Snapshot multispectral oximetry using image replication and birefringent spectrometry

This thesis describes the improvements to the image replicating imaging spectrometer (IRIS) and the development of novel applications in the field of oximetry. IRIS is a snapshot multispectral device with a high transmission output and no need of inversion for data recovering, hence, with high signal-to-noise ratio (SNR). IRIS shows great versatility due to the possibility of choosing multiple contiguous or non-contiguous wavelengths inside its free spectral range. IRIS uses a set of waveplates and Wollaston prisms to demultiplex the spectral information of an object and replicate the image of such object in different wavelengths. The birefringent nature of IRIS means that different wavelengths are separated by the Wollaston prisms with different angles, introducing multiple images of the same object. In addition, the spectral transmission function shows multiple spectral sidelobes that contaminate each IRIS band with light belonging to other wavelengths. These issues can lower the performance of IRIS as a multispectral imaging device. In this thesis, these problems were assessed with the introduction of a filter plate array placed in the image plane of the optical system. This filter array is a set of narrow-band filters (Full Width Half Maximum (FWHM) =10 ± 2 nm ) that removes undesired wavelengths from each IRIS band. Since the spectral transmission of IRIS is replicated along the free spectral range, the filters can be designed to match any of the present spectral lobes in IRIS. The design and fabrication of a filter array enhance the performance of IRIS as a multispectral imaging device: it allows wavelength selection and improves spectral and spatial image quality. The design and manufacture of the corresponding filter holder and camera adapter were critical in terms of offering an easy filter-camera implementation. The filter plate allowed the removal of other dispersed wavelengths by the Wollaston prisms, improving image registration between the set of spectral images created by IRIS, and so, improving the quality of the registered spectral 3-D cube. The implemented improvements on IRIS allow high quality, calibration-free oximetry using eight different wavelengths optimised for oximetry. Two main experiments were performed: 1) Using an inverted microscopy interfaced with IRIS and a linear spectral unmixing technique, we measured the deoxygenation of single horse red blood cells (RBC) in vitro in real time. The oximetry was performed with a subcellular spatial resolution of 0.5 μ m , a temporal resolution of 30 Hz, and an accuracy (standard error of the mean) of ± 1.1% in oxygen saturation. 2) Eight-wavelength calibration-free retinal oximetry performed in nine healthy subjects demonstrated an increase in the stability of the oxygen saturation measurements along retinal vessels when compared with more traditional analysis methods such as two wavelengths oximetry. The stability was measured as the standard deviation along the retinal vessels of the nine subjects and was found to be ∼ 3% in oxygen saturation for eight-wavelengths oximetry and ∼ 5% in oxygen saturation for two-wavelengths oximetry. A modified physical model was used to improve the characterization of light propagation through the eye, retina, and blood vessels by applying a set of feasible physiological assumptions. This model was optimised by an algorithm which solves for the different variables involved in the retinal vessels transmissions in order to accurately calculate the oxygen saturation. The oximetry algorithm was applied in retinal vessels, in collaboration in vivo on rat spinal cord to assess hypoxia in inflammatory diseases such as multiple sclerosis and rheumatoid arthritis and on mice legs to assess hypoxia on autoimmune diseases. A third experiment using a microscope interfaced with IRIS was performed. The experiment aimed to replicate laminar flow conditions observed in retinal vessels and to calculate oxygen diffusion between adjacent streams of blood with different oxygen saturation. For this purpose a PDMS multichannel flow cell with cross sections of 40x100 μm was designed and fabricated allowing us to replicate conditions found in retinal blood vessels. Laminar flow was replicated but the experiment failed in calculating oxygen diffusion due to flaws in the experiment. The experiment with the results and recommendations on how to improve it can be found in Apendix B for future researchers