KEYWORDS: Fluorescence resonance energy transfer, Fluorescence lifetime imaging, Control systems, Luminescence, Molecular interactions, Microscopy, Resonance energy transfer, Proteins, Imaging systems, Breast cancer
Accurate, unambiguous detection of molecular interactions in living cells via measurements of Förster (or fluorescence) resonance energy transfer (FRET) events is experimentally challenging. We develop and apply a physiological fluorescence lifetime imaging microscopy (physiological FLIM) system to significantly improve FRET detection in living cells. Multiple positive and negative cellular controls are implemented to validate the experimental method developed. FLIM measurement techniques were found to remove fluorescence intensity-based artifacts, resulting in a seven-fold improvement in fluorescence measurement precision. The addition of cellular environmental controls, including both temperature and CO2 stabilization, for physiological FLIM eliminates nonspecific FRET in the live-cell system studied. Overall, only physiological FLIM results in statistically significant results that clearly indicated the presence of specific molecular interactions in the live-cell system. This approach can be applied generally to improve the accuracy and precision of FRET measurements in living cells.
Unambiguous FRET detection in living cells is often challenging. Here we describe
how the advantages of fluorescence lifetime sensing with FLIM, fluorophore selection, and critical
environmental controls provide better FRET detection.
In contrast to intensity-based fluorescence microscopy, fluorescence lifetime imaging microscopy (FLIM) bases image contrast on fluorophore excited-state lifetime. This technique is sensitive to the fluorophore's local environment (temperature, ion concentration, dissolved gas concentration, and molecular associations), while being independent of factors impacting fluorescence intensity (fluorophore concentration, photobleaching, scattering, and absorption). We present design features of a novel UV-visible-NIR wide-field time-domain FLIM system with optical sectioning (10 μm), high temporal discrimination (50 ps), and large temporal dynamic range (750 ps - 1 μs), and apply the system to probe cellular metabolic function and detect molecular activity in vivo.