Single Nitrogen-Vacancy Imaging in Nanodiamonds for Multimodal Sensing

Abstract

Overall this analysis highlights the capacity of super-resolved single particle trajectories analysis to provide new insights about the dynamics of cellular structures. 859-PlatChromatin Nanoscale Organization Investigated by FLIM-FRET and STED Superresolution Microscopy Chromatin organization plays an essential role in the regulation of gene activity, which involves the packaging of the genome into transcriptionally active and inactive sites. Chromatin remodeling is generated by the recruitment of nuclear enzymes for a variety of nuclear processes. For instance, chromatin architecture is highly reorganized during DDR (DNA damage response) to promote accurate repair of DNA lesions[1]. However, how the higher-order chromatin structures are formed and then behave in various cellular processes in live cells remains unclear.Here, we adopt a double strategy based on a novel FLIM-FRET assay and super-resolution microscopy to study the nanoscale chromatin organization in intact cells nuclei. The FRET assay is based on the staining of the nuclei with two DNA-binding dyes and the frequency-domain detection of FLIM. We show that the FRET level strongly depends on the relative concentration of the two fluorophores. We describe a method to correct the values of FRET efficiency and demonstrate that, with this correction, the FLIM-FRET assay can be used to quantify variations of nanoscale chromatin compaction in live cells. Super-resolution microscopy is used to investigate, in fixed cells, the nanoscale distribution of specific proteins, like poly(ADP-ribose) polymerase 1 (PARP1), implicated in various DNA repair pathways and in the maintenance of genomic stability [2]. In particular, we apply our recently developed method based on the modulation of the STED power [3]. We show that, by using the phasor approach, we obtain a significant improvement of spatial resolution. We use this double strategy for monitoring changes in nanoscale chromatin organization during the DDR.[1] Hauer HM, Gasser SM, Genes Dev (2017); [2] Chaudhuri AR, Nussenzweig A, Nature Reviews (2017); [3] Sarmento MJ et al, Nat. Commun., (2018);