Razvag Yair, Drori Paz, Klemfner Shalhevet, Meshorer Eran, Lerner Eitan
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Faculty of Mathematics & Science, The Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem, Israel.
Department of Genetics, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem, Israel.
Biophys J. 2025 Feb 19. doi: 10.1016/j.bpj.2025.02.017.
Super-resolution light microscopy techniques facilitate the observation of nm-sized biomolecules, which are 1-2 orders of magnitude smaller than the diffraction limit of light. Using super-resolution microscopy techniques, it is possible to observe fluorescence from two biomolecules in close proximity; however, not necessarily in direct interaction. Using FRETsael, we localize biomolecular interactions exhibiting FRET with nanometer accuracy, from two-color fluorescence lifetime imaging data. The concepts of FRETsael were tested first against simulations, in which the recovered localization accuracy is 20-30 nm for true-positive detections of FRET pairs. Further analysis of the simulation results reports the conditions in which true-positive rates are maximal. We then show the capabilities of FRETsael on simulated samples of actin-vinculin and ER-ribosome interactions, as well as experimental samples of actin-myosin two-color confocal imaging. Overall, the FRETsael approach paves the way toward studying biomolecular interactions with improved spatial resolution from laser scanning confocal two-color fluorescence lifetime imaging.
超分辨率光学显微镜技术有助于观察纳米尺寸的生物分子，这些生物分子比光的衍射极限小1 - 2个数量级。使用超分辨率显微镜技术，可以观察到两个相邻生物分子发出的荧光；然而，它们不一定存在直接相互作用。利用FRETsael，我们可以从双色荧光寿命成像数据中以纳米精度定位表现出荧光共振能量转移（FRET）的生物分子相互作用。FRETsael的概念首先通过模拟进行测试，在模拟中，对于FRET对的真阳性检测，恢复的定位精度为20 - 30纳米。对模拟结果的进一步分析报告了真阳性率最高的条件。然后，我们展示了FRETsael在肌动蛋白 - 纽蛋白和内质网 - 核糖体相互作用的模拟样本以及肌动蛋白 - 肌球蛋白双色共聚焦成像实验样本上的能力。总体而言，FRETsael方法为从激光扫描共聚焦双色荧光寿命成像以更高空间分辨率研究生物分子相互作用铺平了道路。
