Light sheet based fluorescence correlation and cross-correlation spectroscopy for quantitative measurements of bio-molecules in live cells

Abstract

Cellular processes occur over a wide range of spatial (nm - mm) and temporal scales (microsecond -min). However, most microscopy methods do not provide sufficient spatio-temporal resolution to cover this range. Therefore, I introduce here the combination of light sheet microscopy (single plane illumination microscopy - SPIM) and fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS). SPIM-FCS/SPIM-FCCS allows measuring concentration, diffusion, transport, and interactions maps in a true imaging mode with single molecule sensitivity. The method provides diffraction limited spatial resolution with sub-ms temporal resolution which is sufficient to quantify the dynamics of membrane, cytosolic and nuclear proteins in living cells and organisms. In this work I provide guidelines on the system setup, on the suitability of different cameras, on sample preparation and mounting and on the building and characterization of microscope setups, on the suitability of different cameras, on sample preparation and mounting and on the data analysis of this novel imaging FCS and FCCS methods and present applications to demonstrate its suitability for biophysical and biomedical studies.