Oximetry using multispectral imaging: theory and application

Multispectral imaging (MSI) is widely applied across various imaging modalities as a technique for measurement of blood oxygen saturation (OS) in vivo, consequently providing new information about physiology and disease development. This tutorial aims to provide a thorough introduction to the theory and application of MSI oximetry for researchers new to the field, whilst also providing detailed information for more experienced researchers. The optical theory underlying two-wavelength oximetry, three-wavelength oximetry, pulse oximetry, and multispectral oximetry algorithms are described in detail. The varied challenges of applying MSI oximetry to in vivo applications are outlined and discussed, covering: the optical properties of blood and tissue, optical paths in blood vessels, tissue auto-fluorescence, oxygen diffusion, and common oximetry artefacts. Essential image processing techniques for MSI are discussed, in particular image acquisition, image registration strategies, and blood vessel line profile fitting. Approaches to the calibration and validation of MSI oximetry – a fundamental challenge in the field - are described and discussed, including strategies utilizing phantoms, as well as physiological interventions. The optical-principals and unique imaging capabilities of various cutting-edge MSI oximetry techniques are discussed, including photoacoustic, spectroscopic optical coherence tomography, and snapshot MSI techniques.