Actin plays a crucial role in cellular processes ranging from the maintenance of cell structure, motility, signal transduction, and intracellular transport. The advances in structural biology, molecular genetics, and fluorescence microscopy techniques over the last eight decades have enabled scientists to investigate actin extensively. Although a great deal of understanding has been acquired regarding the structure, function, and regulation of actin, further characterisation of actin microenvironments is required, particularly for the elucidation of actin isoforms, their localisation, and differential functions. The use of fluorescent probes in conjunction with advanced microscopy techniques has been instrumental in expanding the knowledge of actin within cellular landscapes. Spectral Phasor analysis is a technique that is capable of the hyperspectral characterisation of the spectral properties of individual fluorescent probes. Shifts in the spectral properties of probes can be linked to changes in the probes local microenvironment. Therefore, the further development of Spectral Phasor analysis for spectral characterisation is expected to lead to greater understanding of actin in cellular processes. This study investigates the use of Spectral Phasor analysis as a hyperspectral tool and its applicability to characterise the microenvironments of actin probes in actin filaments. This approach was applied to fluorescent actin probes, ActinGreen and ACTA1-488, in a fixed cell model where the spectral properties of the probes were characterised and compared in their discrete binding domains on actin in various cellular regions and when influenced by the actin disruptor, Latrunculin A. The application of Spectral Phasor analysis for the characterisation of actin probe microenvironments revealed that ActinGreen and ACTA1-488 exhibit characteristic spectral properties when bound in their respective domains in filament and nuclei regions. Further, distinct spectral properties were revealed for ActinGreen and ACTA1-488 when compared across the plasma membrane, cytoplasm, and nuclear membrane regions of fixed cells. Finally, shifts in spectral properties were observed in ActinGreen and ACTA1-488 microenvironments post treatment with Latrunculin A in both region of interest sets described above. The work conducted here demonstrates that Spectral Phasor analysis can be applied to characterise distinct spectral properties of fluorescent actin probe microenvironments in a fixed cell model based on differences in their binding domains, cellular environment, and in response to an actin disrupting compound. With ongoing development and optimisation, Spectral Phasor analysis can be applied to further hyperspectral characterisation of actin isoform microenvironments in fixed cell models, with the possibility of advancing to live cell characterisation in the future.
Date of Award | 2020 |
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Original language | English |
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- actin
- spectral imaging
- spectrum analysis
Characterisation of actin probe microenvironments using spectral phasor analysis
Westall, J. P. (Author). 2020
Western Sydney University thesis: Master's thesis