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BACKGROUND: Intraocular scatter is an important factor when considering the performance of the human eye as it can negatively affect visual performances (e.g. glare). However, and in contrast to other optical factors that also affect vision such as high order aberrations, there is currently no efficient method to measure accurately and objectively the amount and the angular distribution of forward light scatter in the eye. Various methods and instruments exist to assess forward light scatter (FLS) but the relation between these methods has rarely been quantified. In addition, FLS measurements obtained with existing instruments cannot be related to any physiological factors due to the absence of a valid model. PURPOSE: To investigate the relations between some of the main methods to measure forward light scatter, and to develop an experimental set -up for the objective measurement of forward light scatter that could be ideally related to physiological parameters. METHODS: After a short review of intraocular light scatter, the three main methods used to assess forward light scattering are compared. In this sense, the C-quant (CQ) straylight meter is compared to the van den Berg (VDB) straylight meter and the Hartmann-Shack spot pattern analysis obtained from the Hartmann-Shack aberrometer. The potential of the new Oculus Pentacam functionalities for providing information on backward light scatter (BLS) are also investigated. Finally, an innovative prototype for objective assessment of intraocular light scattering together with a scatter model of the eye is presented. RESULTS and DISCUSSION: Although no significant relationship was found between the different instruments considered (VDB straylight meter, CQ, Pentacam), our results allowed us to clarify some possible confusion introduced by previously published results and to illustrate the fact that existing commercial instruments such as aberrometers and the Pentacam cannot be used to measure FLS without at least some major modifications (hardware or software). Preliminary results with the prototype built in this study suggest that it could be used for the objective measurement of intraocular light scatter. Relating this measurement to physiological parameters stays however elusive, a fact that widens the future scope of this research.

Use of a smartphone as an optical detector for paper microfluidic devices has recently gained substantial attention due to its simplicity, ease of use, and handheld capability. Utilization of a UV light source enhances the optical signal intensities, especially for the particle immunoagglutination assay that has typically used visible or ambient light. Such enhancement is essential for true assimilation of assays to field deployable and point-of-care applications by greatly reducing the effects by independent environmental factors. This work is the first demonstration of using a UV LED (UVA) to enhance the Mie scatter signals from the particle immunoagglutination assay on the paper microfluidic devices and subsequent smartphone detection. Smartphone's CMOS camera can recognize the UVA scatter from the paper microfluidic channels efficiently in its green channel. For an Escherichia coli assay, the normalized signal intensities increased up to 50% from the negative signal with UV LED, compared with the 4% to 7% with ambient light. Detection limit was 10 colony-forming units/mL. Similar results were obtained in the presence of 10% human whole blood.

Spectroscopy and other optical methods can often be employed with limited or no sample preparation, making them well suited for in situ and in vivo analysis. This dissertation focuses on the use of a near-infrared spectroscopy (NIRS) and polarized light scatter for two such applications: the assessment of cardiovascular disease, and the validation of cleaning processes for pharmaceutical equipment.There is a need for more effective in vivo techniques for assessing intravascular disorders, such as aortic aneurysms and vulnerable atherosclerotic plaques. These, and other cardiovascular disorders, are often associated with structural remodeling of vascular walls. NIRS has previously been demonstrated as an effective technique for the analysis of intact biological samples. In this research, traditional NIRS is used in the analysis of aortic tissue samples from a murine knockout model that develops abdominal aortic aneurysms (AAAs) following infusion of angiotensin II. Effective application of NIRS in vivo, however, requires a departure from traditional instrumental principles. Toward this end, the groundwork for a fiber optic-based catheter system employing a novel optical encoding technique, termed molecular factor computing (MFC), was developed for differentiating cholesterol, collagen and elastin through intervening red blood cell solutions. In MFC, the transmission spectra of chemical compounds are used to collect measurements directly correlated to the desired sample information.Pharmaceutical cleaning validation is another field that can greatly benefit from novel analytical methods. Conventionally cleaning validation is accomplished through surface residue sampling followed by analysis using a traditional analytical method. Drawbacks to this approach include cost, analysis time, and uncertainties associated with the sampling and extraction methods. This research explores the development of in situ cleaning validation methods to eliminate these issues. The use of light scatter and polarization was investigated for the detection and quantification of surface residues. Although effective, the ability to discriminate between residues was not established with these techniques. With that aim in mind, the differentiation of surface residues using NIRS and MFC was also investigated.

Introduction and Purpose: Vascular pathologies play an important role in the etiology and progression of number of ocular diseases. Many instruments are developed to monitor retinal hemodynamics, including the Canon Laser Blood Flowmeter (CLBF), in an attempt to better understand the pathophysiology of the disease (Chapter 2). The purpose of this thesis is to determine the impact of light scatter on retinal arteriolar hemodynamic measurement assessed by the CLBF as intraocular light scatter is an inevitable consequence of ageing and particularly cataract. Methodology: Chapter 4 – Artificial light scatter model: One eye from each of 10 healthy young subjects between the ages of 18 and 30 (23.6 ± 3.4) was randomly selected. To simulate light scatter, cells comprising a plastic collar and two plano lenses were filled with solutions of differing concentrations of polystyrene microspheres (Polysciences Inc., USA). 0.002%, 0.004%, 0.006%, 0.008% were prepared, as well as distilled water only. After a preliminary screening to confirm subject eligibility, seven arteriolar hemodynamic measurements were taken by randomly placing the cells between the CLBF objective lens and the subjects’ cornea. Chapter 5 – Ten patients scheduled for extracapsular cataract extraction using phacoemulsification and intraocular lens implantation between the ages of 61 and 84 (mean age 73 years, SD ± 8) were prospectively recruited. Two visits were required to complete the study; One prior to the surgery and one at least six weeks after the surgery to allow for full post-operative recovery. The severity of cataract was documented using the Lens Opacity Classification System (LOCS, III) at the first visit. Each subject underwent visual function assessment at both visits using logMAR Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity charts and the Brightness Acuity Tester (BAT). Retinal arteriolar hemodynamics were measured at both visits using the high intensity setting of the Canon Laser Blood Flowmeter. Results: Chapter 4: Our light scatter model resulted in an artifactual increase of retinal arteriolar diameter (p<0.0001) and thereby increased retinal blood flow (p<0.0001). The 0.006% and 0.008% microsphere concentrations produced significantly higher diameter and flow values than baseline. Centerline blood velocity, however, was not affected by light scatter. Retinal arteriolar diameter values were significantly less with the high intensity laser than with the low intensity laser (p=0.0007). Chapter 5: Group mean retinal arteriolar diameter and blood flow were reduced following extracapsular cataract extraction (Wilcoxon signed-rank test, p=0.022 and p=0.028 respectively); however, centerline blood velocity was unchanged (Wilcoxon signed-rank test, p=0.074). Conclusions: Using an artificial light scatter model (Chapter 3), we demonstrated that the densitometry assessment of vessel diameter is increasingly impacted as the magnitude of artificial light scatter increases; this effect can be partially negated by increasing laser intensity. We showed similar results in the presence of cataract (Chapter 4) by measuring the retinal arteriolar hemodynamics before and after removal of cataract. Care needs to be exercised in the interpretation of studies of retinal vessel diameter that use similar densitometry techniques as cataract is an inevitable consequence of aging.

Introduction and Purpose: Vascular pathologies play an important role in the etiology and progression of number of ocular diseases. Many instruments are developed to monitor retinal hemodynamics, including the Canon Laser Blood Flowmeter (CLBF), in an attempt to better understand the pathophysiology of the disease (Chapter 2). The purpose of this thesis is to determine the impact of light scatter on retinal arteriolar hemodynamic measurement assessed by the CLBF as intraocular light scatter is an inevitable consequence of ageing and particularly cataract. Methodology: Chapter 4 – Artificial light scatter model: One eye from each of 10 healthy young subjects between the ages of 18 and 30 (23.6 ± 3.4) was randomly selected. To simulate light scatter, cells comprising a plastic collar and two plano lenses were filled with solutions of differing concentrations of polystyrene microspheres (Polysciences Inc., USA). 0.002%, 0.004%, 0.006%, 0.008% were prepared, as well as distilled water only. After a preliminary screening to confirm subject eligibility, seven arteriolar hemodynamic measurements were taken by randomly placing the cells between the CLBF objective lens and the subjects’ cornea. Chapter 5 – Ten patients scheduled for extracapsular cataract extraction using phacoemulsification and intraocular lens implantation between the ages of 61 and 84 (mean age 73 years, SD ± 8) were prospectively recruited. Two visits were required to complete the study; One prior to the surgery and one at least six weeks after the surgery to allow for full post-operative recovery. The severity of cataract was documented using the Lens Opacity Classification System (LOCS, III) at the first visit. Each subject underwent visual function assessment at both visits using logMAR Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity charts and the Brightness Acuity Tester (BAT). Retinal arteriolar hemodynamics were measured at both visits using the high intensity setting of the Canon Laser Blood Flowmeter. Results: Chapter 4: Our light scatter model resulted in an artifactual increase of retinal arteriolar diameter (p<0.0001) and thereby increased retinal blood flow (p<0.0001). The 0.006% and 0.008% microsphere concentrations produced significantly higher diameter and flow values than baseline. Centerline blood velocity, however, was not affected by light scatter. Retinal arteriolar diameter values were significantly less with the high intensity laser than with the low intensity laser (p=0.0007). Chapter 5: Group mean retinal arteriolar diameter and blood flow were reduced following extracapsular cataract extraction (Wilcoxon signed-rank test, p=0.022 and p=0.028 respectively); however, centerline blood velocity was unchanged (Wilcoxon signed-rank test, p=0.074). Conclusions: Using an artificial light scatter model (Chapter 3), we demonstrated that the densitometry assessment of vessel diameter is increasingly impacted as the magnitude of artificial light scatter increases; this effect can be partially negated by increasing laser intensity. We showed similar results in the presence of cataract (Chapter 4) by measuring the retinal arteriolar hemodynamics before and after removal of cataract. Care needs to be exercised in the interpretation of studies of retinal vessel diameter that use similar densitometry techniques as cataract is an inevitable consequence of aging.

Tissue biopsy and swab culture are the gold standards for diagnosing tissue infection; these tests require significant time, diagnostic costs, and resources. Towards earlier and specific diagnosis of infection, a non-destructive, rapid, and mobile detection device is described to distinguish bacterial species via light scatter spectra from the surface of an infected tissue, reagent-free. Porcine skin and human cadaveric skin models of wound infection were used with a 650 nm LED and an angular photodiode array to detect bacterial infections on the tissue surface, which can easily be translated to a typical CMOS array or smartphone. Tissue samples were inoculated with Escherichia coli, Salmonella Typhimurium, or Staphylococcus aureus and backscatter was collected from 100 degrees to 170 degrees in 10 degrees increments; each bacterial species resulted in unique Mie scatter spectra. Distinct Mie scatter spectra were obtained from epidermis (intact skin model) and dermis (wound model) samples, as well as from porcine and human cadaveric skin samples. Interactions between bacterial colonies and lipid particles within dermis samples generated a characteristic Mie scatter spectrum, while the lipid itself did not contribute to such characteristic spectrum as corroborated with body lotion experiments. The designed angular photodiode array is able to immediately and non-destructively detect tissue bacterial infection and identify the species of infection within three seconds, which could greatly improve point of care diagnostics and antibiotic treatments.

Abstract This study was carried out to investigate in vivo the changes of the human lens autofluorescence (AF) with aging and cataractogenesis. Measurements were performed in the blue-green AF range (495 nm/520 nm) using a fluorometer designed, built and now clinically tested in our department. 43 random eyes of 43 healthy volunteers aged 6-86 years, five of each decade, were studied for effects of aging and 84 eyes of 84 patients with cortical, nuclear, posterior subcapsular or mixed lens opacities were studied for differences of various cataract types. The results were compared with the back light scatter values obtained by the commercially available Interzeag Lens Opacity Meter 701. Also AF and back light scatter of the lens were measured from 122 smoking males aged 57 to 76 years who participated in a cancer prevention study. The results were compared with the widely used subjective lens opacities classification system, LOCS III. In addition data was collected from 30 randomly chosen eyes of as many subjects with varying degrees of yellow-brown lens coloration in an otherwise healthy eye. We studied the influence of lens yellowing expressed by means of lens AF on visibility of retinal nerve fiber layer in black-and-white images. Lens AF profile consists of anterior and posterior peaks and a central plateau. The height of the anterior peak was used as a measure of the maximum AF value. The square root of the ratio between the posterior and the anterior AF peaks was used for estimating the lens transmission. Our technique was highly reproducible. The coefficient of variation was 3.9% for maximum AF and 2.9% for the lens transmission index. Both the maximum AF and light scatter were exponentially increased with age (r = 0.95 and 0.94, respectively; p &lt; 0.0001). According to the regression line of AF begins to increase in early childhood. It appears by extrapolation to be absent at birth. In contrast light scatter in the lens was present even in young children. The lens transmission for blue-green light, determined from the lens AF curve, was almost unchanging with age up to 60 years. Thereafter it decreased rapidly and the interindividual variation increased.In cataractous lenses the mean AF and scatter values differed statistically significantly from those of age matched healthy controls. The highest AF values were measured in nuclear cataracts where AF was also related to visual acuity and an increasing yellow-brown colour of the nucleus. About half of the total variation of the transmission index values could be accounted for by changes in nuclear colour as assessed by the LOCS III grading system. The transmission index provided a more precise prediction about nuclear colour and opalescence than age or light scatter did. In cortical cataracts the AF curve was low and flattened and the maximum AF value was significantly lower than in the age matched control eyes. The highest light scatter values were measured from cortical cataracts, but the correlation between LOCS III cortical grades and light scatter values was rather weak. Posterior subcapsular cataracts cannot be quantified either with AF or with light scatter measurements. Lens yellowing, expressed as lens AF, had an actual effect on retinal nerve fiber layer visibility. AF measurements provided a better prediction about the visibility score than age or visual acuity did. The results of the present study indicate that the lens autofluorescence measurement may be a useful additional tool together with a subjective grading system in the follow-up of optical changes occurring in the nuclear region of the lens.

This diploma thesis deals with a design modification of detection part of the laboratory instrument for measuring the topography of rough surfaces – laser goniometric scatterometer (SM II). Design modification is based on replacement of so far used detector instead for the detector of higher quality with better measurement parameters. The first part of the diploma thesis contains theoretical basics, which are necessary to understand the relationship between scalar diffraction theory and scattering measurements of monochromatic light. The emphasis is on the importance of choosing appropriate detection coordinates, which are affecting the aberrations of detected diffracted light. The practical part is dedicated to improving the sensitivity of the detection part of the scatterometer SM II that is used in The laboratory of coherence optics at IFE FME BUT. This part justifies the choice of the detector which predetermines both the use of optical elements and the overall design of the detection part as the goniometer.