Three-dimensional spectral phasor analysis characterises nuclear wide chromatin dynamics in living cells

Abstract

Our understanding of the dynamic nature of chromatin and the models that describe it are becoming increasingly complex. This presents a need for live cell analysis techniques that characterise chromatin density in living cells. Since the discovery of the double helical structure of DNA in 1953 by Franklin, Watson and Crick, our understanding of DNA structure and that structure's regulation of cell behaviour has grown increasingly complex. We now realise that this first order structure does not explain the dynamics and functionality of chromatin in the nuclear space. The nucleus is functionally compartmentalised where regions sharing macromolecular activity create spatiotemporal domains that regulate transcription events,5 the diffusion of molecules6, and ultimately, cell behaviour. This nuclear architecture is regulated by scaffold proteins, the post translational modification of histones, cationic interactions with histone proteins and the DNA backbone itself. These regulatory mechanisms suggest that epigenetic control of nuclear architecture is density dependant, where the expression of a particular loci is ultimately determined by the accessibility of that loci to the complex of proteins needed for its transcription. Several living cell applicable, in situ and in vitro techniques have been developed to characterise the density and conformation of chromatin. One such technique utilises a DNA density dependant spectral shift in the non-intercalating chromatin binding dye, Hoechst 33342, (H342). Modern confocal technologies such as spectral and hyperspectral analysis are now able to assemble three dimensional, 3D, replications of fluorescently stained cells by unmixing dyes using mapped spectral characteristics. This thesis utilises the spectral phasor approach to develop a living cell applicable, three dimensional, 3D, DNA density characterisation technique. Density dependant spectral shift in H342 was first confirmed and characterised. The phasor approach was then optimised for 3D analyses and subsequently applied in 3D to living L6 myoblasts to characterise relative DNA density throughout the cell cycle. This research effort demonstrates that the phasor approach to spectral analysis can characterise nuclear wide spectral shift in H342 and that this spectral shift is predictive of chromatin condensation and DNA density increases. Furthermore, spectral phasor analysis can be utilised to isolate discrete spectra in 3D and can be optimised for live cell acquisitions. Spectral phasor analysis is a promising new technology that may further elucidate the dynamic and complex architecture of the nucleus.

Date of Award	2022
Original language	English