Biophysical Analytics, Deutsches Rheumaforschungszentrum - a Leibniz Institute, Berlin, Germany.
Division of Genetics, Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany.
Methods Mol Biol. 2021;2350:145-156. doi: 10.1007/978-1-0716-1593-5_10.
Intravital two-photon microscopy enables monitoring of cellular dynamics and communication of complex systems, in genuine environment-the living organism. Particularly, its application in understanding the immune system brought unique insights into pathophysiologic processes in vivo. Here we present a method to achieve multiplexed dynamic intravital two-photon imaging by using a synergistic strategy combining a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an effective unmixing algorithm based on the calculation of spectral similarities with previously acquired fluorophore fingerprints. Our unmixing algorithm allows us to distinguish 7 fluorophore signals corresponding to various cellular and tissue compartments by using only four detector channels.
活体双光子显微镜能够在真实环境——活体生物中监测细胞动态和复杂系统的通讯。特别是，它在理解免疫系统方面的应用为体内病理生理过程带来了独特的见解。在这里，我们提出了一种通过结合使用荧光团发射光谱范围广、同时激发所有目标荧光团的波混频概念以及基于与先前获得的荧光指纹计算光谱相似性的有效解混算法的协同策略来实现多路复用动态活体双光子成像的方法。我们的解混算法仅使用四个探测器通道即可区分对应于各种细胞和组织隔室的 7 种荧光信号。
