Dissertationen zum Thema „Optical polarization and confocal laser microscopy“

Optical imaging techniques have improved much over the last fifty years since the invention of the laser. With a high brightness source many imaging applications which were once inaccessible to researchers have now become a reality. Among these techniques, the most beneficial one is the use of lasers for both wide-field and confocal imaging systems. The aim of this study was to design a laser imaging system based on the concept of laser scanning confocal microscopy. Specifically the optical system was based on optical fibers allowing the user to image remote areas such as the inner surface of rifled gun barrels and/or pipes with a high degree of precision (+/- 0.01 mm). In order to build such a system, initially the theoretical foundation for a confocal as well as a wide-field imaging system was analyzed. Using this basis a free-space optical confocal system was built and analyzed. The measurements support the fact that both the objective numerical aperture and pinhole size play an important role in the radial and axial resolution of the system as well as the quality of the images obtained. To begin construction of a confocal, optical-fiber based imaging system first an all fiber wide-field imaging system was designed and tested at a working wavelength of 1550 nm. Then an all fiber confocal system was designed at a working wavelength of 808 nm. In both cases results showed that while lateral resolution was adequate, axial resolution suffered since it was found that the design of the optical system needs to take into account under-filling of the objective lens, a result common with the use of laser beams whose divergence is not at all like that of a point source. The work done here will aid technology that will be used in the elimination process of faulty rifling fabrication in defense industry. The reason why the confocal technique is preferred to the conventional wide-field one is the need for better resolution in all directions. Theoretical concepts and mathematical background are discussed as well as the experimental results and the practical advantages of such a system.

The aim of submitted diploma thesis is the study of non-isothermal crystallization kinetics of polylactide (PLA) with selected agents (1 %) and observation of the emerging crystalline structure under polarizing optical microscope. The agents were talc, a mixture of organic salts with the addition of amorphous SiO2 (HPN 68L) and zinc stearate (HPN 20E) and LAK-301 (potassium salt of 5-dimethylsulfoisophtalate), which is a nucleating agent developer for PLA. The PLA matrix served as a reference. Non-isothermal crystallization took place on a differential scanning calorimeter at cooling rates () 0,3; 0,5; 0,7; 1; 1,5; 2 °C/min After non-isothermal crystallization, the crystalline fraction (Xc) od PLA was evaluated from X-ray diffraction analysis, and the supramolecular structure was observed after chemical degradative etching using confocal laser scanning microscope. The crystallization kinetics were evaluated by the methods of Jeziorny and Mo and the activation energy of the crystallization was determined according to the Friedmann method. All prepared materials were amorphous (Xc 40 % for up to 1,5 °C/min). However, for LAK-301, Xc decreased to 30 % already at the = 2 °C/min and it can be assumed that with increasing its nucleation activity will decrease. A spherulitic structure was observed in all samples, but the number and size of spherulites decreased with increasing and the appearance varied according to the type of agent. Both kinetic models proved to be unsuitable for materials with low Xc and the highest because the rate of crystallization did not change. With the Jeziorny method, it was possible to evaluate the kinetics only for the relative crystallinity Xt = 29–50 % and with the Mo method it was not possible to evaluate the data for the highest for PLA matrix and sample with HPN 68L. The samples with LAK-301 and HPN 68L showed the lowest activation energy.

Thesis (M.S.)--Missouri University of Science and Technology, 2009.
Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed August 14, 2009) Includes bibliographical references (p. 16-17).

Proposed are novel sensors for extreme environment power plants, laser beam analysis and biomedicine. A hybrid wireless-wired extreme environment temperature sensor using a thick single-crystal Silicon Carbide (SiC) chip embedded inside a sintered SiC probe design is investigated and experimentally demonstrated. The sensor probe employs the SiC chip as a Fabry-Perot (FP) interferometer to measure the change in refractive index and thickness of SiC with temperature. A novel temperature sensing method that combines wavelength-tuned signal processing for coarse measurements and classical FP etalon peak shift for fine measurements is proposed and demonstrated. This method gives direct unambiguous temperature measurements with a high temperature resolution over a wide temperature range. An alternative method using blackbody radiation from a SiC chip in a two-color pyrometer configuration for coarse temperature measurement and classical FP laser interferometry via the same chip for fine temperature measurement is also proposed and demonstrated. The sensor design is successfully deployed in an industrial test rig environment with gas temperatures exceeding 1200 C. This sensor is proposed as an alternate to all-electrical thermocouples that are susceptible to severe reliability and lifetime issues in such extreme environments. A few components non-contact thickness measurement system for optical quality semi-transparent samples such as Silicon (Si) and 6H SiC optical chips such as the one used in the design of this sensor is proposed and demonstrated. The proposed system is self-calibrating and ensures a true thickness measurement by taking into account material dispersion in the wavelength band of operation. For the first time, a 100% repeatable all-digital electronically-controlled pinhole laser beam profiling system using a Texas Instruments (TI) Digital Micro-mirror Device (DMD) commonly used in projectors is experimentally demonstrated using a unique liquid crystal image generation system with non-invasive qualities. Also proposed and demonstrated is the first motion-free electronically-controlled beam propagation analyzer system using a TI DMD and a variable focus liquid lens. The system can be used to find all the parameters of a laser beam including minimum waist size, minimum waist location and the beam propagation parameter M2. Given the all-digital nature of DMD-based profiling and all-analog motion-free nature of the Electronically Controlled Variable Focus Lens (ECVFL) beam focus control, the proposed analyzer versus prior-art promises better repeatability, speed and reliability. For the first time, Three Dimensional (3-D) imaging is demonstrated using an electronically controlled Liquid Crystal (LC) optical lens to accomplish a no-moving parts depth section scanning in a modified commercial 3-D confocal microscope. The proposed microscopy system within aberration limits has the potential to eliminate the sample or objective motion-caused mechanical forces that can distort the original sample structure and lead to imaging errors. A signal processing method for realizing high resolution three dimensional (3-D) optical imaging using diffraction limited low resolution optical signals is also proposed.
Ph.D.
Optics and Photonics
Optics and Photonics
Optics PhD

La polarimétrie de Mueller est une technique optique qui mesure la réponse polarimétrique complète d’un milieu sous la forme d’une seule matrice de Mueller afin de remonter à ses propriétés optiques comme le dichroïsme, la biréfringence et la dépolarisation. Le couplage avec la microscopie non-linéaire (SHG par exemple) permet d’avoir accès à des informations précises sur un milieu biologique (structure, organisation, . . .). Cela impose de passer à une modalité d’imagerie à balayage laser, qui nécessite de mesurer la réponse polarimétrique du milieu pixel-par-pixel en des temps relativement courts (de l’ordre de la microseconde). Le but de cette thèse est de mettre en oeuvre un polarimètre de Mueller dont les cadences d’acquisition sont compatibles avec l’imagerie à balayage laser. Dans un premier temps, un polarimètre de Mueller inédit est proposé, basé sur le codage spectral de la polarisation dont toute l’information polarimétrique de l’échantillon est mesurée sous la forme d’un seul signal d’intensité en un temps record (10 μs). Ce dispositif est constitué d’une source à balayage rapide en longueur d’onde à 100 kHz (ou swept-source), de lames de phase d’ordre élevé et d’un détecteur monocanal. Les erreurs systématiques qui entachent la mesure sont évaluées et des méthodes de correction permettent de les prendre en compte dans une étape d’étalonnage qui utilise la réponse de deux milieux étalons.Ensuite, le polarimètre est implémenté dans un microscope commercial à balayage laser, utilisé initialement pour réaliser de l’imagerie non-linéaire (SHG). Cela requiert un redimensionnement du montage, ainsi que la synchronisation entre les deux systèmes. Par ailleurs, un protocole de calibration du dispositif est développé et permet de tenir compte de l’ensemble des erreurs systématiques du polarimètre indépendamment des anisotropies optiques engendrées par le microscope. Enfin, les premières images polarimétriques de Mueller en microscopie à balayage laser ont été acquises sur des échantillons inhomogènes spatialement (rubans adhésifs et cristaux de roches). La potentialité de la microscopie multimodale est démontrée sur des échantillons de fibroses de foie, en couplant l’imagerie polarimétrique de Mueller et la microscopie non-linéaire au sein d’un seul instrument
Mueller polarimetry is an optical technique allowing the acquisition of the full polarimetric signature of a medium with a single Mueller matrix, and leading to its polarimetric parameters such as dichroism, birefringence and depolarization. Coupling Mueller polarimetry with nonlinear microscopy techniques (SHG for example), more precise information about the medium could be obtained (structure, organization . . .). This imaging technique uses a laser scanning system to measure the Mueller matrix of a medium point-to-point quickly (of the order of the microsecond). The aim of this thesis is to develop a Mueller polarimeter compatible with the laser scanning system. First, a new Mueller polarimeter is proposed using spectral encoding of the polarization and measuring the full polarimetric signature of a sample with a single channeled spectrum in a fast way (10 μs). This setup is composed of a 100 kHz swept-source laser, high order retarders and a single channel detector. Systematic errors on the Mueller matrix measurement are evaluated and correction methods take into account these errors in a calibration step that uses polarimetric signature of two references medium. Then, the polarimeter is implemented on a commercial laser scanning microscope that usually images non-linear contrasts (SHG). The update needs to reduce the dimension of the polarimeter and ensure an electronic synchronization between these two systems. However, a new calibration step is proposed and takes into account all the systematic errors of the polarimeter, independently of the optical anisotropy induced by the microscope. Finally, the images with the first Mueller scanning microscope are obtained with spatially inhomogeneous samples (cellophane tapes, rocks). The potentiality of the multimodal scanning microscopy Mueller/SHG on the same instrument is demonstrated in the case of hepatic fibrosis

Abstract This thesis reports on studies of plasmonic nanoparticles and particularly gold nanostars as signal enhancers and contrast agents for biophotonic applications including visualisation, treatment of living cells and chemical sensing. In this thesis, the optical properties of nanoparticles of different size and morphology and their silica composites were compared. Because they are the most suitable plasmonic nanostructures, gold nanostars were utilised for optical imaging modalities such as confocal microscopy and Doppler optical coherence tomography. The ability of gold nanoparticles to enhance the signal in surface-enhanced vibrational spectroscopy, including Raman and Fourier transform infrared spectroscopy was additionally studied. Finally, various gold nanoparticles were applied for cell optoporation to increase the penetration ability of exogeneous substances. In summary, significant advantages of nanostars such as their low-toxicity, high scattering and contrast abilities, in addition to a broad, tunable, plasmon resonance wavelength range, as well as the capability to enhance the signal of analyte molecules in vibrational spectroscopy were demonstrated in this thesis. The results of this study on the effectiveness of nanostars have a broad scope of utility and open a wide perspective for their utilisation in nanobiophotonics and biomedicine
Tiivistelmä Tämä opinnäytetyö kertoo tutkimuksista, joissa plasmoninanopartikkeleita ja erityisesti kultananotähtiä on käytetty signaalinvahvistimina biofotoniikan sovelluksissa, kuten visualisointi, elävien solujen käsittely ja kemiallinen tunnistus. Tässä työssä verrattiin eri kokoisten ja muotoisten nanopartikkeleiden ja niiden piioksidikomposiittien optisia ominaisuuksia. Sopivimpina plasmoninanorakenteina kultananotähtiä käytettiin optisiin kuvantamismenetelmiin, kuten konfokaalimikroskopiaan ja Doppler-optiseen koherenssitomografiaan. Lisäksi kuvattiin myös kultananopartikkelien kykyä parantaa pinta-aktivoidun värähtelevän spektroskopian signaalia, mukaan lukien Raman- ja Fourier-muunnos-infrapuna-spektroskopia. Lopuksi, eri kultananopartikkeleita käytettiin soluoptoporaatioon eksogeenisten aineiden läpäisevyyden lisäämiseksi. Yhteenvetona, työssä osoitettiin nanotähtien merkittävät edut, kuten matala-myrkyllisyys, suuret sironta- ja kontrastiominaisuudet, laaja plasmoniresonanssin aallonpituusalue ja sen viritettävyys, sekä kyky parantaa analyyttimolekyylien signaalia värähtelyspektroskopiassa. Niinpä tutkimustulokset nanotähtien tehokkuudesta ovat laajasti käyttökelpoisia ja ne avaavat laajan näkökulman niiden hyödyntämiseen nanobiofotoniikassa ja biolääketieteessä

This dissertation proposes, studies and experimentally demonstrates novel liquid crystal (LC) optics to solve challenging problems in RF and photonic signal processing, freespace and fiber optic communications and microscopic imaging. These include free-space optical scanners for military and optical wireless applications, variable fiber-optic attenuators for optical communications, photonic control techniques for phased array antennas and radar, and 3-D microscopic imaging. At the heart of the applications demonstrated in this thesis are LC devices that are non-pixelated and can be controlled either electrically or optically. Instead of the typical pixel-by-pixel control as is custom in LC devices, the phase profile across the aperture of these novel LC devices is varied through the use of high impedance layers. Due to the presence of the high impedance layer, there forms a voltage gradient across the aperture of such a device which results in a phase gradient across the LC layer which in turn is accumulated by the optical beam traversing through this LC device. The geometry of the electrical contacts that are used to apply the external voltage will define the nature of the phase gradient present across the optical beam. In order to steer a laser beam in one angular dimension, straight line electrical contacts are used to form a one dimensional phase gradient while an annular electrical contact results in a circularly symmetric phase profile across the optical beam making it suitable for focusing the optical beam. The geometry of the electrical contacts alone is not sufficient to form the linear and the quadratic phase profiles that are required to either deflect or focus an optical beam. Clever use of the phase response of a typical nematic liquid crystal (NLC) is made such that the linear response region is used for the angular beam deflection while the high voltage quadratic response region is used for focusing the beam. Employing an NLC deflector, a device that uses the linear angular deflection, laser beam steering is demonstrated in two orthogonal dimensions whereas an NLC lens is used to address the third dimension to complete a three dimensional (3-D) scanner. Such an NLC deflector was then used in a variable optical attenuator (VOA), whereby a laser beam coupled between two identical single mode fibers (SMF) was mis-aligned away from the output fiber causing the intensity of the output coupled light to decrease as a function of the angular deflection. Since the angular deflection is electrically controlled, hence the VOA operation is fairly simple and repeatable. An extension of this VOA for wavelength tunable operation is also shown in this dissertation. A LC spatial light modulator (SLM) that uses a photo-sensitive high impedance electrode whose impedance can be varied by controlling the light intensity incident on it, is used in a control system for a phased array antenna. Phase is controlled on the Write side of the SLM by controlling the intensity of the Write laser beam which then is accessed by the Read beam from the opposite side of this reflective SLM. Thus the phase of the Read beam is varied by controlling the intensity of the Write beam. A variable fiber-optic delay line is demonstrated in the thesis which uses wavelength sensitive and wavelength insensitive optics to get both analog as well as digital delays. It uses a chirped fiber Bragg grating (FBG), and a 1xN optical switch to achieve multiple time delays. The switch can be implemented using the 3-D optical scanner mentioned earlier. A technique is presented for ultra-low loss laser communication that uses a combination of strong and weak thin lens optics. As opposed to conventional laser communication systems, the Gaussian laser beam is prevented from diverging at the receiving station by using a weak thin lens that places the transmitted beam waist mid-way between a symmetrical transmitter-receiver link design thus saving prime optical power. LC device technology forms an excellent basis to realize such a large aperture weak lens. Using a 1-D array of LC deflectors, a broadband optical add-drop filter (OADF) is proposed for dense wavelength division multiplexing (DWDM) applications. By binary control of the drive signal to the individual LC deflectors in the array, any optical channel can be selectively dropped and added. For demonstration purposes, microelectromechanical systems (MEMS) digital micromirrors have been used to implement the OADF. Several key systems issues such as insertion loss, polarization dependent loss, wavelength resolution and response time are analyzed in detail for comparison with the LC deflector approach. A no-moving-parts axial scanning confocal microscope (ASCM) system is designed and demonstrated using a combination of a large diameter LC lens and a classical microscope objective lens. By electrically controlling the 5 mm diameter LC lens, the 633 nm wavelength focal spot is moved continuously over a 48 [micro]m range with measured 3-dB axial resolution of 3.1 [micro]m using a 0.65 numerical aperture (NA) micro-objective lens. The ASCM is successfully used to image an Indium Phosphide twin square optical waveguide sample with a 10.2 [micro]m waveguide pitch and 2.3 [micro]m height and width. Using fine analog electrical control of the LC lens, a super-fine sub-wavelength axial resolution of 270 nm is demonstrated. The proposed ASCM can be useful in various precision three dimensional imaging and profiling applications.
Ph.D.
Optics and Photonics
Optics

碩士
國立臺灣大學
光電工程學研究所
92
In this thesis, we study of optical characteristics of AgOx nanostructure thin films with different thickness on the glass substrate using confocal laser scanning microscope and use surface plasmon properties to explain all the optical phenomenon of confocal images. Finally, we demonstrate successfully that the strong evanescence field resulting from surface plasmon resonance of AgOx nanostructure thin films will enhance the lateral resolution of the confocal microscope in the near field. Also, we observe that surface plasmon resonance will interrupt by objects in the near field.

碩士
國立交通大學
照明與能源光電研究所
108
This study uses an advanced confocal microscope to achieve non-invasive optical measurements, In order to pursuit the demand of miniaturization and rapid detection of modern semiconductor industry. This microscope is provided with properties such as horizontal submicron resolutions and longitudinal nanometer resolutions, which can measure the three-dimensional image of the surface of the sample, and the minor change of the nanoscale optical path difference caused by refractive index rapidly. It is helpful for yield detection of the product and the understanding of the operational status. This research while applying voltage operation of the luminescent electrochemical component, the tiny variations of refractive index of the sample can be measured by the change of the ion concentration distribution of the internal. On the other hand, this research also uses the electrically assisted liquid crystal to analyze the characteristics of optical path difference at different polarizations, when operating at various voltages.

Glaucoma is characterized by progressive optic nerve head (ONH) deformation and retinal nerve fiber layer (RNFL) thinning but the relative sequence of ONH and RNFL changes in glaucoma remains largely uncertain. It has been proposed that structural damage of the optic nerve can often be detected before detectable functional loss. Therefore, investigating structural changes of the ONH and RNFL is of importance and relevance in the monitoring and management of glaucoma progression. Spectral domain optical coherence tomography (OCT), scanning laser polarimetry (SLP) and confocal scanning laser ophthalmoscopy (CSLO) are the three prevailing digital imaging technologies for measurement of RNFL thickness, RNFL retardance and ONH parameters, respectively. Although these instruments have been extensively investigated for detection of glaucomatous damage, less is known about their relative performance for detection of change in glaucoma progression. Although previous studies on non- human primates showed that disruption of the microtubule structure of the retinal ganglion cell axons detected by SLP as reduction of RNFL retardance, as well as ONH surface deformation detected by CSLO, could be detected prior to reduction of RNFL thickness measured with OCT, clinical data corroborating this observation are lacking. The sequence of change of RNFL thickness, RNFL retardance and ONH parameters has not been investigated in human glaucoma.
This research project aimed to investigate the performance of OCT, SLP and CLSO for change detection of RNFL and ONH damages, determine the relative sequence of change of RNFL retardance and RNFL thickness and ONH deformation, and evaluate if ocular biomechanical properties, measured as corneal hysteresis by the ocular response analyzer (ORA, Reichert Inc.), influence the detection of ONH and RNFL progression in glaucoma patients. We hypothesized that ONH deformation and loss of RNFL retardance could be detected before detectable RNFL thinning and that the baseline corneal hysteresis would be a risk factor for ONH and RNFL damage in glaucoma.
In the first study, we analyzed 184 eyes of 116 patients with glaucoma and 43 normal eyes of 23 healthy individuals followed for a mean of 4.6 years. All subjects had RNFL retardance and RNFL thickness measurements obtained with GDx ECC (Carl Zeiss Meditec) and Cirrus HD-OCT (Carl Zeiss Meditec), respectively, at 4-month intervals. Progressive reduction of RNFL retardance and RNFL thickness were evaluated with Guided Progression Analysis (GPA, Carl Zeiss Meditec) with reference to the RNFL retardance change map and RNFL thickness change map, respectively. Twenty seven eyes of 26 patients showed progressive RNFL thinning whereas 8 eyes of 8 patients had RNFL retardance reduction in the latest follow-up visit. Seven eyes of 7 patients had progressive RNFL thinning and reduction of RNFL retardance detected by both instruments; all had progressive RNFL thinning evident before reduction of RNFL retardance and the mean lag time was 13.4 months (range: 4.0-37.6 months). The survival time of eyes detected with RNFL thinning was significantly shorter than the survival time of eyes detected with reduction of RNFL retardance (P=0.001). No eyes in the normal group showed progressive RNFL changes during follow-up. Collectively, we showed that at a comparable specificity (100%, 95% confidence interval: 96.3%-100%), progressive RNFL thinning was detected more often than progressive reduction of RNFL retardance and the former preceded the latter in eyes with both progressive RNFL thinning and reduction of RNFL retardance.
In the second study using a similar study design, we investigated the sequence of change of ONH surface depression detected by CSLO (HRT 3, Heidelberg Engineering) and RNFL thinning detected by OCT (Cirrus HD-OCT, Carl Zeiss Meditec) in 146 eyes of 90 glaucoma patients followed at approximately 4-month intervals for an average of 5.4 years. Significant ONH surface depression and RNFL thinning were defined with reference to Topographic Change Analysis (TCA, Heidelberg Engineering) and Guided Progression Analysis (GPA, Carl Zeiss Meditec), respectively. At a specificity of 94.3% (95% confidence interval: 86.2%-97.8%) for both RNFL thinning and ONH surface depression (determined in a normal group comprising 70 eyes from 35 normal subjects), 57 eyes (39.0%) had ONH surface depression, 46 eyes (31.5%) had RNFL thinning, and 23 eyes (15.8%) had both in the glaucoma group. Among these 23 eyes, 19 (82.6%) had ONH surface depression detected prior to RNFL thinning and the median lag time was 15.8 months (range, 4.0-40.8 months). Although only 7.0% of eyes (4/57) had RNFL thinning at the onset of ONH surface depression, 45.7% (21/46) had ONH surface depression at the onset of RNFL thinning. The survival time of eyes with ONH surface depression was significantly shorter than the survival time of eyes detected with RNFL thinning (P=0.002). With reference to the HRT TCA and OCT GPA, ONH surface depression occurred before RNFL thinning in a significant proportion of patients with glaucoma at a comparable specificity.
Of note, a significant proportion of eyes had ONH surface depression without any detectable progressive RNFL thinning in the second study, and vice versa. Investigating whether the risk factors for ONH surface depression and RNFL progression are different is therefore important. In the final study, we investigated if baseline corneal hysteresis is a risk factor for progressive ONH surface depression and RNFL thinning. Following the same cohort of 146 eyes of 90 glaucoma patients for an average of 6.8 years, we detected that 65 eyes (44.5%) had progressive ONH surface depression, 55 eyes (37.7%) had progressive RNFL thinning and 20 eyes (13.7%) had visual field progression (based on the EMGT criteria). After adjusting for ages, CCT, baseline diastolic IOP, average IOP during follow-up, baseline disc area and baseline MD in the cox proportional hazards model, baseline corneal hysteresis was significantly associated with ONH surface depression (HR=0.70, P=0.008), visual field progression (HR=0.56, P=0.019), but not with progressive RNFL thinning (HR=0.96, P=0.751). For each 1-mmHg decrease of baseline CH, the hazards for ONH surface depression and visual field progression increased by 30% and 44%, respectively.
In summary, at a comparable level of specificity, progressive ONH surface depression detected by CSLO could be observed prior to progressive RNFL thinning detected by OCT, which preceded identified reduction of RNFL retardance detected by SLP. For eyes with concomitant ONH surface depression, RNFL thinning and visual field progression, ONH surface depression always preceded visual field progression. Our finding indicates that a time window for therapeutic intervention may exist upon detection of ONH surface depression before irreversible RNFL and visual field loss and that measurement of CH would be useful to predict ONH surface depression and visual field progression.
Further studies are required to investigate the sequence of optic nerve head change and RNFL progression with the same instrument. Whether IOP lowering treatment initiated at the time of ONH deformation would be effective to prevent or slow down RNFL and visual field loss needs to be further investigated. A more reliable and accurate measure of the ocular biomechanical properties is necessary for evaluation of their contribution to glaucoma progression.
青光眼是一種進展性視神經病變，其特徵為﹕視神經乳頭變形，神經纖維層（RNFL）的變薄以及相應的視野缺損。然而，青光眼結構性改變和功能性變化發生的相對順序仍不清楚。視神經結構性改變被認為要早於功能性改變的發生。因此，研究視乳頭的結構性改變具有重要意義，有助於早期診斷青光眼的進展及隨訪青光眼患者。目前主要用於RNFL厚度，RNFL阻滯性以及視乳頭參數的影像學掃描儀器為頻域OCT，鐳射偏振光掃描器（SLP）和共聚焦鐳射掃描眼底鏡（CSLO）。儘管這三種儀器已經廣泛用於青光眼損傷的檢測，但在青光眼患者結構性變化的應用並不常見。既往在非人靈長類動物的實驗中，通過破壞神經節細胞軸突中的微小管結構，從而發現RNFL的阻滯性以及視乳頭的變化要先於RNFL厚度變化的發生。然而在臨床研究中並未得到證實。同時，在青光眼患者中，RNFL厚度變化，RNFL阻滯減少以及視神經頭參數改變之間的先後順序並未得到證實。
本次實驗研究的目的在於探討OCT，SLP及CSLO在診斷青光眼病人RNFL及視乳頭進展的能力，確定RNFL厚度變化，RNFL阻滯性減少以及視神經頭參數改變之間的相對順序，以及評估眼反應分析儀（ORA）測得的角膜粘滯性（CH）是否影響視乳頭及RNFL厚度的進展。我們假設：視神經乳頭的變形，RNFL阻滯性的減少要先於RNFL厚度的變化，基線角膜粘滯性的測量會影響視乳頭及RNFL進展的檢測。116個青光眼病人的184隻眼以及23個正常對照的43隻眼被納入第一個研究中。所有受試物件均接受每4個月一次的OCT以及SLP RNFL的掃描，平均隨訪時間為4.6年。通過OCT及SLP中Guided Progression Analysis（GPA, Carl Zeiss Meditec）程式，一系列RNFL厚度及粘滯性圖被自動分析從而獲得RNFL厚度及粘滯性的變化結果。26個青光眼患者的27隻眼表現為RNFL厚度的進行性變薄，8個患者的8隻眼表現為RNFL粘滯性的減少。其中7個患者的7隻眼同時表現為RNFL厚度變薄及粘滯性的減少，所有這7隻眼的RNFL變薄的發生要早於RNFL粘滯性的減少，兩者間隔時間平均為13.4月（4.0-37.6月）。RNFL厚度變薄者的生存概率明顯小於RNFL粘滯性減少的青光眼患者（P=0.001）。隨訪中，我們未發現正常對照組中RNFL厚度變薄或者粘滯性改變者。總體說來，在同一特異性水準（100%），RNFL厚度的變化頻率高於粘滯性的改變，RNFL厚度的變薄要早於粘滯性減少的發生。
採用相同於第一個研究的研究方法，我們研究CSLO測得的視乳頭表面凹陷以及測得OCT的RNFL厚度變化發生的相對順序。90個青光眼患者的146隻眼以及35個正常對照物件的70隻眼被納入第二個研究中。所有受試物件均接受4個月一次的CSLO及OCT掃描從而獲得一系列的視神經頭表面的拓撲圖像以及RNFL厚度圖。CSLO TCA及OCT GPA程式自動對比基線及隨訪中所獲得的視神經頭表面的拓撲圖像以及RNFL厚度圖，從而獲得視乳頭表面凹陷及RFNL進展報告。平均隨訪5.4年後，CSLO及OCT在診斷視神經頭及RNFL進展的特異性為94.3%，57只青光眼患眼（39.0%）表現為顯著性視乳頭表面凹陷，46隻眼（31.5%）表現為RFNL厚度的進行性變薄，而23隻眼（15.8%）同時表現為視乳頭面凹陷和RFNL的進行性變薄。在這23只眼中，19隻眼（82.6%）變現為視乳頭表面凹陷先於RFNL厚度變薄的發生，間隔時間的中值為15.8個月（4.0-40.8月）。儘管在顯著性視乳頭表面凹陷發生時，僅有7.0%的患眼表現為RNFL厚度的變薄；但是，在RNFL厚度發生顯著性變薄時已有45.7%的患眼表現為視乳頭表面凹陷。視乳頭表面凹陷患眼的生存概率差於RNFL厚度變薄患眼（P=0.002）。在青光眼患者的隨訪中，CLSO TCA測得的視乳頭表面凹陷要早於OCT GPA測得的RNFL厚度的變化。
最後的一個研究目在於評估眼反應分析儀（ORA）測得的基線角膜粘滯性（CH）是否為視乳頭表面凹陷及RNFL厚度變薄的危險因素。平均隨訪同一人群即第二個研究中的90個青光眼患者的146眼6.8年，65隻眼（44.5%）被檢測出具有進行性視乳頭表面凹陷，55隻眼（37.7%）表現為進行性RNFL厚度的變薄，20隻眼（13.7%）表現為進行性視野的缺損（基於EMGT標準）。基線CH與視乳頭表面凹陷，視野進展間具有顯著性相關關係（HR=0.70，P=0.008及HR=0.56，P=0.019），但CH與進行性RNFL厚度變薄間並無顯著性相關關係（HR=0.96，P=0.751）。每1毫米汞柱基線CH的降低，發生視乳頭表面凹陷及視野缺損的危險性將增加30%及44%。CH的測量值與青光眼進展的危險性具有顯著相關關係。
總之，在具有可比性特異性水準下，CSLO檢測的進展性視乳頭表面凹陷的發生要先於OCT檢測的進行性RNFL厚度的變薄，後者的發生早於SLP測得的RNFL粘滯性的改變。對於同時有視乳頭表面凹陷，RNFL厚度變薄及RNFL粘滯性改變的青光眼患眼，視乳頭表面凹陷的發生要早於視野的進展。我們的實驗研究表明了在青光眼患者發生視乳頭表面凹陷時，治療的時間窗的存在有助於避免不可逆的RNFL缺失及視野的缺損。角膜粘滯性的測量對於預測視乳頭表面凹陷及視野進展具有重要意義。
展望未來的研究中，用同一種儀器進行視乳頭及神經纖維層的隨訪，從而得出相對的變化次序很有必要。研究在視乳頭或者神經纖維層發生變化時進行眼壓的干預是否能避免視功能的進一步損傷顯得尤為重要。用於測量角膜生物學特性的更為準確，可信度更高的儀器真正研發中，以及進一步探討角膜生物學特性與青光眼進展之間的關係。
Xu, Guihua.
Thesis Ph.D. Chinese University of Hong Kong 2015.
Includes bibliographical references (leaves 116-145).
Abstracts also in Chinese.
Title from PDF title page (viewed on 18, October, 2016).
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)
Biofilm formation remains a serious concern in industry and in the medical field. They are responsible for huge economical losses in many industrial sectors and for serious infections associated with biomaterial implants and medical devices. These infections are hard to treat because bacteria within the biofilm are more resistant to antibiotics and to the host immune system. Therefore, studying biofilm properties and mechanisms is of utmost relevance to develop efficient approaches to deal with this drawback. Biofilm thickness is an important parameter in biofilm characterization. The main purpose of this thesis was to assess the suitability of Optical Coherence Tomography (OCT) to measure biofilm thickness by comparing the results with the values obtained with the Low Load Compression Test (LLCT) and Confocal Laser Scanning Microscopy (CLSM). OCT is a nondestructive technique, which is increasingly applied on the medical field and allows the acquisition of images in real-time, in situ and with high resolution. Biofilms were grown onto glass slides and the measurements were done in the following order: first OCT, since it is a non-destructive technique, second LLCT and last CLSM. In order to study if slime could influence the measurements, different strains and bacteria exhibiting distinct extracellular polymeric substance (EPS) production capacities were used. Staphylococcus aureus ATCC 12600 is an EPS producer, while Staphylococcus aureus 5298 is not and Streptococcus mutans use extracellular glucans in the presence of sucrose to build its protective matrix, so different treatments with different percentages of sucrose were used in the growth medium during biofilm development. The results obtained proved that OCT is suitable to measure biofilm thicknesses and the presence of slime does not influence the results. In contrast, LLCT measurements showed to be affected by slime and biofilm density. CLSM showed limitations relatively to stain and laser penetration. Additionally, the results obtained emphasize the potential applicability of OCT to investigate the mesoscopic structure of the biofilm.
A formação de biofilmes é um problema sério na industria e na área médica, sendo responsáveis por enormes prejuízos económicos em muitos sectores industriais e por sérias infeções associadas a implantes e dispositivos médicos. Estas infeções são difíceis de tratar, visto que as bactérias quando presentes no biofilme ganham maior resistência a antibióticos e ao sistema imunitário. Por este motivo, estudar as propriedades e mecanismos dos biofilmes é de extrema importância para conseguir desenvolver abordagens eficazes que permitam ultrapassar estes problemas. A espessura dos biofilmes é um parâmetro importante para a sua caracterização. O principal objectivo desta tese foi avaliar a adequação do OCT para medir a espessura dos biofilmes, comparando os resultados com os valores medidos com o LLCT e CLSM. O OCT é uma técnica não destrutiva que tem sido utilizada na área da medicina e permite a aquisição de imagens em tempo real, in-situ e com elevada resolução. Os Biofilmes cresceram em lâminas de vidro e as medições foram feitas na seguinte ordem: primeiro OCT, visto ser uma técnica não destrutiva, seguido do LLCT e por fim o CLSM. Neste trabalho também foi estudado se a presença de EPS pode influenciar as medições em alguma destas técnicas. Para isso foram utilizadas diferentes bactérias e estirpes com capacidades distintas de produção de matrizes extracelulares. A Staphylococcus aureus ATCC 12600 tem capacidade de produzir EPS naturalmente, ao contrário da Staphylococcus aureus 5298. Por outro lado a Streptococcus mutans usa glucanos extracelulares para construir a sua matriz extracelular quando o meio de crescimento possui sacarose. Deste modo foram utilizadas diferentes percentagens deste glícido com o objectivo de obter diferentes tipos de matrizes. Os resultados obtidos provam que o OCT é uma técnica que pode ser usada para medir a espessura de biofilmes. Por outro lado as medições obtidas pelo LLCT são afectadas pela matriz extracelular dos biofilmes e pela sua densidade. O CLSM mostrou limitações relativas a capacidade de penetração do laser e dos corantes. Adicionalmente os resultados obtidos sublinham o potencial do OCT para estudar a estrutura do biofilme ao nível da mesoescala.

Cancer is a world menace. After years of endeavor seeking the end of it, people started to realize that no matter how powerful the therapy could be, detection at early stage is always a cheaper, easier and more successful solution compared with curative methods for cancer developed onto its advanced stage. However, relatively few early-detection approaches have proven sufficiently effective and practical for mass use as a point-of-care tool. An early-cancer screening tool integrating the desired features of sensitive, informative, portable, and cost-effective is in need for the doctors. The progress in optical imaging and Micro-electro-mechanical system (MEMS) technology offers a promise for an innovative cancer screening alternative that is non-invasive, radiation-free, portable and potentially cost-effective. This dissertation investigates handheld instrumentation as multi-modalities of miniature imaging probes with various designs of MEMS devices, to obtain real-time images of epithelial tissue optical and physiological properties, combining the quantitative advantages of spectral analysis with the qualitative benefits of imaging to distinguish early cancer. This dissertation in sequence presents the handheld instruments in the fashions of Laser-scanning confocal microscopy (LSCM), optical diffuse reflectance imaging, nonlinear optical imaging modalities with their subsequent image-guided managements in oral cancer, skin cancer detection, circulating tumor cell (CTC) imaging, and imaging guided surgeries. One of the main challenges facing miniaturization lies in the mechanism of beam deflection across the sample. This dissertation introduces two generations of MEMS devices desgined, fabricated and incorporated in the imaging probes. A two-axis vertical comb driven silicon micromirror was used in the development of a handheld LSCM for oral cancer detection. Though obtaining numerous advantages, this first generation silicon MEMS micromirror suffers from small aperture size and high voltage requirement for actuation, which result in low collection efficiency in fluorescence imaging and medial safety concerns, respectively. Therefore a stainless steel scanner compatible with electrical discharge machining (EDM) process was fabricated with simplified process, low-voltage magnetic actuation and large fluorescence collection efficiency, with its capability demonstrated in the incorporation and embodiment of a handheld hyperspectral nonlinear imaging probe. Besides, software and controlling innovations for handheld imaging modalities are presented. A feedback controlling system for MEMS scanning status monitoring was developed for stabilized imaging rendering. For the sake of further improved imaging stability in handheld imaging and to enable on-site mosaic for large field viewing, a handheld mosaic system was developed and presented.
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Dissertação de mestrado em Biotecnologia, Engenharia de Bioprocessos.
De acordo com as estimativas mais recentes produzem-se mundialmente cerca de 286 milhares de toneladas de soro de leite por dia. O soro produzido diariamente pode providenciar, em média, 2,4 milhares de toneladas de proteínas que, potencialmente, podem satisfazer as necessidades proteicas diárias a cerca de 35 milhões de pessoas. Durante muito tempo o soro foi encarado como um resíduo industrial ou produto com valor comercial residual. Só nas três últimas décadas foram desenvolvidos processos de separação que permitem obter ingredientes alimentares com alto teor proteico a partir do soro de leite. As proteínas do soro de leite bovino estão entre as mais equilibradas fontes de aminoácidos da dieta humana; para além disso, foram já descritas muitas actividades biológicas, essenciais para o desenvolvimento harmonioso da cria, com repercussões benéficas também no ser humano. Muitas dessas propriedades estão encriptadas nas moléculas proteicas manifestando-se após proteólise que pode ocorrer durante o processo de digestão ou durante o processamento enzimático e/ou fermentativo. A tripsina, enzima seleccionada para o trabalho desenvolvido, revela-se uma enzima interessante para a libertação de péptidos bioactivos, dada a sua especificidade, mas também para o aumento da digestibilidade e diminuição da alergenicidade das proteínas do soro de leite bovino (capítulo 1). As proteínas do soro podem ser usadas numa grande variedade de aplicações na indústria alimentar. Uma das funcionalidades das proteínas resulta da capacidade que possuem de responder a alterações ambientais alterando a sua conformação nativa e, por agregação, formar redes elásticas tridimensionais – gelificação. Os géis conferem estrutura, textura e estabilidade aos alimentos; possibilitam, ainda, a retenção de grandes quantidades de água e outras pequenas moléculas dentro da matriz alimentar (capítulo 2). No capítulo 3 do trabalho que se apresenta, estudaram-se as alterações do comportamento reológico (a baixa deformação) que ocorrem durante o processo de gelificação por aquecimento de suspensões de proteínas de soro, a pH neutro e força iónica de 220 mM. As propriedades reológicas dos géis finais foram estudadas em função da concentração de proteínas e do grau de hidrólise enzimática a que, previamente, as proteínas do soro de leite foram sujeitas. O aumento do grau de hidrólise reduz drasticamente a capacidade de gelificação das proteínas do soro e, ao contrário da variação da concentração, altera o balanço de forças que suportam a estrutura tridimensional do gel, provavelmente, diminuindo a contribuição das ligações covalentes e hidrofóbicas e aumentando a contribuição das pontes de hidrogénio. No capítulo 4, estudaram-se as alterações do comportamento reológico que ocorrem durante o processo de gelificação por aquecimento de sistemas mistos de proteínas de soro (nativas ou hidrolisadas) e polissacarídeos neutros (galactomananos), a pH neutro e força iónica de 220 mM. Para os vários sistemas estudados, as propriedades reológicas dos géis finais foram estudadas em função da concentração de polissacarídeos. Recorrendo a técnicas microscópicas (microscopia óptica e microscopia confocal de varrimento laser), verificou-se que, da incompatibilidade termodinâmica entre os dois tipos de biopolímeros, resultou uma separação de fases do tipo segregativo. Este fenómeno, por ser simultâneo ao processo de gelificação, condiciona fortemente as propriedades reológicas dos géis, pois, se por um lado favorece a concentração da fase proteica gelificante, por outro, diminui a conectividade dessa mesma fase. A quantificação da massa de água (e solutos), excluída durante o processo de gelificação (sinérese), permitiu verificar que a presença de polissacarídeos diminui a severidade do fenómeno.
Accordingly to the last estimates, the daily production of milk whey is about 286 thousand tons. The produced whey can provide 2.4 thousand tons of proteins that, potentially, can assure the daily protein requirements of about 35 million people. In the past, whey was looked upon as an industrial residue or a low value product. Only in the last three decades separation processes were developed that allow the production of food ingredients with high protein content from milk whey. Proteins from bovine milk whey are between the more equilibrated human dietary sources of amino acids; they have been described as possessing many essential biological activities which are important for the harmonious development of the calf and have also beneficial repercussions for humans. Many of these bioactivities are encrypted in the native protein molecules and are only revealed after proteolysis during the natural digestion process or during enzymatic and/or fermentative treatments. For this work, trypsin was selected to hydrolyse whey proteins since, due to its specificity, trypsin is an interesting enzyme for the liberation of bioactive peptides and, also, for the improvement of protein digestibility and for decreasing protein allergenicity (chapter 1). Whey proteins can be used for a wide range of industrial food applications. One of proteins functionalities results from their capacity to respond to changes in their physicochemical environment by modifying their native conformation and forming, after aggregation, tridimensional elastic networks – gelling. Gels confer structure, texture and stability to food products; they also allow the retention of large quantities of water and other small molecules inside the food matrix (chapter 2). In the 3rd chapter of the present work, the changes in the rheological behaviour during the heat-induced gelling process of whey protein dispersions were studied by small deformation rheology (neutral pH and ionic force 220 mM). The rheological properties of the final gels were studied as a function of protein concentration and as a function of the degree of enzymatic hydrolysis. Hydrolysis reduces dramatically the gelling ability of whey proteins and alters the balance of the different forces that sustain the elastic network, probably, increasing the proportion of hydrogen bonds and decreasing the proportion of covalent and hydrophobic interactions. In the 4th chapter, the changes in the rheological behaviour during the heat-induced gelling process of mixed systems of whey proteins (native or hydrolysed) and neutral polysaccharides (galactomannans) were studied at neutral pH and ionic force 220 mM. The rheological properties of final gels were studied as a function of polysaccharide concentration. Using microscopic techniques (optical microscopy and confocal laser scanning microscopy), thermodynamic incompatibility between the two polymers, inducing segregative phase separation, was observed. When phase separation and gelling processes occur simultaneously the rheological properties of mixed gels are greatly influenced because phase separation favour the protein gelling phase concentration but, at the same time, decreases its connectivity. The quantification of the water (and solutes) excluded during the gelling process (syneresis) permits to verify that, when polysaccharide is present during the gelling process, syneresis is less severe.
Fundação para a Ciência e a Tecnologia (FCT) - Projecto (POCTI/QUI/36452/2000) - Instituto de Ciências e Tecnologia Agrárias e Agro-Alimentares.

The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations.