German Cancer Research Center (DKFZ), Heidelberg, Germany.
Department of Chemistry, National University of Singapore, Singapore.
Nat Protoc. 2015 Dec;10(12):1948-74. doi: 10.1038/nprot.2015.100. Epub 2015 Nov 5.
Single-plane illumination (SPIM) or total internal reflection fluorescence (TIRF) microscopes can be combined with fast and single-molecule-sensitive cameras to allow spatially resolved fluorescence (cross-) correlation spectroscopy (FCS or FCCS, hereafter referred to FCS/FCCS). This creates a powerful quantitative bioimaging tool that can generate spatially resolved mobility and interaction maps with hundreds to thousands of pixels per sample. These massively parallel imaging schemes also cause less photodamage than conventional single-point confocal microscopy-based FCS/FCCS. Here we provide guidelines for imaging FCS/FCCS measurements on commercial and custom-built microscopes (including sample preparation, setup calibration, data acquisition and evaluation), as well as anticipated results for a variety of in vitro and in vivo samples. For a skilled user of an available SPIM or TIRF setup, sample preparation, microscope alignment, data acquisition and data fitting, as described in this protocol, will take ∼1 d, depending on the sample and the mode of imaging.
单平面照明（SPIM）或全内反射荧光（TIRF）显微镜可与快速和单分子敏感的相机相结合，以允许空间分辨荧光（交叉）相关光谱学（FCS 或 FCCS，以下简称 FCS/FCCS）。这创建了一种强大的定量生物成像工具，可生成具有数百到数千个像素/样品的空间分辨迁移率和相互作用图。这些大规模并行成像方案比传统的基于单点共焦显微镜的 FCS/FCCS 造成的光损伤更小。在这里，我们为商业和定制显微镜上的 FCS/FCCS 测量提供了指南（包括样品制备、设置校准、数据采集和评估），以及各种体外和体内样品的预期结果。对于现有 SPIM 或 TIRF 设备的熟练使用者，根据样品和成像方式的不同，本协议中描述的样品制备、显微镜对准、数据采集和数据拟合将需要约 1 天的时间。
