Multi-Wavelength in vivo Photon Absorption Remote Sensing: Towards Non-Contact Label-Free Functional Vascular Imaging

Abstract

Blood oxygen saturation (SO2) is an important functional metric in the diagnosis and monitoring of blinding eye diseases and cancer. Additionally, SO2 imaging has high value in illustrating changes in blood oxygenation within a vascular network, particularity when changes are demonstrated within the context of surrounding biological structures. This has promising potential to provide valuable information to researchers and clinicians on the mechanisms of disease progression and the efficacy of treatment.

Various techniques have been explored for SO2 imaging, however limitations of inaccuracy in measurement, a requirement of contact with the tissue and the reliance on exogenous labels have prevented the clinical adoption of these approaches. Photon absorption remote sensing (PARS) is a novel imaging technique that is label-free, non-contact and absorption-based. When a photon is absorbed by a biomolecule, energy can be released through radiative or non-radiative relaxation. Most imaging modalities are limited to capturing one form of relaxation contrast, however PARS is capable of capturing both simultaneously. The unique PARS approach has promising potential as an SO2 imaging modality. This thesis explores work which furthers efforts towards accurate, non-contact, label-free SO2 imaging using PARS.

First, system developments are implemented to demonstrate the first multi-wavelength in-vivo PARS system. The use of independent excitation paths, power compensation, and the improvement of the secondary excitation generation enables the reliable and consistent in-vivo multi-wavelength PARS imaging of chicken embryo vasculature. Additionally, the power compensation of incident excitation pulses is critical for quantitative SO2 measurements to ensure that measured SO2 is not impacted by power variations in the excitation source.

This is followed by the development of techniques for in-vitro phantom studies. A blood oxygenation and deoxygenation protocol is developed and tested, enabling the time-efficient and low-cost preparation of blood samples at various oxygenation levels. Additionally, a flow phantom is developed with a 50 micrometer channel which successfully enables PARS signal to be captured from blood in an in-vitro flow phantom. This experimental setup was unable to demonstrate a change in PARS signal across various blood samples at differing oxygenation levels. Simulation is used to demonstrate that the blood preparation and samples are not the cause of the unsuccessful result. This result is determined to be a consequence of the flow phantom design. The knowledge gained through the iterative design process provides valuable insight to guide future flow phantom developments.

Finally, in-vivo experimentation of the multi-wavelength PARS system successfully demonstrated the variation in blood oxygenation during the hypoxia and recovery of a chicken embryo. The hypoxia holder was designed to modulate the ambient oxygen inside the holder and induce states of hypoxia and recovery. This highlights the success of the PARS multi-wavelength system in demonstrating a relative change in SO2 in-vivo.

The presented work furthers efforts towards accurate, non-contact, label-free PARS SO2 imaging through the development of the first multi-wavelength in-vivo PARS system, in-vitro blood and flow phantom developments and the in-vivo demonstration of relative change in SO2 measured using PARS.