Leray Aymeric, Riquet Franck B, Richard Elodie, Spriet Corentin, Trinel Dave, Héliot Laurent
Interdisciplinary Research Institute, Science and Technology University of Lille, 1 rue du Prof. Calmette, Lille Cedex, France.
Microsc Res Tech. 2009 May;72(5):371-9. doi: 10.1002/jemt.20665.
Frequency-domain fluorescence lifetime imaging microscopy (FLIM) has become a commonly used technique to measure lifetimes in biological systems. However, lifetime measurements are strongly dependent on numerous experimental parameters. Here, we describe a complete calibration and characterization of a FLIM system and suggest parameter optimization for minimizing measurement errors during acquisition. We used standard fluorescent molecules and reference biological samples, exhibiting both single and multiple lifetime components, to calibrate and evaluate our frequency domain FLIM system. We identify several sources of lifetime precision degradation that may occur in FLIM measurements. Following a rigorous calibration of the system and a careful optimization of the acquisition parameters, we demonstrate fluorescence lifetime measurements accuracy and reliability. In addition, we show its potential on living cells by visualizing FRET in CHO cells. The proposed calibration and optimization protocol is suitable for the measurement of multiple lifetime components sample and is applicable to any frequency domain FLIM system. Using this method on our FLIM microscope enabled us to obtain the best fluorescence lifetime precision accessible with such a system.
频域荧光寿命成像显微镜（FLIM）已成为测量生物系统中荧光寿命的常用技术。然而，寿命测量强烈依赖于众多实验参数。在此，我们描述了一个FLIM系统的完整校准和特性分析，并提出了参数优化方法，以在采集过程中最小化测量误差。我们使用标准荧光分子和具有单寿命及多寿命成分的参考生物样品，来校准和评估我们的频域FLIM系统。我们识别出了FLIM测量中可能出现的几个导致寿命精度下降的来源。在对系统进行严格校准并仔细优化采集参数后，我们展示了荧光寿命测量的准确性和可靠性。此外，我们通过在CHO细胞中可视化荧光共振能量转移（FRET）展示了其在活细胞中的潜力。所提出的校准和优化方案适用于测量具有多寿命成分的样品，并且适用于任何频域FLIM系统。在我们的FLIM显微镜上使用这种方法使我们能够获得该系统所能达到的最佳荧光寿命精度。
