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Fluorescence Correlation and Cross-Correlation Spectroscopy in Zebrafish

  • Xue Wen Ng
  • Karuna Sampath
  • Thorsten Wohland
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1863)

Abstract

There has been increasing interest in biophysical studies on live organisms to gain better insights into physiologically relevant biological events at the molecular level. Zebrafish (Danio rerio) is a viable vertebrate model to study such events due to its genetic and evolutionary similarities to humans, amenability to less invasive fluorescence techniques owing to its transparency and well-characterized genetic manipulation techniques. Fluorescence techniques used to probe biomolecular dynamics and interactions of molecules in live zebrafish embryos are therefore highly sought-after to bridge molecular and developmental events. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are two robust techniques that provide molecular level information on dynamics and interactions respectively. Here, we detail the steps for applying confocal FCS and FCCS, in particular single-wavelength FCCS (SW-FCCS), in live zebrafish embryos, beginning with sample preparation, instrumentation, calibration, and measurements on the FCS/FCCS instrument and ending with data analysis.

Key words

FCS FCCS Single molecule Biomolecular interactions Dissociation constant and affinity 

Notes

Acknowledgment

X.W.N. is supported by the NUS graduate research scholarship. T.W. acknowledges funding by the Ministry of Education of Singapore (grant number MOE2016-T3-1-005). Work in the laboratory of K.S. is supported by Warwick Medical School and the BBSRC. K.S. thanks Andreas Zaucker and Scott Clarke for the image of the experimental setup for mounting zebrafish embryos.

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Xue Wen Ng
    • 1
  • Karuna Sampath
    • 2
  • Thorsten Wohland
    • 1
    • 3
  1. 1.Department of Chemistry and Centre for Bioimaging SciencesNational University of SingaporeSingaporeSingapore
  2. 2.Division of Biomedical Sciences, Warwick Medical SchoolUniversity of WarwickCoventryUK
  3. 3.Department of Biological SciencesNational University of SingaporeSingaporeSingapore

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