J Biomed Opt. 2009 Nov-Dec;14(6):060502. doi: 10.1117/1.3257254.
Accurate, unambiguous detection of molecular interactions in living cells via measurements of Forster (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 CO(2) 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.
通过测量Förster（或荧光）共振能量转移（FRET）事件，在活细胞中准确、明确地检测分子相互作用在实验上具有挑战性。我们开发并应用了一种生理荧光寿命成像显微镜（physiological FLIM）系统，以显著提高活细胞中的 FRET 检测水平。实施了多个正、负细胞对照，以验证所开发的实验方法。FLIM 测量技术被发现可以消除基于荧光强度的伪影，从而将荧光测量精度提高了七倍。添加细胞环境控制，包括温度和 CO2 稳定，用于生理 FLIM，可以消除活细胞系统中无特异性的 FRET。总的来说，只有生理 FLIM 才能得到具有统计学意义的结果，清楚地表明在活细胞系统中存在特定的分子相互作用。这种方法可以广泛应用于提高活细胞中 FRET 测量的准确性和精度。
