Fluorescence Correlation and Cross-Correlation Spectroscopy in Zebrafish.

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.