Spectral phasor analysis of live cell lipid microenvironments

Abstract

Physically and biochemically distinct membrane microenvironments modulate cell behaviour. There is a need to develop non-destructive techniques capable of elucidating domain change simultaneously over a broad range of environments in live cells. Doing so will significantly improve our understanding of these spatially and temporally transient domains, direct observation of which has been limited. Spectral Phasor Analysis detects small shifts in exogenous and endogenous fluorophore emissions caused by domain change with high spatial resolution, without requiring reference spectra. This project was designed to ascertain the suitability of Spectral Phasor Analysis for characterizing intra- and inter-environmental spatial relationships of live cell lipid domains. For this thesis, Spectral Phasor Analysis was used to characterize Nile red emissions associated with discrete cell regions. Nile red spectral data was transformed to polar plots using the first harmonic and masks were applied to cell images to isolate phasor points associated with regions of interest. Spectral width and wavelength characteristics were determined for each region of interest to identify areas of environmental change due to ionomycin treatment. Cursors were used to select phasor populations associated with unique lipid microenvironments within isolated regions of interest to assess whether there were changes in spatial distribution associated with spectral change. Shifts in the Nile red spectral profile were detected between discrete cell regions in live cells. Decreased spectral width in cytosolic membrane associated environments following ionomycin treatment indicated change in domain characteristics and it was observed that ionomycin treatment modulates spatial organization of live cell lipid membrane environments. Spectral Phasor Analysis can be used to identify regions undergoing environmental change in live cells and elucidate the spatial relationship between unique cell microenvironments. Future development of the analytical model presented here will improve its spatial and temporal resolution. It can then be applied in conjunction with other experimental models, to increase our understanding of cellular processes involved in lipid domain formation, cell signalling and signal transduction, protein sorting and transport, and disease states resulting in, or caused by lipid dysregulation.

Date of Thesis submission	2023
Original language	English
Awarding Institution	Western Sydney University
Supervisor	Mark Jones (Supervisor)