First and second harmonic spectral phasor analysis of a calcium fluorophore in glucose stimulated live cells

Abstract

The characterisation of cellular processes across the cell is pivotal to understanding responses to growth, differentiation and changes in the cellular environment. In many cases, analysis on the effects of cellular stimulation have been isolated to specific regions, rather than across the cell. Several stimuli are known to elicit cell-wide responses including voltage changes and hormonal regulation. Changes in the extracellular milieu, including the availability of substrates such as glucose, are likely to affect the cell-wide responses to stimuli. For example, insulin-mediated glucose uptake has been well characterised using fluorescence intensity measurements of calcium signalling events within a minute-based timeframe. However, varying the levels of glucose in the extracellular matrix may likely affect the cellular response to insulin stimulation. Characterisation of the potential cell-wide calcium responses to changes in glucose availability may further elucidate the subsequent responses to stimuli such as insulin.

Spectral phasor analysis is a technique that takes spectrally acquired data and generates a first and second harmonic phasor plot through Fourier transformation. It has been shown to detect changes in a fluorophore’s microenvironment across the cell such as sodium, DNA and lipid membranes. While first harmonic analysis has been routinely applied in these studies, usage in concert with second harmonic analysis may provide a more complete understanding of fluorophore dynamics. Here, first and second harmonic spectral phasor analysis was used to develop a model to characterise calcium microenvironments across progenitor myoblasts to ascertain the glucose response from basal activity.

First and second harmonic spectral phasor analysis may potentially be a useful tool to characterise cell-wide responses to changes in the extracellular milieu. Future studies may incorporate this approach using fluorophores targeting other cellular components to elicit similar responses to changes in the extracellular milieu. This research may enable a better understanding of how these changes affect the cell-wide responses to stimuli such as voltage changes and hormonal stimulation.

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