In vivo fluorescence correlation and cross-correlation spectroscopy

Abstract

In this manuscript, we describe the application of Fluorescence Correla- tion Spectroscopy (FCS), Fluorescence Cross-Correlation Spectroscopy (FCCS), and scanning FCS (sFCS) to two in vivo systems. In the first part, we describe the appli- cation of two-photon standard and scanning FCS in Caenorhabditis elegans embryos. The differentiation of a single fertilized egg into a complex organism in C. ele- gans is regulated by a number of protein-dependent processes. The oocyte divides asymmetrically into two daughter cells of different developmental fate. Two of the involved proteins, PAR-2 and NMY-2, are studied. The second investigated system is the mechanism of RNA interference in human cells. An EGFP based cell line that allows to study the dynamics and localization of the RNA-induced silencing complex (RISC) with FCS in vivo is created, which has so far been inaccessible with other experimental methods. Furthermore, Fluorescence Cross-Correlation Spectroscopy is employed to highlight the asymmetric incorporation of labeled siRNAs into RISC.