We describe a visible-light multi-spectral system for vascular oximetry studies that can be implemented in lowand middle-income countries, using a low-cost electronics and optical elements, for instance a Raspberry Pi, a Pi camera under a resolution of 5-megapixel, 2592x1944-pixel resolution, and four different light sources at 480nm, 532nm, 593nm and 610nm on a singular structured illumination area. It is designed to quantify the vascular oxygen saturation change of the rat dorsal spinal cord, which uses a Phyton custom application that synchronize all elements to execute the imaging process in one system, powered by a portable rechargeable 5V battery pack. Aimed for drug discovery, tracking disease progression and understanding of progressive and degenerative diseases. By replacing expensive and bulky imaging systems

Cooper, JM

Harvey, AR

Konda, PC

Motaghian, S

Ochoa-Gutierrez, V

Reboud, J

English

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Multi-spectral vascular oximetry of rat dorsal spinal cord

UCL Discovery

PROCEEDINGS OF SPIESPIEDigitalLibrary.org/conference-proceedings-of-spieMulti-spectral vascular oximetry of ratdorsal spinal cordOchoa-Gutierrez, Victor, Konda, Pavan, Motaghian, Sara,Reboud, Julien, Cooper, Jonathan, et al.Victor Ochoa-Gutierrez, Pavan C. Konda, Sara Motaghian, Julien Reboud,Jonathan M. Cooper, Andrew R. Harvey, "Multi-spectral vascular oximetry ofrat dorsal spinal cord," Proc. SPIE 11230, Optics and Biophotonics in Low-Resource Settings VI, 1123012 (21 February 2020); doi: 10.1117/12.2558281Event: SPIE BiOS, 2020, San Francisco, California, United StatesDownloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 09 Nov 2020  Terms of Use: https://www.spiedigitallibrary.org/terms-of-useMulti-spectral vascular oximetry of rat dorsal spinal cordVictor Ochoa-Gutierreza,b, Pavan C. Kondaa, Sara Motaghiana, Julien Reboudb, Jonathan M.Cooperb, and Andrew R. HarveyaaSchool of Physics and Astronomy, University of Glasgow, Glasgow, UKbJames Watt School of Engineering, University of Glasgow, Glasgow, UKABSTRACTWe describe a visible-light multi-spectral system for vascular oximetry studies that can be implemented in low-and middle-income countries, using a low-cost electronics and optical elements, for instance a Raspberry Pi, aPi camera under a resolution of 5-megapixel, and four light sources at 480nm, 532nm, 593nm and 610nm. Itis designed to quantify the vascular oxygen saturation of the rat dorsal spinal cord, Python custom applicationsynchronizes all elements to execute the imaging process in one system. The device is suitable offers a replacementfor high cost bulky systems in drug discovery, tracking disease progression and understanding of progressive anddegenerative diseases.Keywords: Multispectral Imaging, Oximetry, Imaging System, Gradient-Index Lenses, Low-resource Settings,Biomedical Engineering.1. INTRODUCTIONThe aim of this visible-light multi-spectral system is to achieve a solution capable of multispectral imaginganalysis in real time that potentially can be adapted or modified according the especial needs of a particularresearch and researchers in low- and middle-income countries or remote places, by simply using a rechargeablebattery pack to power the portable imaging system. In-vivo microscopic imaging techniques [1,2], i.e. oximetryon mice and rats is widespread and increasingly animal models are used in biomedicine, for drug discovery andtracking disease progression. The number of animals used for experimental purposes continues to rise every year,in 2018 an estimated of 3.6 million procedures were made using mice and rats [3]. We propose an in-vivo imagingsystem that has the promise to reduce the number of animals by collecting of high-resolution imaging data inlongitudinal repeated-measures during one single study especially the oxygen consumption in deep tissue thathas been proved that has a relationship to different diseases.4–92. LOW-COST SYSTEM OVERVIEWAn imaging system was assembled and designed using; red LED (625nm), amber LED (590nm), green LED(525nm) and blue LED (475nm), dichroic optical blocks (Thorlabs - CM1-DCH/M), longpass dichroic mirrors(Thorlabs), focusing lenses, heat sinks (Figs. 1, 2 and 3). Universal port that holds a GRIN lens (GRINtech),structured illumination is used by a fiber-array adapted to Raspberry Pi camera, these elements were used to builda low-cost system for imaging.10 In addition, the system is capable of directing the illumination into an annularillumination pattern, allowing a single pass illumination of the vasculature,7 i.e. reducing the complexity of theoxygenation measurement in deep tissue and blood vessels for a better understanding of axonal remyelination,multiple sclerosis research, immune cells in lymph nodes and towards a cure for rheumatoid arthritis. Theuniversal imaging sensor; is constituted by a Pi camera (resolution 5-megapixel, 2592x1944-pixel static images)and 3D printed holder for the GRIN lens and the illumination fibers, with a locking mechanism to improvecoupling between the camera and the GRIN lens, see Fig. 1. The system uses a Python custom applicationto control the imaging and illumination elements synchronizing all elements at once for oximetry measurementprospectuses.Further author information: (Send correspondence to A.R.H.)A.R.H.: E-mail: Andy.Harvey@glasgow.ac.ukOptics and Biophotonics in Low-Resource Settings VI, edited by David Levitz, Aydogan Ozcan, Proc. of SPIEVol. 11230, 1123012 · © 2020 SPIE · CCC code: 1605-7422/20/$21 · doi: 10.1117/12.2558281Proc. of SPIE Vol. 11230  1123012-1Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 09 Nov 2020Terms of Use: https://www.spiedigitallibrary.org/terms-of-usedicroic beamsplittersPi cameraTissueor sampleFiber illuminationGRIN lens480nm593nm 610nm 532nmFiber illuminationFigure 1. Principal imaging system elements; optical illumination, image acquisition and optical magnification using aGRIN lens, tissue representation.Figure 2. Optical block for structured illumination, dichroic mirrors in position (Thorlabs).3. RESEARCH APPLICATIONS FOR IMAGING TECHNIQUES INLOW-RESOURCE SETTINGSThe developed system will enable a longitudinal vascular oximetry study using a structured illumination techniqueon a single animal over a period of four or six weeks. An important contribution is the reduction of use of rodentsby a factor four or five. a second important benefit is the reduction of variability inherent in longitudinal study ofone single animal, this will enable high quality data related to the degenerative diseases reducing the uncertaintyof using more rodents in short periods of time.Figure 3. Imaging System completely assembled and operational for research studies.Proc. of SPIE Vol. 11230  1123012-2Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 09 Nov 2020Terms of Use: https://www.spiedigitallibrary.org/terms-of-use4. INITIAL DEMONSTRATIONSRecent evidence has implicated mitochondrial dysfunction, and hypoxia, in the aetiology and pathology ofmultiple sclerosis (MS) and experimental autoimmune encephalomyelitis, an animal model of MS.6,7 The role ofthese pathological changes in neurological dysfunction, lesion formation, and disease progression remains unclear.However, obvious differences in the vasculature (such as diameter, and oximetry variations, especially hypoxiapatterns) have been observed that correlate with measures of disease severity and neurological dysfunction inrats with experimental autoimmune encephalomyelitis (EAE) during the early stages of disease. For preliminarypurposes images were recorded from a hyperspectral image sequence of the exposed rat spinal cord shown inFig. 4. This initial demonstration shows the optical and imaging performance of the system, that provides thevital information for the oximetry signal extraction.Figure 4. Rat spinal cord vasculature imaged at 480nm, 532nm, 593nm and 610nm. Oximetry is based on varyingintensity of blood vessels with wavelength where the changes on oxi-haemoglobin can be measure.5. CONCLUSIONWe described a visible-light multi-spectral microscope system for longitudinal oximetry studies in small animals.This device will continue to be improved and continue tested in the field, that includes modifications at themathematical model establish by [7,11] for oximetry calculations, avoiding some errors when using two oximetrywavelengths [12, 13] for multi imaging vasculature studies. Enabling a low-resource settings tool that will leadto better understanding of the progression of degenerative diseases based on the oxygenation levels, especiallyduring the absence of oxygenation or hypoxia stages [14] .ACKNOWLEDGMENTSWe are grateful for our funding sources. This research is supported by NC3Rs, IAA-EPSRC, CONACyT-SENER.REFERENCES[1] Desjardins, M., Sylvestre, J.-P., Jafari, R., Kulasekara, S., Rose, K., Trussart, R., Arbour, J. D., Hudson,C., and Lesage, F., “Preliminary investigation of multispectral retinal tissue oximetry mapping using ahyperspectral retinal camera,” Experimental Eye Research 146, 330 – 340 (2016).[2] Rouse, A. R., Udovich, J. A., and Gmitro, A. F., “In-vivo multi-spectral confocal microscopy,” (2005).[3] “Animal research numbers in 2018 — understanding animal research — understanding ani-mal research.” http://www.understandinganimalresearch.org.uk/news/communications-media/animal-research-numbers-in-2018/. (Accessed on 01/07/2020).[4] Davies, A. L., Desai, R. A., Bloomfield, P. S., McIntosh, P. R., Chapple, K. J., Linington, C., Fairless, R.,Diem, R., Kasti, M., Murphy, M. P., and Smith, K. J., “Neurological deficits caused by tissue hypoxia inneuroinflammatory disease,” Annals of Neurology 74(6), 815–825 (2013).Proc. of SPIE Vol. 11230  1123012-3Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 09 Nov 2020Terms of Use: https://www.spiedigitallibrary.org/terms-of-use[5] Dyson, A., Stidwill, R., Taylor, V., and Singer, M., “Tissue oxygen monitoring in rodent models of shock,”American Journal of Physiology-Heart and Circulatory Physiology 293(1), H526–H533 (2007). PMID:17384135.[6] Mahad, D. J., Ziabreva, I., Campbell, G., Lax, N., White, K., Hanson, P. S., Lassmann, H., and Turnbull,D. M., “Mitochondrial changes within axons in multiple sclerosis,” Brain 132, 1161–1174 (03 2009).[7] van der Putten, M. A., MacKenzie, L. E., Davies, A. L., Fernandez-Ramos, J., Desai, R. A., Smith, K. J.,and Harvey, A. 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Express 8, 2896–2905 (Jun 2017).[12] Rodriguez, D. A., Pfefer, T. J., Wang, Q., Lopez, P. F., and Ramella-Roman, J. C., “A Monte Carlo Analysisof Error Associated With Two-Wavelength Algorithms for Retinal Oximetry,” Investigative Ophthalmology& Visual Science 57, 6474–6481 (11 2016).[13] Hashimoto, R., Kurata, T., Sekine, M., Nakano, K., Ohnishi, T., and Haneishi, H., “Two-wavelength oxime-try of tissue microcirculation based on sidestream dark-field imaging,” Journal of Biomedical Optics 24(3),1 – 8 (2018).[14] Shi, Y., Oeh, J., Hitz, A., Hedehus, M., Eastham-Anderson, J., Peale, F. V., Hamilton, P., O’Brien, T.,Sampath, D., and Carano, R. A., “Monitoring and targeting anti-vegf induced hypoxia within the viabletumor by 19f–mri and multispectral analysis,” Neoplasia 19(11), 950 – 959 (2017).Proc. of SPIE Vol. 11230  1123012-4Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 09 Nov 2020Terms of Use: https://www.spiedigitallibrary.org/terms-of-use

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Multi-spectral vascular oximetry of rat dorsal spinal cord

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