Advancing imaging tools to study the 3D nuclear organization

DNA spatial organization is supposed to play a key role in nuclear functions. This internship, in line with the global aims of the LOCCO team, therefore investigates this organization in 3D. Two complementary approaches to image this nuclear organization were carried out in this study. On the one hand, a 3D super-resolution (STORM) experiment would allow to image directly and precisely the nucleus and its components, with the high lateral and axial resolutions that STORM and MFM set-up together allow to reach. However, to properly carry out this study, a drift correction system had to be implemented. This drift correction system was therefore developed and implemented on the MFM set-up. Its performance was tested for lateral and axial precision, and a physical example of drift correction was performed in post-treatment. Each time, the desired precision of 5 nm (axially and laterally) was obtained. For real time drift correction, the speed of the whole process remains to be improved, essentially through the camera acquisition time. MatLab compiler to call the different functions in C-language was thus tested, but the final implementation of the camera’s image acquisition in C was not profitable in terms of complexity, compared to its direct implementation in MatLab. On the other hand, we studied the micro-rheology of the cell nucleus. Fluorescent nano-beads were injected with a micro-tip directly in U2OS cell nucleus. With the passivation of those beads we were able to show that they indeed diffuse more in the nucleus (despite their larger size) compared to non-passivated beads. This behavior, as for the reduced trajectory length of non-passivated beads compared to passivated ones, was attributed to the passivation process that results in less DNA/beads interactions. The visualization of the detections in 3D and their projection on one plane in 2D showed well excluded zones, thus regions of high concentration of DNA. We therefore managed to make, by contrast, an image of the fluid part of the nucleus.