Nanoscale investigation of chromatin organization by structured illumination microscopy

This thesis work aims to present a novel approach to reconstruct Structured Illumination Microscopy, SIM, raw data and to analyze SIM reconstructed images. These new approaches will be demonstrated in the study of chromatin organization. The dissertation will be articulated as follows: Chapter 1 provides an introduction to chromatin nanoscale organization and optical fluorescence microscopy, which is one of the main tools involved in life sciences studies. Indeed, optical microscopy allowed investigating, with high specificity and sensitivity, living samples such as cells, and even tissues. The reader will be presented with a summary on the fluorescence optical microscopy and on the super-resolution, SR, techniques available today including SIM, which is the microscope used in this thesis work. In Chapter 2 the focus is on the introduction of a new reconstruction tool for specific SR-SIM microscopy powered by the Separation of Photons by Lifetime Tuning, SPLIT, method. The introduction of the concept, applied in other works to different SR techniques, will be followed by the practical implementation of the method on the SIM microscope. Then, the applicability of the technique, which we called SPLIT-SIM, will be demonstrated on several different samples. Indeed, it will be used on Simulated data, on test experimental beads, on biological samples both in one and two-color staining. In Chapter 3 the focus will move on the coupling of SIM reconstructed data to colocalization analysis. In particular, for the first time, SIM was coupled to Image Cross-Correlation Spectroscopy, ICCS, in the study of two-color images of a model sample. DNA origami-based structures were chosen as a model sample with precise distances allowing for evaluation of the analysis results. Moreover, all the images analyzed by the pixel-based technique, SIM-ICCS, were analyzed also with an object-based technique as a comparison to evaluate which could be the best choice in SIM acquisitions. Finally, Chapter 4 will be focused on the application of the analysis, performed in chapter 3, to two-color SIM images of nuclear structure. The analysis will be performed on ‘positive control’ in which the target structures will be colocalized and on a negative control in which the structured are spatially segregated within the nucleus. Both object-based and pixel-based analysis will be able to extract coherent results thus showing how SIM-ICCS can become an interesting and useful tool to analyze SIM multicolor acquisitions.