[Truncated abstract] Purpose: To improve our ability to interpret and validate Heidelberg Retina Flowmeter (HRF) flow images by recording flow measurements from specific regions of the retinal vasculature in in-vitro and in-vivo eye models in which retinal blood flow can be manipulated. Methods: The first experiments utilised an isolated perfused pig eyes perfused with a 50%/50% Krebs/RBC solution at known flow rates ranging from zero to 300 μl/min. After HRF image acquisition, the retinal vasculature was perfused with fluorescein isothiocyanate (FITC), for fluorescence microscopy. Using the standard HRF software and a 10×10 pixel measurement window, flow rates were measured from a retinal artery, vein, arteriole, venule, and the retinal capillary bed and a capillary-free-zone. The relationship between HRF measured flow and perfusion flow in the different measurement regions was determined. For the second study, HRF flow images were acquired at retinal sites in Brown Norway rats over a range of focus levels before and after cessation of retinal blood flow by laser-induced central retinal artery occlusion. Using the 10×10 pixel and 4×4 pixel measurement windows, HRF measured flow was performed in retinal artery, vein, arteriole, capillary and choroidal vessel locations. The relationship between HRF measured flow and focus depth was determined for each location before and after central retinal artery occlusion. At the conclusion of each experiment the effect of reduction of systemic blood pressure (by exsanguination) on HRF flow measurements in choroidal vessels and in locations without visible choroidal vessels was assessed. Finally, HRF flow measurements were acquired after euthanasia, to determine the HRF flow signal which was still present under zero biological flow conditions (the zerooffset). Results: In the isolated perfused pig eye experiments it was found that the HRF flow response varied according to vascular location. At zero perfusate flow, HRF flow was consistently greater than zero at all locations, averaging 171.9 ± 44.7 AU (n=97), representing background noise. Arteries and veins yielded the highest HRF measured flow values, but the relationship between HRF measured flow and perfusate flow was not linear. In arterioles the HRF flow was more linear over a broader range of perfusate flow rates but the peak flow signal was an order of magnitude smaller than that in arteries and veins. Both the linearity and magnitude of the flow signal in venules was less than that in arterioles.

Thesis (M.S.)--Rochester Institute of Technology, 2005. / Typescript. Includes bibliographical references (leaves 54-56).

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17826号 / 医博第3824号 / 新制||医||999(附属図書館) / 30641 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 村井 俊哉, 教授 柳田 素子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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<p>Optical coherence tomography (OCT) is a noninvasive three-dimensional interferometric imaging technique capable of achieving micrometer scale resolution. It is now a standard of care in ophthalmology, where it is used to improve the accuracy of early diagnosis, to better understand the source of pathophysiology, and to monitor disease progression and response to therapy. In particular, retinal imaging has been the most prevalent clinical application of OCT, but researchers and companies alike are developing OCT systems for cardiology, dermatology, dentistry, and many other medical and industrial applications. </p><p>Adaptive optics (AO) is a technique used to reduce monochromatic aberrations in optical instruments. It is used in astronomical telescopes, laser communications, high-power lasers, retinal imaging, optical fabrication and microscopy to improve system performance. Scanning laser ophthalmoscopy (SLO) is a noninvasive confocal imaging technique that produces high contrast two-dimensional retinal images. AO is combined with SLO (AOSLO) to compensate for the wavefront distortions caused by the optics of the eye, providing the ability to visualize the living retina with cellular resolution. AOSLO has shown great promise to advance the understanding of the etiology of retinal diseases on a cellular level.</p><p>Broadly, we endeavor to enhance the vision outcome of ophthalmic patients through improved diagnostics and personalized therapy. Toward this end, the objective of the work presented herein was the development of advanced techniques for increasing the imaging speed, reducing the form factor, and broadening the versatility of OCT and AOSLO. Despite our focus on applications in ophthalmology, the techniques developed could be applied to other medical and industrial applications. In this dissertation, a technique to quadruple the imaging speed of OCT was developed. This technique was demonstrated by imaging the retinas of healthy human subjects. A handheld, dual depth OCT system was developed. This system enabled sequential imaging of the anterior segment and retina of human eyes. Finally, handheld SLO/OCT systems were developed, culminating in the design of a handheld AOSLO system. This system has the potential to provide cellular level imaging of the human retina, resolving even the most densely packed foveal cones.</p> / Dissertation

The focus of this thesis is the improvement of acquisition and processing of Mueller matrix polarimetry using a confocal scanning laser ophthalmoscope (CSLO) and the application of Mueller matrix polarimetry to image the retina. Stepper motors were incorporated into a CSLO to semi-automate Mueller matrix polarimetry and were used in retinal image acquisition. Success rates of Fourier transform based edge detection filters, designed to improve the registration of retinal images, were compared. The acquired polarimetry images were used to reassess 2 image quality enhancement techniques, Mueller matrix reconstruction (MMR) and Stokes vector reconstruction (SVR), focusing on the role of auto-contrasting or normalization within the techniques and the degree to which auto-contrasting or normalization is responsible for image quality improvement of the resulting images. Mueller matrix polarimetry was also applied to find the retardance image of a malaria infected retinal blood vessel imaged in a confocal scanning laser microscope (CSLM) to visualize hemozoin within the vessel. Image quality enhancement techniques were also applied and image quality improvement was quantified for this blood vessel. The semi-automation of Mueller matrix polarimetry yielded a significant reduction in experimental acquisition time (80%) and a non-significant reduction in registration time (44%). A larger sample size would give higher power and this result might become significant. The reduction in registration time was most likely due to less movement of the eye, particularly in terms of decreased rotation seen between registered images. Fourier transform edge detection methods increased the success rate of registration from 73.9% to 92.3%. Assessment of the 2 MMR images (max entropy and max signal-to-noise ratio (SNR)) showed that comparison to the best CSLO images (not auto-contrasted) yielded significant average image quality improvements of 158% and 4% when quantified with entropy and SNR, respectively. When compared to best auto-contrasted CSLO images, significant image quality improvements were 11% and 5% for entropy and SNR, respectively. Images constructed from auto-contrasted input images were of significantly higher quality than images reconstructed from original images. Of the 2 other images assessed (modified degree of polarization (DOPM) and the first element of the Stokes vector (S0)), DOPM and S0 yielded significant average image quality improvements quantified by entropy except for the DOPM image of the RNFL. SNR was not improved significantly when either SVR image was compared to the best CSLO images. Compared to the best auto-contrasted CSLO images, neither DOPM nor S0 improved average image quality significantly. This result might change with a larger number of participants. When MMR were applied to images of malaria infected retinal slides, image quality was improved by 19.7% and 15.3% in terms of entropy and SNR, respectively, when compared to the best CSLO image. The DOPM image yielded image quality improvements of 8.6% and -24.3% and the S0 image gave improvements of 9.5% and 9.4% in entropy and SNR, respectively. Although percent increase in image quality was reduced when images were compared to initial auto-contrasted CSLO images, the final image quality was improved when auto-contrasting occurred prior to polarimetry calculations for max SNR and max entropy images. Quantitative values of retardance could not be found due to physical constraints in the CSLM that did not allow for characterization of its polarization properties and vibrational noise. Mueller matrix polarimetry used to find the retardance image of a malaria infected retina sample did yield visualization of hemozoin within the vessel but only qualitatively. In conclusion, improvements in the acquisition and registration of CSLO images were successful in leading to considerably shorter experimentation and processing times. In terms of polarimetric image quality improvement techniques, when compared to the best CSLO image. A large proportion of the improvement was in fact due to partially or completely stretching the pixel values across the dynamic range of the images within the algorithm of each technique. However, in general the image quality was still improved by the Mueller matrix reconstruction techniques using both entropy and SNR to generate the CSLO retinal images and the CSLM imaged malaria infected sample. In the malaria sample, retinal blood vessel visualization was also qualitatively improved. The images yielded from Mueller matrix polarimetry applied to a malaria infected retinal sample localized hemozoin within the blood vessel, but a quantitative image of the phase retardance could not be achieved.

The focus of this thesis is the improvement of acquisition and processing of Mueller matrix polarimetry using a confocal scanning laser ophthalmoscope (CSLO) and the application of Mueller matrix polarimetry to image the retina. Stepper motors were incorporated into a CSLO to semi-automate Mueller matrix polarimetry and were used in retinal image acquisition. Success rates of Fourier transform based edge detection filters, designed to improve the registration of retinal images, were compared. The acquired polarimetry images were used to reassess 2 image quality enhancement techniques, Mueller matrix reconstruction (MMR) and Stokes vector reconstruction (SVR), focusing on the role of auto-contrasting or normalization within the techniques and the degree to which auto-contrasting or normalization is responsible for image quality improvement of the resulting images. Mueller matrix polarimetry was also applied to find the retardance image of a malaria infected retinal blood vessel imaged in a confocal scanning laser microscope (CSLM) to visualize hemozoin within the vessel. Image quality enhancement techniques were also applied and image quality improvement was quantified for this blood vessel. The semi-automation of Mueller matrix polarimetry yielded a significant reduction in experimental acquisition time (80%) and a non-significant reduction in registration time (44%). A larger sample size would give higher power and this result might become significant. The reduction in registration time was most likely due to less movement of the eye, particularly in terms of decreased rotation seen between registered images. Fourier transform edge detection methods increased the success rate of registration from 73.9% to 92.3%. Assessment of the 2 MMR images (max entropy and max signal-to-noise ratio (SNR)) showed that comparison to the best CSLO images (not auto-contrasted) yielded significant average image quality improvements of 158% and 4% when quantified with entropy and SNR, respectively. When compared to best auto-contrasted CSLO images, significant image quality improvements were 11% and 5% for entropy and SNR, respectively. Images constructed from auto-contrasted input images were of significantly higher quality than images reconstructed from original images. Of the 2 other images assessed (modified degree of polarization (DOPM) and the first element of the Stokes vector (S0)), DOPM and S0 yielded significant average image quality improvements quantified by entropy except for the DOPM image of the RNFL. SNR was not improved significantly when either SVR image was compared to the best CSLO images. Compared to the best auto-contrasted CSLO images, neither DOPM nor S0 improved average image quality significantly. This result might change with a larger number of participants. When MMR were applied to images of malaria infected retinal slides, image quality was improved by 19.7% and 15.3% in terms of entropy and SNR, respectively, when compared to the best CSLO image. The DOPM image yielded image quality improvements of 8.6% and -24.3% and the S0 image gave improvements of 9.5% and 9.4% in entropy and SNR, respectively. Although percent increase in image quality was reduced when images were compared to initial auto-contrasted CSLO images, the final image quality was improved when auto-contrasting occurred prior to polarimetry calculations for max SNR and max entropy images. Quantitative values of retardance could not be found due to physical constraints in the CSLM that did not allow for characterization of its polarization properties and vibrational noise. Mueller matrix polarimetry used to find the retardance image of a malaria infected retina sample did yield visualization of hemozoin within the vessel but only qualitatively. In conclusion, improvements in the acquisition and registration of CSLO images were successful in leading to considerably shorter experimentation and processing times. In terms of polarimetric image quality improvement techniques, when compared to the best CSLO image. A large proportion of the improvement was in fact due to partially or completely stretching the pixel values across the dynamic range of the images within the algorithm of each technique. However, in general the image quality was still improved by the Mueller matrix reconstruction techniques using both entropy and SNR to generate the CSLO retinal images and the CSLM imaged malaria infected sample. In the malaria sample, retinal blood vessel visualization was also qualitatively improved. The images yielded from Mueller matrix polarimetry applied to a malaria infected retinal sample localized hemozoin within the blood vessel, but a quantitative image of the phase retardance could not be achieved.

This diploma thesis is dealing with the detection of blood vessels pulsation in retinal sequences. The goal is to create an algorithm for objective evaluation of pulsation in retinal video sequences.

Biofilms of Actinobacillus succinogenes, grown in a biofilm reactor system, were investigated for structure and cell viability, through microscopic visualisation with a confocal scanning laser microscope (CSLM) and a scanning electron microscope (SEM). Biofilms were sampled and visualised at steady state conditions with the broth containing succinic acid titres between 15 and 21 g/L. All sampled biofilm was 6 days old. Six-day-old biofilms of A. succinogenes showed a heterogeneous biofilm architecture composed of cell micro-colony pillars which varied considerably in thickness, area and shape. Microcolony pillars consisted of a densely packed entanglement of sessile cells. Quantitative analysis revealed that the pillars were mostly large, with a mean pillar diameter of 170 m and a mean thickness of 92 m, although pillar diameter and thickness were variable as they ranged from 25 – 500 m and 30 – 300 m, respectively. In the regions close to the substratum surface, pillars were characterised by having defined borders with a network of channels ranging from 40 – 200 m in width separating them. However, towards the middle of the biofilm depth some of the pillars coalesced. For this reason low cross sectional area coverage of biofilm consistently occurred at the bottom portion of the biofilm whilst the highest coverage was in the middle portion of the biofilm. Regarding cell morphology, very large differences were observed. Planktonic cells were rod-shaped, whereas sessile cells expressed an elongated rod morphology and thus were much longer and thinner compared with planktonic cells. Planktonic cells were 1 – 2 m thick and 4 – 5 m long, while sessile cells were 0.5 – 1 m thick and 5 – 100 m long. Long sessile cells resulted in extensive tangling in microcolony pillars, which may have contributed to the structural stability of the pillars. Fibre-like connections of constant diameter were observed between cells, and between the cells and surface. The diameter of these connections was approximately 20 – 30 nm. Viability stains showed that in the bottom portion (from 0 - 20 m above the substratum surface) of the biofilm, most of the cells were dead. However, the portion of covered area attributed to living cells increased past the middle of the biofilm towards the top part of the biofilm. A high percentage of living cells was thus found towards the top part of the biofilm. Overall, 65% (with 2% standard deviation) of the entire biofilm was composed of dead cells. In this way, the results show that operation at high acid conditions comes at a cost of low overall biomass productivity due to decreased active biomass. / Dissertation (MEng)--University of Pretoria, 2017. / Chemical Engineering / MEng / Unrestricted

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