Optical coherence tomography systems are used to image the retina in 3D to allow ophthalmologists diagnose ocular disease. These systems yield large data sets that are often labor-intensive to analyze and require significant expertise in order to draw conclusions, especially when used over time to monitor disease progression. Spectral Domain Optical Coherence Tomography (SD-OCT) instantly acquires depth profiles at a single location with a broadband source. These systems require mechanical scanning to generate two- or three-dimensional images. Instead of mechanically scanning, a beamlet array was used to permit multiple depth measurements on the retina with a single snapshot using a 3x 3 beamlet array. This multi-channel system was designed, assembled, and tested using a 1 x 2 beamlet lens array instead of a 3 x 3 beamlet array as a proof of concept prototype. The source was a superluminescent diode centered at 840nm with a 45nm bandwidth. Theoretical axial resolution was 6.92um and depth of focus was 3.45mm. Glass samples of varying thickness ranging from 0.18mm to 1.14mm were measured with the system to validate that correct depth profiles can be acquired for each channel. The results demonstrated the prototype system performed as expected, and is ready to be modified for in vivo applicability.

Amblyopia is the leading cause of monocular visual impairment in children. Therapy for amblyopia is extremely beneficial in some children but ineffective in others. It is critical that the reasons for this discrepancy are understood. Emerging evidence indicates that current clinical protocols for the diagnosis of amblyopia may not be sufficiently sensitive in identifying individuals who, on more detailed examination, exhibit subtle structural defects of the eye. Presently, the magnitude of this problem is unknown. The aim of this study was to establish the prevalence of subtle retinal/optic nerve head defects in eyes diagnosed with amblyopia, to distinguish between possible explanations for the origin of such defects and to investigate the relationship between quantitative measures of retinal structure, retinal nerve fibre layer thickness and optic nerve head dimensions. Using the imaging technique of Optical Coherence Tomography (OCT) retinal structure has been investigated in detail, following the visual pathway across the retina from the fovea, via the paramacular bundle to the optic disc, where peripapillary retinal nerve fibre thickness has been imaged and subjected to detailed measures along with optic disc size and shape. The study formed two phases, the first imaging the eyes of visually normal adults and children, comparing them to amblyopes, both adults and children who had completed their treatment. The second phase, a longitudinal study, investigated retinal structure of amblyopic children undertaking occlusion therapy for the first time. By relating pre-therapy quantitative measures to the visual outcome the second phase of the study aimed to examine whether OCT imaging could identify children achieving a poor final outcome. The results show a clear picture of inter-ocular symmetry structure in all individuals, visually normal and amblyopic. Optic disc characteristics revealed no structural abnormalities in amblyopes, in any of the measured parameters, nor was there any association between the level of visual acuity and the measured structure. At the fovea differences were shown to occur in the presence of amblyopia, with thickening of the fovea and reduction of the foveal pit depth. The structural changes were found to be both bilateral and symmetrical with the fellow eye also affected. In the longitudinal phase of the study these changes were demonstrated to a greater extent in children who 'failed' to respond to treatment. This bilateral, symmetrical structural change found at the fovea, which has not been previously reported, cannot therefore be the primary cause of the visual loss which has been diagnosed as amblyopia.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Glaucoma is an ophthalmic pathology that is the second leading cause of blindness. The laboratory design of a Polarization Sensitive Spectral Domain Optical Coherence Tomographic System aims to detect early glaucoma symptoms and prevent vision loss that occurs due to late or no glaucoma diagnosis. In order to perform human clinical trials at partner hospitals across the country, a miniaturized and portable version of the laboratory system was developed. The system facilitates easy transportation and clinical testing of the otherwise voluminous laboratory system across different eye centers. Significant consideration was given for performance optimization, cost reduction, design improvements and providing a friendly user-patient interface. / text

Optical Coherence Tomography (OCT) is a non-invasive three-dimensional imaging technique. OCT synthesizes a cross-sectional image from a series of lateral adjacent depth scans, and with a two-dimensional scanning scheme, three-dimensional intensity image of sample can be constructed. Due to its non-invasive capability, OCT has been widely applied in ophthalmology, cardiology and dermatology; and in addition to three-dimensional intensity image construction, various functional OCT imaging techniques have been developed for clinical application. My research is focused on developing functional OCT systems for application in ophthalmology, including polarization-sensitive optical coherence tomography (PS-OCT) for retinal nerve fiber layer (RNFL) birefringence measurement and dual-wavelength photothermal optical coherence tomography (DWP-OCT) for microvasculature blood oxygen saturation (SO2) measurement. In the study, a single-mode-fiber based polarization-sensitive swept-source OCT (PS-SS-OCT) with polarization modulator, polarization-sensitive bulk-optics balanced detection module is constructed and polarization processing methods based on Stokes vectors are applied to determine birefringence. PS-OCT is able to provide human subject's RNFL thickness, phase retardation, and birefringence information. Degradation in the degree of polarization (DOP) along depth is investigated and its difference between four quadrants of RNFL (superior, temporal, inferior and nasal) indicates the structural property difference. DWP-OCT is a novel functional OCT system consisting of a phase-sensitive optical coherence tomography system (PhS-OCT) and two photothermal excitation lasers. PhS-OCT is based on a swept-source laser operating in the 1060 nm wavelength range; the two photothermal excitation lasers with wavelength 770 nm and 800 nm are intensity modulated at different frequencies. PhS-OCT probe beam and two photothermal excitation beams are combined and incident on the sample, optical pathlength (op) change on the sample introduced by two photothermal excitation beams are measured and used for blood SO2 estimation. A polarization microscope is proposed for future study. The polarization microscope is an imaging technique providing molecular structure and orientation based on probe light's polarization state information. The polarization microscope uses a wavelength tunable light source, and can achieve any incident polarization state by a retarder-rotator combination. Specimen's birefringence can be determined based on the changing of detected light amplitude. / text

C1QTNF5 is a 25kDa short chain collagen of unknown function which is mutated in late-onset retinal macular degeneration (L-ORMD). L-ORMD is an autosomal dominant disease characterised by sub-retinal pigment epithelial deposits leading to photoreceptor death and visual loss and shows several similarities to age-related macular degeneration (AMD). A Tyr402His polymorphism in complement factor H (CFH), a regulatory protein in the innate immune system, has been associated with increased risk of AMD. C1QTNF5 and CFH are both expressed and secreted by the retinal pigment epithelium (RPE) which supports photoreceptors and is responsible for phagocytosis of shed rod photoreceptor outer segments (ROS). The properties of the normal C1QTNF5 and disease-associated Ser163Arg mutation were examined in detail, including protein characterisation, cellular processing and function. Recombinant wild type and mutant C1QTNF5 were produced and their multimerisation and solubility functions compared. Both proteins were found to be soluble and to form similar multimeric species which were resistant to reducing conditions, as seen in other short chain collagens. Due to the similarities between LORMD and AMD, a proposed interaction between C1QTNF5 and CFH was investigated. CFH is composed of 20 short consensus repeats (SCR) and interactions were confirmed between C1QTNF5 and both CFH and SCR modules 7-8 and 19-20. CFH showed a greater affinity for mutant C1QTNF5 compared with wild type on the basis of surface plasmon resonance assays. Stably transfected RPE-derived cell lines were created which expressed either wild type or mutant C1QTNF5. Both proteins were found to be secreted and showed similar cellular processing with no evidence of aggregation or retention of the mutant protein within the endoplasmic reticulum. In order to investigate C1QTNF5 function, phagocytosis of ROS by the stably transfected cell lines was carried out. Cells expressing wild type C1QTNF5 showed greater ROS phagocytosis compared with mutant C1QTNF5-expressing or untransfected cells. Addition of anti-C1QTNF5 antibody increased ROS phagocytosis further. In summary, it is proposed that wild type and mutant C1QTNF5 are secreted by the RPE where they interact with CFH. C1QTNF5 is also shown to have a role in ROS phagocytosis, with mutation in C1QTNF5 affecting phagocytosis efficiency, which may contribute to sub-RPE deposit formation. The results suggest that CFH may also be involved in this process, suggesting a common pathogenic pathway between L-ORMD and AMD.

Purpose Intrinsically photosensitive retinal ganglion cells (ipRGCs) express a unique photopigment called melanopsin. Capable of direct phototransduction, the ipRGCs are also influenced by rods and cones via synaptic inputs.  Thus, the photoinput that mediates the pupil light reflex derives from both outer (rods and cones) and inner (melanopsin-mediated) retinal photoreception. This thesis has aimed to develop a pupillometric test that provides quantitative information about the functional status of outer and inner retinal photoreception in healthy eyes and in eyes with retinal degeneration. In addition to regulating the pupil light reflex, the ipRGCs signal light information for the circadian rhythm, thus, these two non-visual physiologic responses to inner retinal photoreception were examined simultaneously. Methods Pupil responses to a long and short wavelength light over a range of intensities (under conditions of light, mesopic and dark adaptation) were recorded using a customized infrared computerized pupillometer. Results were compared for two groups: patients with retinitis pigmentosa and controls. The response function threshold intensity and a half-max intensity was determined from the rod-weighted and cone-weighted pupil responses and correlated to extent of visual loss. The pupil response to light offset was assessed as a measure of direct melanopsin activation. Lastly, pupil responses to red and blue light at equal photo flux were recorded hourly during a 24-hour period and correlated to salivary melatonin concentrations in healthy subjects. Results In normal eyes, the blue light evoked greater pupil responses compared to equiluminant red light. With increasing intensity, pupil contraction became more sustained which was most apparent with the brightest blue light. In patients with retinitis pigmentosa, the pupil responses mediated predominantly by rod and cone activation were significantly reduced compared to controls, (p<0.001) and the relative decrease in their contribution resulted in a greater influence of melanopsin on the post-stimulus response. Even at endstage retinal degeneration, pupil responses that derived predominantly from residual cone activity were detectable. The threshold intensity of the rod-mediated, but not cone-mediated, pupil response was also significantly reduced (p=0.006) in patients and the half-maximal intensity of rods correlated with severity of visual loss (r2=0.7 and p=0.02). In healthy controls, the melanopsin-mediated pupil response demonstrated a circadian modulation whereas the cone-mediated pupil response did not. Conclusion Early and progressive loss of rod function in mild-moderate stages of retinitis pigmentosa is detectable and quantifiable as a progressive loss of pupillary sensitivity to extremely dim blue lights obtained under conditions of dark adaptation. In advanced stages of retinal degeneration, chromatic pupillometry is more sensitive than standard electroretinography for detecting residual levels of rod and especially cone activity. In addition, selective wavelength pupillometry can assess non-visual light-dependent functions. The timing of the post-stimulus pupil response to blue light is in phase with melatonin secretion, suggesting a circadian regulation of this pupil parameter. / Bakgrund Jätteganglieceller (intrinsically photosensitive retinal ganglion cells, ipRGCs) är en klass av fotoreceptorer som utnyttjar ett unikt vitamin-A-baserat fotopigment som kallas melanopsin. Utöver deras direkta ljuskänslighet, mottar ipRGCs stimulerande och hämmande synaptiska signaler från andra fotoreceptorer (tappar och stavar) som därigenom kan modulera aktiviteten hos ipRGCs. Ögats pupillreflex medieras alltså av ljus både via yttre (stavar och tappar) och inre (melanopsin-medierad) retinal fotoreception, och den gemensamma afferenta pupillomotor-signalen leds till den pretectala nucleus olivarius via axoner från ipRGCs. Arbetet i denna avhandling syftar till att utveckla ett kliniskt pupilltest som ger kvantitativ information om yttre och inre retinala fotoreceptorers funktionella status hos friska försökspersoner och patienter med retinal degeneration. Förutom att styra pupillreflexen, skickar ipRGCs även impulser som påverkar kroppens dygnsrytm. Därför ingår även en delstudie i vilken ipRGCs aktivitet studeras genom att avläsa icke-visuella fysiologiska reaktioner på inre retinal fotoreception. Metoder Ljus av lång (röd) respektive kort (blå) våglängd presenterades med stegvis ökad ljusstyrka för att selektivt stimulera stavar, tappar eller melanopsin. Pupillreaktionerna registrerades med en infraröd datoriserad pupillometer och jämfördes mellan friska kontroller och patienter med retinitis pigmentosa. I uppföljande experiment gjordes mer noggranna tester i syfte att isolera aktiveringen av varje ljusmottagande element. Tröskelintensiteten för stav- eller tapp-medierad pupillreaktion bestämdes med linjär regressionsanalys. Reaktionskurvan för stavmedierad pupillreflex kvantifierades (halv-maximal intensitet) och jämfördes med svårighetsgraden av sjukdomen i två familjer med samma sjukdomsframkallande mutation för retinitis pigmentosa. För att undersöka icke-visuella reaktioner på inre fotoreception från ipRGCs, undersöktes pupillreaktion på rött och blått ljus varje timme under en 24-timmarsperiod och korrelerades till melatoninkoncentration i saliv hos friska personer med normal syn. Resultat I normala ögon, gav blått ljus en kraftigare pupillreaktion jämfört med rött ljus av samma ljusstyrka. Med ökande intensitet, blev pupillkontraktionen mer ihållande, vilket var tydligast med starkt blått ljus. Hos patienter med retinitis pigmentosa, var både tapp- och stav-medierad pupillreaktion signifikant reducerad jämfört med kontroller, (p<0,001). Patienter med avancerad sjukdom och icke-reaktivt elektro-retinogram hade fortfarande mätbar pupillreflex, huvudsakligen härrörande från kvarvarande stavaktivitet. I två familjer med retinitis pigmentosa beroende på en enda missense-mutation av NR2E3 genen, var tröskelvärdet för stavmedierad pupillreflex signifikant reducerat (p= 0,006) och korrelerade till sjukdomens svårighetsgrad. Tappmedierad pupillreflex hos dessa patienter skilde sig dock inte signifikant från kontroller, trots att fotopiskt (tapp) elektroretinogram var klart avvikande. Hos friska kontroller visade melanopsinmedierat pupillsvar en dygnsvariation medan tapp-medierat pupillsvar inte gjorde det. Slutsatser Som tillägg till standardundersökningar kan selektiv våglängds-pupillometri (kromatisk pupillometri) vara användbart för utvärdering av funktionen hos stavar och tappar. Denna avhandling visar att tidig och gradvis förlust av stav-funktion i milt-måttligt stadium av retinitis pigmentosa är detekterbar och mätbar som en progressiv förlust av pupillens känslighet för mycket svagt blått ljus, efter mörkeradaptation. I avancerade stadier av retinal degeneration är kromatisk pupillometri känsligare än standardelektroretinografi för att detektera kvarvarande nivåer av stav- och speciellt tapp-aktivitet. Hos unga patienter, där elektroretinografi kan vara tekniskt svårt, är pupillometri en lovande teknik för att värdera yttre retinal fotoreception relaterad till synfunktion. Dessutom kan selektiv våglängdspupillometri ge information om icke-visuella ljusberoende funktioner. Pupillreaktionen på blått ljus varierar med melatoninsekretionen, vilket tyder på en cirkadisk reglering. Ytterligare studier krävs för att undersöka om selektiv våglängds-pupillometri även kan användas i samband med sjukdomar relaterade till störd dygnsrytm, som sömnlöshet och årstidsbunden depression.