Department of Infectious Diseases, King's College London, Faculty of Life Sciences & Medicine, London, UK.
Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.
Nat Commun. 2023 Aug 17;14(1):4994. doi: 10.1038/s41467-023-40731-x.
Simultaneous imaging of nine fluorescent proteins is demonstrated in a single acquisition using fluorescence lifetime imaging microscopy combined with pulsed interleaved excitation of three laser lines. Multicolor imaging employing genetically encodable fluorescent proteins permits spatio-temporal live cell imaging of multiple cues. Here, we show that multicolor lifetime imaging allows visualization of quadruple labelled human immunodeficiency viruses on host cells that in turn are also labelled with genetically encodable fluorescent proteins. This strategy permits to simultaneously visualize different sub-cellular organelles (mitochondria, cytoskeleton, and nucleus) during the process of virus entry with the potential of imaging up to nine different spectral channels in living cells.
本文展示了如何在单个采集过程中，利用荧光寿命成像显微镜结合三条激光线的脉冲交错激发，同时对 9 种荧光蛋白进行成像。采用遗传编码的荧光蛋白进行多色成像，可以对多个线索进行时空活细胞成像。在这里，我们表明，多色寿命成像可以可视化标记有遗传编码荧光蛋白的宿主细胞上的四重标记的人类免疫缺陷病毒。这种策略允许在病毒进入过程中同时可视化不同的亚细胞细胞器（线粒体、细胞骨架和细胞核），并有可能在活细胞中对多达 9 个不同的光谱通道进行成像。
