Padilla-Parra Sergi, Auduge Nicolas, Coppey-Moisan Maite, Tramier Marc
Department of Pediatrics, Infectious Diseases, Emory University, 2015 Uppergate Dr, Atlanta, GA, 30322, USA.
Institut Jacques Monod, UMR 7592, CNRS, Université Paris-Diderot, 75013, Paris, France.
Biophys Rev. 2011 Jun;3(2):63-70. doi: 10.1007/s12551-011-0047-6. Epub 2011 May 17.
New imaging methodologies in quantitative fluorescence microscopy and nanoscopy have been developed in the last few years and are beginning to be extensively applied to biological problems, such as the localization and quantification of protein interactions. Fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) is currently employed not only in biophysics or chemistry but also in bio-medicine, thanks to new advancements in technology and also new developments in data treatment. FRET-FLIM can be a very useful tool to ascertain protein interactions occurring in single living cells. In this review, we stress the importance of increasing the acquisition speed when working in vivo employing Time-Domain FLIM. The development of the new mathematical-based non-fitting methods allows the determining of the fraction of interacting donor without the requirement of high count statistics, and thus allows the performing of high speed acquisitions in FRET-FLIM to still be quantitative.
近年来，定量荧光显微镜和纳米显微镜领域开发了新的成像方法，并开始广泛应用于生物学问题，如蛋白质相互作用的定位和定量。通过荧光寿命成像显微镜（FLIM）检测的荧光共振能量转移（FRET），由于技术的新进展以及数据处理的新发展，目前不仅应用于生物物理学或化学领域，还应用于生物医学领域。FRET-FLIM可以成为确定单个活细胞中发生的蛋白质相互作用的非常有用的工具。在这篇综述中，我们强调了在体内使用时域FLIM时提高采集速度的重要性。基于新数学的非拟合方法的发展使得无需高计数统计就能确定相互作用供体的比例，从而使得在FRET-FLIM中进行高速采集仍能保持定量。
