Introduction The ocular surface provides a unique opportunity to study hemodynamics since the vessels can be visualized directly, without treatment and non-invasively. The availability of instruments to measure various hemodynamic parameters on the ocular surface in an objective manner are lacking. The quantification of red blood cell velocity, blood oxygen saturation and conjunctival redness on the ocular surface using novel, validated techniques has the potential of providing useful information about vascular physiology. The specific aims of each chapter are as follows: Chapter 3: The objective was to design, develop and validate a system that would non-invasively quantify the red blood cell velocity in the conjunctival vessels. A tool was developed to automatically analyze video sequences of conjunctival vessels, digitally imaged with high enough magnification to resolve movement of the blood within the vessel. Chapter 4: The objective was to: a) design and develop a method in order to non-invasively quantify the changes in blood oxygen saturation (SO2) in the conjunctival vessels and demonstrate reliability of the measures and, b) demonstrate the application of the method by showing a response to an isocapnic hyperoxic provocation and compare those values to the results from a valid instrument. Chapter 5: The aim of this experiment was to examine variations in ocular redness levels, red blood cell velocities and oxygen saturation levels over time in clinically healthy participants and also to compare differences between two age groups. Chapter 6: The aim of this experiment was to examine the ocular redness levels, red blood cell velocities and oxygen saturation levels in clinically healthy participants when a topical ophthalmic decongestant was instilled onto the eye and to demonstrate the validity of the use of two novel techniques. Chapter 7: The aim of this experiment was to examine ocular redness, red blood cell velocity and oxygen saturation in participants who were habitual soft contact lens wearers (study) compared to those that did not (control) and also to compare differences in silicone (SH) and non-silicone hydrogel wearers. Methods Chapter 3: Simulations representing moving RBCs within a vessel and the random variation of each cell in terms of speed, shape and intensity were created in order to evaluate the performance of the algorithm. For each vessel, a signal that correlated to blood cell position was extracted from each frame, and the inter-frame displacement was estimated through a modified dynamic time warping (DTW) algorithm. This provided the red blood cell velocity over time in each point of the vessels. Thus, from these estimates, the mean red blood cell velocity for each vessel was easily evaluated. The true mean velocity from the simulation with the one estimated by the algorithm was compared and the system accuracy was determined. Chapter 4: a) Conjunctival vessels were imaged with two narrow-band interference filters with O2-sensitive and O2-insensitive peak transmissions using a Zeiss slit lamp at 32x magnification. Optical densities were calculated from vascular segments using the average reflected intensities inside and outside the vessels. Optical density ratios were used to calculate relative oxygen saturation values. Video images of the bulbar conjunctiva were recorded at three times of the day. Measurement repeatability was assessed over location at each time and across consecutive frames. b) Subjects initially breathed air for 10 minutes followed by pure oxygen (O2) for 20 minutes, and then air for a final 10 minute period using a sequential re-breathing circuit. Simultaneously, SO2 values measured with a pulse oximeter ear clip and finger clip were recorded. The validity of the dual wavelength method was demonstrated by comparing the values to those from the ear clip pulse oximeter. Chapter 5: Participants attended eight separate visits over the course of a day. Levels of bulbar conjunctival redness, red blood cell velocity and blood oxygen saturation were measured on a vessel of interest. Chapter 6: Participants attended three separate visits during an allotted 60 minute session. Bulbar conjunctival redness, red blood cell velocity and blood oxygen saturation were measured on a vessel of interest, pre-insertion, just after insertion and, 10 minutes after insertion of a topical ocular decongestant. Significant differences between the three measures were assessed and correlations between the three parameters were reported. Chapter 7: Participants were measured 8 times over the course of a day with their contact lenses in place. Bulbar conjunctival redness, red blood cell velocity and blood oxygen saturation were measured. Results Chapter 3: Results for the simulated videos demonstrated a very good concordance between the estimated and actual velocities supporting its validity. The mean relative error for the modified Dynamic Time Warping (DTW) method is 6%. Chapter 4: The intraclass correlations (ICCs) between the three locations at each time point were 0.93, 0.56 and 0.86 respectively. Measurements across 5 consecutive frames showed no significant difference for all subjects (ICC = 0.96). The ICCs between the two methods at each time point were 0.45, 0.10 and 0.11 respectively. a) There was no significant difference in SO2 between the three locations measured using the dual wavelength method for all subjects. There was also no significant difference between the three locations at any of the time points for the dual wavelength method. b) In response to isocapnic hyperoxic provocation using the dual wavelength method, blood oxygen saturation was increased from control values and subsequently recovered after withdrawal of hyperoxia. Blood oxygen saturation values recorded from the ear clip and finger clip of the pulse oximeter also showed an increase from control values and subsequently recovered after withdrawal of hyperoxia. SO2 comparison between the dual wavelength method and the ear-clip pulse oximeter method did not show a significant difference. The interaction between the two methods and time on SO2 was not significant. Chapter 5: From baseline, the group mean redness and oxygen saturation did not change significantly over time. There was a significant difference in the group mean red blood cell velocity values over time. There was no significant difference between age strata for all three measures. Chapter 6: After drop instillation redness values decreased significantly. There was no change in red blood cell velocity and oxygen saturation over time. There was a moderate significant correlation between SO2 and red blood cell velocity just after drop insertion. Chapter 7: When comparing the study and control groups, no significant difference in redness or SO2 over time was found. RBC velocity over time was found to be significantly different between groups. When comparing the two study groups (SH vs. hydrogel) no significant difference across either measure over time was found. Conclusions Chapter 3: Signal displacement estimation through the DTW algorithm can be used to estimate mean red blood cell velocity. Successful application of the algorithm in the estimation of RBC velocity in conjunctival vessels was demonstrated. Chapter 4: The application of the dual wavelength method was demonstrated and optical density ratios can be used in a reliable manner for relative oxygen saturation measurements. This valid method promises to enable the study of conjunctival O2 saturation under various experimental and physiological conditions. Chapter 5: The results of this study support the theory of metabolic regulation. The lack of any significant change across time for redness and oxygen saturation along with significant changes in red blood cell velocity substantiates this notion. Chapter 6: This study supports the literature regarding metabolic regulation of the microvasculature during the use of various stimuli. The results demonstrated that oxygen saturation levels remain stable even when a significant decrease in ocular redness is measured. The novel techniques used in this experiment demonstrated the expected action of the decongestant further contributing to their application and validity. Chapter 7: In summary, the participants in the study group were habitual contact lens wearers that had lower RBC velocities when compared to the control group supporting the notion that contact lenses initiate a hypoxic response. The lack of change in SO2 in either group supports the theory of metabolic regulation.

Introduction The ocular surface provides a unique opportunity to study hemodynamics since the vessels can be visualized directly, without treatment and non-invasively. The availability of instruments to measure various hemodynamic parameters on the ocular surface in an objective manner are lacking. The quantification of red blood cell velocity, blood oxygen saturation and conjunctival redness on the ocular surface using novel, validated techniques has the potential of providing useful information about vascular physiology. The specific aims of each chapter are as follows: Chapter 3: The objective was to design, develop and validate a system that would non-invasively quantify the red blood cell velocity in the conjunctival vessels. A tool was developed to automatically analyze video sequences of conjunctival vessels, digitally imaged with high enough magnification to resolve movement of the blood within the vessel. Chapter 4: The objective was to: a) design and develop a method in order to non-invasively quantify the changes in blood oxygen saturation (SO2) in the conjunctival vessels and demonstrate reliability of the measures and, b) demonstrate the application of the method by showing a response to an isocapnic hyperoxic provocation and compare those values to the results from a valid instrument. Chapter 5: The aim of this experiment was to examine variations in ocular redness levels, red blood cell velocities and oxygen saturation levels over time in clinically healthy participants and also to compare differences between two age groups. Chapter 6: The aim of this experiment was to examine the ocular redness levels, red blood cell velocities and oxygen saturation levels in clinically healthy participants when a topical ophthalmic decongestant was instilled onto the eye and to demonstrate the validity of the use of two novel techniques. Chapter 7: The aim of this experiment was to examine ocular redness, red blood cell velocity and oxygen saturation in participants who were habitual soft contact lens wearers (study) compared to those that did not (control) and also to compare differences in silicone (SH) and non-silicone hydrogel wearers. Methods Chapter 3: Simulations representing moving RBCs within a vessel and the random variation of each cell in terms of speed, shape and intensity were created in order to evaluate the performance of the algorithm. For each vessel, a signal that correlated to blood cell position was extracted from each frame, and the inter-frame displacement was estimated through a modified dynamic time warping (DTW) algorithm. This provided the red blood cell velocity over time in each point of the vessels. Thus, from these estimates, the mean red blood cell velocity for each vessel was easily evaluated. The true mean velocity from the simulation with the one estimated by the algorithm was compared and the system accuracy was determined. Chapter 4: a) Conjunctival vessels were imaged with two narrow-band interference filters with O2-sensitive and O2-insensitive peak transmissions using a Zeiss slit lamp at 32x magnification. Optical densities were calculated from vascular segments using the average reflected intensities inside and outside the vessels. Optical density ratios were used to calculate relative oxygen saturation values. Video images of the bulbar conjunctiva were recorded at three times of the day. Measurement repeatability was assessed over location at each time and across consecutive frames. b) Subjects initially breathed air for 10 minutes followed by pure oxygen (O2) for 20 minutes, and then air for a final 10 minute period using a sequential re-breathing circuit. Simultaneously, SO2 values measured with a pulse oximeter ear clip and finger clip were recorded. The validity of the dual wavelength method was demonstrated by comparing the values to those from the ear clip pulse oximeter. Chapter 5: Participants attended eight separate visits over the course of a day. Levels of bulbar conjunctival redness, red blood cell velocity and blood oxygen saturation were measured on a vessel of interest. Chapter 6: Participants attended three separate visits during an allotted 60 minute session. Bulbar conjunctival redness, red blood cell velocity and blood oxygen saturation were measured on a vessel of interest, pre-insertion, just after insertion and, 10 minutes after insertion of a topical ocular decongestant. Significant differences between the three measures were assessed and correlations between the three parameters were reported. Chapter 7: Participants were measured 8 times over the course of a day with their contact lenses in place. Bulbar conjunctival redness, red blood cell velocity and blood oxygen saturation were measured. Results Chapter 3: Results for the simulated videos demonstrated a very good concordance between the estimated and actual velocities supporting its validity. The mean relative error for the modified Dynamic Time Warping (DTW) method is 6%. Chapter 4: The intraclass correlations (ICCs) between the three locations at each time point were 0.93, 0.56 and 0.86 respectively. Measurements across 5 consecutive frames showed no significant difference for all subjects (ICC = 0.96). The ICCs between the two methods at each time point were 0.45, 0.10 and 0.11 respectively. a) There was no significant difference in SO2 between the three locations measured using the dual wavelength method for all subjects. There was also no significant difference between the three locations at any of the time points for the dual wavelength method. b) In response to isocapnic hyperoxic provocation using the dual wavelength method, blood oxygen saturation was increased from control values and subsequently recovered after withdrawal of hyperoxia. Blood oxygen saturation values recorded from the ear clip and finger clip of the pulse oximeter also showed an increase from control values and subsequently recovered after withdrawal of hyperoxia. SO2 comparison between the dual wavelength method and the ear-clip pulse oximeter method did not show a significant difference. The interaction between the two methods and time on SO2 was not significant. Chapter 5: From baseline, the group mean redness and oxygen saturation did not change significantly over time. There was a significant difference in the group mean red blood cell velocity values over time. There was no significant difference between age strata for all three measures. Chapter 6: After drop instillation redness values decreased significantly. There was no change in red blood cell velocity and oxygen saturation over time. There was a moderate significant correlation between SO2 and red blood cell velocity just after drop insertion. Chapter 7: When comparing the study and control groups, no significant difference in redness or SO2 over time was found. RBC velocity over time was found to be significantly different between groups. When comparing the two study groups (SH vs. hydrogel) no significant difference across either measure over time was found. Conclusions Chapter 3: Signal displacement estimation through the DTW algorithm can be used to estimate mean red blood cell velocity. Successful application of the algorithm in the estimation of RBC velocity in conjunctival vessels was demonstrated. Chapter 4: The application of the dual wavelength method was demonstrated and optical density ratios can be used in a reliable manner for relative oxygen saturation measurements. This valid method promises to enable the study of conjunctival O2 saturation under various experimental and physiological conditions. Chapter 5: The results of this study support the theory of metabolic regulation. The lack of any significant change across time for redness and oxygen saturation along with significant changes in red blood cell velocity substantiates this notion. Chapter 6: This study supports the literature regarding metabolic regulation of the microvasculature during the use of various stimuli. The results demonstrated that oxygen saturation levels remain stable even when a significant decrease in ocular redness is measured. The novel techniques used in this experiment demonstrated the expected action of the decongestant further contributing to their application and validity. Chapter 7: In summary, the participants in the study group were habitual contact lens wearers that had lower RBC velocities when compared to the control group supporting the notion that contact lenses initiate a hypoxic response. The lack of change in SO2 in either group supports the theory of metabolic regulation.

The use of grading scales is common in clinical practice and research settings. A number of grading scales are available to the practitioner, however, despite their frequent use, they are only poorly understood and may be criticised for a number of things such as the variability of the assessments or the inequality of scale steps within or between scales. Hence, the global aim of this thesis was to study the McMonnies/Chapman-Davies (MC-D), Institute for Eye Research (IER), Efron, and validated bulbar redness (VBR) grading scales in order to (1) get a better understanding and (2) attempt a cross-calibration of the scales. After verifying the accuracy and precision of the objective and subjective techniques to be used (chapter 3), a series of experiments was conducted. The specific aims of this thesis were as follows: • Chapter 4: To use physical attributes of redness to determine the accuracy of the four bulbar redness grading scales. • Chapter 5: To use psychophysical scaling to estimate the perceived redness of the four bulbar redness grading scales. • Chapter 6: To investigate the effect of using reference anchors when scaling the grading scale images, and to convert grades between scales. • Chapter 7: To grade bulbar redness using cross-calibrated versions of the MC-D, IER, Efron, and VBR grading scales. Methods: • Chapter 4: Two image processing metrics, fractal dimension (D) and % pixel coverage (% PC), as well as photometric chromaticity (u’) were selected as physical measures to describe and compare redness in the four bulbar redness grading scales. Pearson correlation coefficients were calculated between each set of image metrics and the reference image grades to determine the accuracy of the scales. • Chapter 5: Ten naïve observers were asked to arrange printed copies of modified versions of the reference images (showing vascular detail only) across a distance of 1.5m for which only start and end point were indicated by 0 and 100, respectively (non-anchored scaling). After completion of scaling, the position of each image was hypothesised to reflect its perceived bulbar redness. The averaged perceived redness (across observers) for each image was used for comparison to the physical attributes of redness as determined in chapter 4. • Chapter 6: The experimental setup from chapter 5 was modified by providing the reference images of the VBR scale as additional, unlabelled anchors for psychophysical scaling (anchored scaling). Averaged perceived redness from anchored scaling was compared to non-anchored scaling, and perceived redness from anchored scaling was used to cross-calibrate grades between scales. • Chapter 7: The modified reference images of each grading scale were positioned within the 0 to 100 range according to their averaged perceived redness from anchored scaling, one scale at a time. The same 10 observers who had participated in the scaling experiments were asked to represent perceived bulbar redness of 16 sample images by placing them, one at a time, relative to the reference images of each scale. Perceived redness was taken as the measured position of the placed image from 0 and was averaged across observers. Results: • Chapter 4: Correlations were high between reference image grades and all sets of objective metrics (all Pearson’s r’s≥0.88, p≤0.05); each physical attribute pointed to a different scale as being most accurate. Independent of the physical attribute used, there were wide discrepancies between scale grades, with sometimes little overlap of equivalent levels when comparing the scales. • Chapter 5: The perceived redness of the reference images within each scale was ordered as expected, but not all consecutive within-scale levels were rated as having different redness. Perceived redness of the reference images varied between scales, with different ranges of severity being covered by the images. The perceived redness was strongly associated with the physical attributes of the reference images. • Chapter 6: There were differences in perceived redness range and when comparing reference levels between scales. Anchored scaling resulted in an apparent shift to lower perceived redness for all but one reference image compared to non-anchored scaling, with the rank order of the 20 images for both procedures remaining fairly constant (Spearman’s ρ=0.99). • Chapter 7: Overall, perceived redness depended on the sample image and the reference scale used (RM ANOVA; p=0.0008); 6 of the 16 images had a perceived redness that was significantly different between at least two of the scales. Between-scale correlation coefficients of concordance (CCC) ranged from 0.93 (IER vs. Efron) to 0.98 (VBR vs. Efron). Between-scale coefficients of repeatability (COR) ranged from 5 units (IER vs. VBR) to 8 units (IER vs. Efron) for the 0 to 100 range. Conclusions: • Chapter 4: Despite the generally strong linear associations between the physical characteristics of reference images in each scale, the scales themselves are not inherently accurate and are too different to allow for cross-calibration based on physical redness attributes. • Chapter 5: Subjective estimates of redness are based on a combination of chromaticity and vessel-based components. Psychophysical scaling of perceived redness lends itself to being used to cross calibrate the four clinical scales. • Chapter 6: The re-scaling of the reference images with anchored scaling suggests that redness was assessed based on within-scale characteristics and not using absolute redness scores, a mechanism that may be referred to as clinical scale constancy. The perceived redness data allow practitioners to modify the grades of the scale they commonly use so that comparisons of grading estimates between calibrated scales may be made. • Chapter 7: The use of the newly calibrated reference grades showed close agreement between grading estimates of all scales. The between-scale variability was similar to the variability typically observed when a single scale is repeatedly used. Perceived redness appears to be dependent upon the dynamic range of the reference images of the scale. In conclusion, this research showed that there are physical and perceptual differences between the reference images of all scales. A cross-calibration of the scales based on the perceived redness of the reference images provides practitioners with an opportunity to compare grades across scales, which is of particular value in research settings or if the same patient is seen by multiple practitioners who are familiar with using different scales.

The use of grading scales is common in clinical practice and research settings. A number of grading scales are available to the practitioner, however, despite their frequent use, they are only poorly understood and may be criticised for a number of things such as the variability of the assessments or the inequality of scale steps within or between scales. Hence, the global aim of this thesis was to study the McMonnies/Chapman-Davies (MC-D), Institute for Eye Research (IER), Efron, and validated bulbar redness (VBR) grading scales in order to (1) get a better understanding and (2) attempt a cross-calibration of the scales. After verifying the accuracy and precision of the objective and subjective techniques to be used (chapter 3), a series of experiments was conducted. The specific aims of this thesis were as follows: • Chapter 4: To use physical attributes of redness to determine the accuracy of the four bulbar redness grading scales. • Chapter 5: To use psychophysical scaling to estimate the perceived redness of the four bulbar redness grading scales. • Chapter 6: To investigate the effect of using reference anchors when scaling the grading scale images, and to convert grades between scales. • Chapter 7: To grade bulbar redness using cross-calibrated versions of the MC-D, IER, Efron, and VBR grading scales. Methods: • Chapter 4: Two image processing metrics, fractal dimension (D) and % pixel coverage (% PC), as well as photometric chromaticity (u’) were selected as physical measures to describe and compare redness in the four bulbar redness grading scales. Pearson correlation coefficients were calculated between each set of image metrics and the reference image grades to determine the accuracy of the scales. • Chapter 5: Ten naïve observers were asked to arrange printed copies of modified versions of the reference images (showing vascular detail only) across a distance of 1.5m for which only start and end point were indicated by 0 and 100, respectively (non-anchored scaling). After completion of scaling, the position of each image was hypothesised to reflect its perceived bulbar redness. The averaged perceived redness (across observers) for each image was used for comparison to the physical attributes of redness as determined in chapter 4. • Chapter 6: The experimental setup from chapter 5 was modified by providing the reference images of the VBR scale as additional, unlabelled anchors for psychophysical scaling (anchored scaling). Averaged perceived redness from anchored scaling was compared to non-anchored scaling, and perceived redness from anchored scaling was used to cross-calibrate grades between scales. • Chapter 7: The modified reference images of each grading scale were positioned within the 0 to 100 range according to their averaged perceived redness from anchored scaling, one scale at a time. The same 10 observers who had participated in the scaling experiments were asked to represent perceived bulbar redness of 16 sample images by placing them, one at a time, relative to the reference images of each scale. Perceived redness was taken as the measured position of the placed image from 0 and was averaged across observers. Results: • Chapter 4: Correlations were high between reference image grades and all sets of objective metrics (all Pearson’s r’s≥0.88, p≤0.05); each physical attribute pointed to a different scale as being most accurate. Independent of the physical attribute used, there were wide discrepancies between scale grades, with sometimes little overlap of equivalent levels when comparing the scales. • Chapter 5: The perceived redness of the reference images within each scale was ordered as expected, but not all consecutive within-scale levels were rated as having different redness. Perceived redness of the reference images varied between scales, with different ranges of severity being covered by the images. The perceived redness was strongly associated with the physical attributes of the reference images. • Chapter 6: There were differences in perceived redness range and when comparing reference levels between scales. Anchored scaling resulted in an apparent shift to lower perceived redness for all but one reference image compared to non-anchored scaling, with the rank order of the 20 images for both procedures remaining fairly constant (Spearman’s ρ=0.99). • Chapter 7: Overall, perceived redness depended on the sample image and the reference scale used (RM ANOVA; p=0.0008); 6 of the 16 images had a perceived redness that was significantly different between at least two of the scales. Between-scale correlation coefficients of concordance (CCC) ranged from 0.93 (IER vs. Efron) to 0.98 (VBR vs. Efron). Between-scale coefficients of repeatability (COR) ranged from 5 units (IER vs. VBR) to 8 units (IER vs. Efron) for the 0 to 100 range. Conclusions: • Chapter 4: Despite the generally strong linear associations between the physical characteristics of reference images in each scale, the scales themselves are not inherently accurate and are too different to allow for cross-calibration based on physical redness attributes. • Chapter 5: Subjective estimates of redness are based on a combination of chromaticity and vessel-based components. Psychophysical scaling of perceived redness lends itself to being used to cross calibrate the four clinical scales. • Chapter 6: The re-scaling of the reference images with anchored scaling suggests that redness was assessed based on within-scale characteristics and not using absolute redness scores, a mechanism that may be referred to as clinical scale constancy. The perceived redness data allow practitioners to modify the grades of the scale they commonly use so that comparisons of grading estimates between calibrated scales may be made. • Chapter 7: The use of the newly calibrated reference grades showed close agreement between grading estimates of all scales. The between-scale variability was similar to the variability typically observed when a single scale is repeatedly used. Perceived redness appears to be dependent upon the dynamic range of the reference images of the scale. In conclusion, this research showed that there are physical and perceptual differences between the reference images of all scales. A cross-calibration of the scales based on the perceived redness of the reference images provides practitioners with an opportunity to compare grades across scales, which is of particular value in research settings or if the same patient is seen by multiple practitioners who are familiar with using different scales.

This project has provided a better understanding of the human conjunctiva, the glistening tissue covering the white of the eye, at the cellular level. The observations of this study may serve as a useful marker against which changes in conjunctival tissue due to disease, surgery, drug therapy or contact lens wear can be assessed. Laser scanning confocal microscopy was used to observe and measure characteristics the conjunctiva of healthy human volunteer subjects. It was concluded that this technique is a powerful tool for studying the human conjunctiva and assessing key aspects of the structure of this tissue. The effects of contact lens wear on the conjunctiva can be investigated effectively at a cellular level using this technology.