MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK.
Kennedy Institute for Rheumatology, University of Oxford, Oxford, OX3 7LF, UK.
Small Methods. 2022 Jun;6(6):e2200149. doi: 10.1002/smtd.202200149. Epub 2022 Mar 28.
Quantifying molecular dynamics within the context of complex cellular morphologies is essential toward understanding the inner workings and function of cells. Fluorescence recovery after photobleaching (FRAP) is one of the most broadly applied techniques to measure the reaction diffusion dynamics of molecules in living cells. FRAP measurements typically restrict themselves to single-plane image acquisition within a subcellular-sized region of interest due to the limited temporal resolution and undesirable photobleaching induced by 3D fluorescence confocal or widefield microscopy. Here, an experimental and computational pipeline combining lattice light sheet microscopy, FRAP, and numerical simulations, offering rapid and minimally invasive quantification of molecular dynamics with respect to 3D cell morphology is presented. Having the opportunity to accurately measure and interpret the dynamics of molecules in 3D with respect to cell morphology has the potential to reveal unprecedented insights into the function of living cells.
在复杂的细胞形态学背景下定量分子动力学对于理解细胞的内部运作和功能至关重要。光漂白后荧光恢复（FRAP）是测量活细胞中分子反应扩散动力学最广泛应用的技术之一。由于 3D 荧光共聚焦或宽场显微镜的时间分辨率有限和不希望的光漂白，FRAP 测量通常仅限于亚细胞大小的感兴趣区域的单平面图像采集。在这里，提出了一种结合晶格光片显微镜、FRAP 和数值模拟的实验和计算管道，可快速、微创地定量 3D 细胞形态学中分子动力学。有机会准确测量和解释 3D 中分子相对于细胞形态的动力学，有可能揭示对活细胞功能的前所未有的见解。
