Spectral phasor characterisation of sodium microenvironments in live myoblast stem cells

Abstract

Sodium is implicated in many cellular process including, muscle contraction, proliferation, and differentiation. To date, studies investigating the role of sodium primarily focus on cell-wide changes or specific membrane pores. Characterisation of sodium microenvironments within live cells is expected to lead to a better understanding of the role of sodium in cellular processes including, dynamic interactions with proteins, DNA and RNA in stem cell differentiation. The advent of cell permeant dyes that fluoresce when bound to specific ions provides one element in which to investigate sodium microenvironments in live cells. In order to characterise the spectral properties of sodium bound dye, there needs to be an approach that can acquire data, from live cells, in real time. More recently, Spectral Phasor, a spectral imaging analysis technique that overcomes the need for complex analysis algorithms has been used to demonstrate microenvironment changes with fluorescent proteins. The aim of this thesis was to determine the applicability and potential development of the Spectral Phasor Approach (SPA) for the study of sodium microenvironments in live cells. This thesis reports the application of the sodium ion dye CoroNa Green, and initial modifications of Spectral Phasor, particularly in the context of characterising microenvironment changes in live progenitor myoblast stem cells undergoing differentiation. The SPA was developed by isolating regions of the spectrum exhibiting the largest spectral shifts for spectral acquisition. Acquiring data in the 520 "" 550 nm range enabled the identification of distinct emissions in differentiating cells versus undifferentiated cells. Acquiring spectral data in this range also reduced the acquisition time which consequently appeared to enable minute-interval spectral analysis, and three-dimensional spectral characterisation of live cells. Initial experiments utilising full range scans of the spectrum revealed most significant spectral shifts at 10 minutes. Utilising the more 'optimised' parameters made apparent that the largest spectral shifts occurred at 3 minutes post induction of differentiation, but increasingly became similar overtime. These data confirms previous research which demonstrated sodium influxes as an early antecedent event to proliferation, DNA synthesis and differentiation, and in vitro research which reports the involvement of sodium ions in regulating gene expressions. Moreover, spectral shifts reported here appear to match the initial temporal wave of gene expression reportedly involved in differentiation. This indicates a potential correlation between the early sodium microenvironment changes and the expression of genes involved in the early stages of differentiation. Future research may focus on perturbing sodium influxes to determine the role of sodium microenvironments in the expression of the initial wave of gene expression. This study demonstrates the utility of the Spectral Phasor approach in characterising sodium microenvironments in live cells.

Date of Award	2017
Original language	English