Advanced Fluorescence Correlation Spectroscopy for The Study of Nuclear Dynamics

Chromatin is a macromolecule mainly composed by DNA and histones. Chromatin not only has the function of compacting the DNA in order to make it fit into the nucleus, but also plays an active role in the regulation of all biological processes using DNA as template in eukaryotes, such as transcription, DNA replication and DNA repair. The architectural organization of chromatin can play an important role in gene expression by regulating the diffusion of molecules via binding interactions and molecular crowding. In this respect, understanding how variations of chromatin architecture affect nuclear dynamics is of fundamental importance. Among the techniques that are able to probe the nuclear interior, fluorescence microscopy is sensitive, specific and does not require strong manipulation of the sample, allowing also measurements to be performed in living samples. But not all the fluorescence microscopy techniques have the adequate temporal resolution to follow the dynamics of the nuclear environment. Fluorescence Correlation Spectroscopy (FCS) is able to probe chromatin accessibility and molecular crowding in live cells by measuring fast diffusion of molecules in the range between microseconds and milliseconds. In particular, single point FCS (spFCS) has a high temporal resolution but lacks spatial information. Conversely, spatially-resolved methods, like scanning FCS, have in general limited temporal resolution. The aim of this thesis is to overcome these limitations through the Intensity Sorted FCS technique. This technique is able to probe fast molecular diffusion in nuclear environment distinguishing between different regions of the space at a high temporal resolution. This achievement is due to the idea of dividing a whole FCS measurement, performed through a slow scan of the beams, into short temporal segment: each segment is analyzed and for each one an ACF is calculated. Then the ACFs are sorted into two populations basing on the intensity of a reference trace, that specifically distinguish the two nuclear regions probed: for each set of ACFs corresponding to a region, the average ACF is calculated. In this way it is possible to retrieve statistically robust information about diffusion in two distinct nuclear regions in the same measurement.