PUBLICATION

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

Authors
Krieger, J.W., Singh, A.P., Bag, N., Garbe, C.S., Saunders, T.E., Langowski, J., Wohland, T.
ID
ZDB-PUB-170214-84
Date
2015
Source
Nature Protocols   10: 1948-74 (Journal)
Registered Authors
Saunders, Timothy Edward
Keywords
Fluorescence imaging, Fluorescence spectroscopy, Molecular biophysics, Single-molecule biophysics
MeSH Terms
  • Algorithms
  • Animals
  • Cell Survival
  • Drosophila/embryology
  • Equipment Design
  • Optical Imaging/instrumentation*
  • Optical Imaging/methods
  • Software
  • Spectrometry, Fluorescence/instrumentation*
  • Spectrometry, Fluorescence/methods
  • Zebrafish/embryology
PubMed
26540588 Full text @ Nat. Protoc.
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.
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping