FRET efficiency measurement in a molecular tension probe with a low-cost frequency-domain fluorescence lifetime imaging microscope.
J Biomed Opt
; 24(12): 1-11, 2019 12.
Article
en En
| MEDLINE
| ID: mdl-31884745
ABSTRACT
We demonstrate the possibility of measuring FRET efficiency with a low-cost frequency-domain fluorescence lifetime imaging microscope (FD-FLIM). The system utilizes single-frequency-modulated excitation, which enables the use of cost-effective laser sources and electronics, simplification of data acquisition and analysis, and a dual-channel detection capability. Following calibration with coumarin 6, we measured the apparent donor lifetime in mTFP1-mVenus FRET standards expressed in living cells. We evaluated the system's sensitivity by differentiating the short and long lifetimes of mTFP1 corresponding to the known standards' high and low FRET efficiency, respectively. Furthermore, we show that the lifetime of the vinculin tension sensor, VinTS, at focal adhesions (2.30 ± 0.16 ns) is significantly (p < 10 - 6) longer than the lifetime of the unloaded TSMod probe (2.02 ± 0.16 ns). The pixel dwell time was 6.8 µs for samples expressing the FRET standards, with signal typically an order of magnitude higher than VinTS. The apparent FRET efficiency (Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Sondas Moleculares
/
Transferencia Resonante de Energía de Fluorescencia
/
Microscopía Fluorescente
Tipo de estudio:
Health_economic_evaluation
Límite:
Animals
Idioma:
En
Revista:
J Biomed Opt
Asunto de la revista:
ENGENHARIA BIOMEDICA
/
OFTALMOLOGIA
Año:
2019
Tipo del documento:
Article
País de afiliación:
Estados Unidos