  
Keyword(s):  
Frequency Domain ◽  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Optimized Protocol
Sign in / Sign up

Export Citation Format

Share Document