Spectral phasor analysis of calcium in progenitor myoblast cells

Abstract

Calcium is involved in many cellular processes including muscular contraction, proliferation and cell differentiation. Current studies have investigated the role of calcium concentrating on the distribution and localisation of calcium ions across cellular compartments. However, current analysis techniques are limited in their capacity to characterise calcium microenvironments within live cells to further elucidate the role of calcium focused on the interactions experienced by calcium and the influence of environmental conditions. Advancements in fluorescent cell permeant dyes that bind specifically to calcium have permitted the investigation of calcium within live cells. Despite this, an approach to characterise changes in the spectral properties of these fluorescent dyes within live cells had not been available. The recent advent of the Spectral Phasor approach, a spectral imaging analysis technique, offers a method for examining shifts in the spectral profile of a fluorescent probe indicative of changes in cellular microenvironments. Hence, the aim of this thesis was to determine the applicability of Spectral Phasor analysis to study calcium microenvironments in fixed and live cells. This thesis presents the adaptation and application of Spectral Phasor analysis to calcium specific fluorescent probe Fura Red to characterise spectral shifts indicative of microenvironment changes in fixed and live undifferentiated L6 progenitor myoblast cells. Initial experimentation revealed spectral shifts in the Fura Red fluorescent probe bound to calcium within the live and fixed myoblast cells. Discrete regions exhibiting unique shifts in the spectral profile of Fura Red were spatially mapped onto the cell distinguishing between sections of the cell that either shared similarities in the alteration to Fura Red or were shown to be spectrally distinct. Furthermore, this thesis also reports the application of this approach to fixed and live myoblast cells undertaking the early stages of differentiation to characterise changes in calcium microenvironments during the differentiation process. Finally, we also report the application of Spectral Phasor analysis to characterise spectral changes in Fura Red within live differentiating myoblast cells treated with either Ethyl-3,4-dihydroxybenzoate (EDHB) or N-(6- Aminohexyl)-5-chloro-1- naphthalenesulfonamide hydrochloride (W-7). The findings of this research supports previous studies that convey the diverse role of calcium within cellular processes and cell signalling pathways. This research also indicates a potential correlation between shifts in the spectral profile of Fura Red and discrete calcium microenvironments within fixed and live cells. Future research effort will focus on the characterisation of calcium changes throughout the full differentiation process as well as the optimisation and further development of this technique to be applied to the numerous cell processes that involve calcium.

Date of Award	2017
Original language	English