title: Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms.
abstract: 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.
authors.affiliation: German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Jan W
authors.initials: JW
authors.lastname: Krieger
authors.affiliation: Department of Chemistry, National University of Singapore, Singapore.|National University of Singapore (NUS) Centre for BioImaging Sciences, National University of Singapore, Singapore.|Mechanobiology Institute, National University of Singapore, Singapore.
authors.forename: Anand P
authors.initials: AP
authors.lastname: Singh
authors.affiliation: Department of Chemistry, National University of Singapore, Singapore.|National University of Singapore (NUS) Centre for BioImaging Sciences, National University of Singapore, Singapore.|Department of Biological Sciences, National University of Singapore, Singapore.
authors.forename: Nirmalya
authors.initials: N
authors.lastname: Bag
authors.affiliation: Interdisciplinary Center for Scientific Computing, University of Heidelberg, Heidelberg, Germany.
authors.forename: Christoph S
authors.initials: CS
authors.lastname: Garbe
authors.affiliation: Mechanobiology Institute, National University of Singapore, Singapore.|Department of Biological Sciences, National University of Singapore, Singapore.|Institute for Molecular and Cell Biology, A*Star, Singapore.
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Saunders
authors.affiliation: German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Jörg
authors.identifier: http://orcid.org/0000-0001-8600-0666
authors.initials: J
authors.lastname: Langowski
authors.affiliation: Department of Chemistry, National University of Singapore, Singapore.|National University of Singapore (NUS) Centre for BioImaging Sciences, National University of Singapore, Singapore.|Department of Biological Sciences, National University of Singapore, Singapore.
authors.forename: Thorsten
authors.identifier: http://orcid.org/0000-0002-0148-4321
authors.initials: T
authors.lastname: Wohland
mesh_terms: D000465:Algorithms; D000818:Animals; D002470:Cell Survival; D004330:Drosophila / Q000196:embryology; D004867:Equipment Design; D061848:Optical Imaging / Q000295:instrumentation* / Q000379:methods; D012984:Software; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods; D015027:Zebrafish / Q000196:embryology