Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms

被引：147

作者：

Krieger, Jan W. ^{[1
]}

Singh, Anand P. ^{[2
,3
,4
]}

Bag, Nirmalya ^{[2
,3
,5
]}

Garbe, Christoph S. ^{[6
]}

Saunders, Timothy E. ^{[4
,5
,7
]}

Langowski, Joerg ^{[1
]}

Wohland, Thorsten ^{[2
,3
,5
]}

机构：

[1] German Canc Res Ctr, Heidelberg, Germany

[2] Natl Univ Singapore, Dept Chem, Singapore, Singapore

[3] Natl Univ Singapore, Ctr BioImaging Sci, Singapore 117548, Singapore

[4] Natl Univ Singapore, Mechanobiol Inst, Singapore 117548, Singapore

[5] Natl Univ Singapore, Dept Biol Sci, Singapore 117548, Singapore

[6] Heidelberg Univ, Interdisciplinary Ctr Sci Comp, Heidelberg, Germany

[7] ASTAR, Inst Mol & Cell Biol, Singapore, Singapore

来源：

NATURE PROTOCOLS | 2015年 / 10卷 / 12期

基金：

新加坡国家研究基金会;

关键词：

PLANE ILLUMINATION MICROSCOPY; COUPLED-DEVICE CAMERA; LIVING CELLS; MEMBRANE ORGANIZATION; CONFOCAL MICROSCOPE; BAYESIAN-APPROACH; PLASMA-MEMBRANE; DIFFUSION; DYNAMICS; RESOLUTION;

D O I：

10.1038/nprot.2015.100

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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 similar to 1 d, depending on the sample and the mode of imaging.

引用

页码：1948 / 1974

页数：27

共 80 条

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