Monitoring phytoplankton species composition and their abundance are routine tasks in marine ecological research and environmental monitoring. As phytoplankton populations are highly heterogeneous in terms of size, morphology, and most significantly, their abundance can change drastically in a very short time, it is extremely difficult to quantify and monitor them and there are demands on the instrumentation. Conventional optical microscopy and flow cytometry are the main tools to enumerate and identify phytoplankton, but they have a compromise between spatial information and acquisition speed. While imaging flow cytometry has the potential to integrate the benefit of high spatial resolution from optical microscopy and the advantage of high throughput from flow cytometry, two intrinsic blur sources, motion blur and out-of-focus blur, prevent imaging flow cytometers from obtaining high spatial resolution images with high throughput. To address these limitations, in this work, a novel light sheet based fluorescence imaging flow cytometer has been proposed, constructed, and tested for phytoplankton analysis. Both 2D and 3D imaging mode of the light sheet based fluorescence imaging flow cytometer have been investigated. In the 2D imaging mode, the instrument can screen untreated costal water samples at a volumetric throughput up to 1 ml/min. The instrument demonstrated shows a high immunity to motion blur, and all-in-focus fluorescence images are captured with a lateral resolution of 0.75 ± 0.06 µm for a wide size range ~ 1 µm to ~ 200 µm that includes pico-, nano-and microphytoplankton. This is made possible by suppressing the out-of-focus blur using thin light sheet illumination and image deconvolution, and by precluding the motion blur with a unique flow configuration. With these abilities, the instrument demonstrated has high potential as a practical field instrument for monitoring phytoplankton. In the 3D imaging mode, the instrument can scan a large number of phytoplankton cells in a short time with spatial resolution as achieved by light sheet microscopy. The lateral resolution is 0.81 ± 0.07 µm, and axial resolution in terms of FWHM of the axial scattering PSF is 1.42 ± 0.15 µm. The volumetric throughput of the instrument is 0.5 µl/min. This is benefitted from the improvement that 3D images can be acquired without the need of sample immobilization, in contrast to existing 3D imaging approaches, such as confocal fluorescence microscopy. Preliminary results from untreated coastal water samples and cultured samples show promising potentials of the instrument for phytoplankton monitoring and scientific research.

Thesis (MTech(Electrical Engineering))--Cape Technikon, Cape Town,1999 / This thesis describes the design and implementation of a new feedback controller for a scanning tunneling microscope or STM. The previous controller had several shortcomings when it came to the data throughput rate of the data acquisition system, the scan rate, and the way the data was stored and displayed. The initial investigation was done to determine the most cost effective way to implement the data acquisition system. Various approaches such as DSP systems, analogue systems and microcontroller systems were looked at. The investigation also looked at the best way to get the data from the Z directional control loop to the PC for displaying the data. The final choice was to use an ultra fast microcontroller for the control loop implementation and to change the DOS based software for Windows based software. The embedded system was divided into two parts. The first was the controller for the X and Y scan directions, and the second was for the Z scan direction. A digital PI control loop was implemented on the Z controller to control the height of the scan tip above the specimen surface. The microcontroller that was chosen for this was the Microchip PIC17c43. The data transfer to the PC was done with a PC-14 programmable digital input/output card. Two options for the implementation of the PC-14 software were considered. The first option was the software that was bundled with the card. This software proved to be very slow, so special device-driver-based software was developed to control the PC-14 card and the data transfer to and from the Pc. The PC software was implemented using Visual C++. Both the XY and the Z controllers proved to be working satisfactorily in the existing STM arrangement. It was discovered that the XY controller was overloaded with the many tasks that it has to perform, and a suitable alternative system to replace the XY controller is proposed. The selection of the PC that will be used for the data acquisition system is also discussed. It was found that this choice had a very big influence on the design of the final system because of the difference in PC bus design. Several proposals to increase the functionality of the PC software are also made.

Orientador: Luis Rogério de Oliveira Hein / Banca: George de Paula Bernardes / Banca: Carlos Renato Zacharias / Banca: Arnaldo Homobono Paes de Andrade / Banca: Durval Rodrigues Júnior / Resumo: O objetivo deste trabalho é desenvolver rotinas para a reconstrução de relevos de superfícies irregulares, particularmente aquelas observadas por microscopia óptica ou eletrônica de varredura, e estudar os conceitos de imageamento envolvidos, visando definir critérios para uma futura padronização dos métodos de processamento na área de estudo de relevos. As imagens obtidas em um microcoscópio óptico Nikon Epiphot 200 e em um microscópio eletrônico de varredura 5600 LV, respectivamente, foram processadas por rotinas de reconstrução a partir do foco e a partir do paralaxe. Essas rotinas implementadas apresentam a inovação, respectivamente, de permitir que o usuário aumente o tamanho da região onde o critério de foco é empregado e a diminuição do tempo de processamento através da análise da transformada de Hartley somente na direção horizontal. Os resultados mostram que a rotina de reconstrução a partir do foco implementada neste trabalho apresentou as mesmas tendências obtidas em um rugosímetro Mitutoyo Surtest 301 e menor quantidade de ruídos quando é utilizado polarizadores; e que a rotina de reconstrução a partir do paralaxe apresenta um erro médio de 5 ± 2% para pontos do par estéreo que são epipolares. Finalmente, o menor tempo de processamento, no caso da reconstrução a partir do paralaxe, permite aumentar a resolução espacial (maior quantidade de pixels) visando diminuir a rotação usada para obter o par estéreo e consequentemente reduzir a oclusão de área e o problema de pontos não epipolares. / Abstract: The objective of this work is to develop routines for the reconstruction of irregular surfaces relieves, particularly those observed by optical microscopy or scanning electron microscopy, and to study the imaging concepts, seeking to define criteria for a future standardization of the processing methods in the area of study of relieves. The images were obtained in an optical microscope Nikon Epiphot 200 and in an scanning electron microscope 5600. They were, respectively, processed by depth from focus routine and parallax measurement. Those implemented routines present the innovation, respectively, of allowing the user to increase the size of the area where the focus criterion is used and the decrease of the time of processing through the analysis of the transformed of Hartley only in the horizontal direction. The results show that the depth from focus routine implemented in this work it presented the same tendencies obtained in a Mitutoyo Surtest 301 contact profilometer and smaller amount of noises when polarizers is used. The parallax routine presents error of 5 ± 2% for epipolar points from the stereo pair. Finally, in the case of the reconstruction from parallax is necessary to reduce the rotation used to obtain the stereo pair and to increase the space resolution (larger amount of pixels) to reduce the area occlusion and the problem of non-epipolar points.

Die vorliegende Arbeit befasst sich mit zwei neuen methodischen Ansätzen auf dem Gebiet der Fluoreszenzmikroskopie. Im ersten Teil der Arbeit wir eine Methode vorgestellt, mit der die Winkelselektivität der Fluoreszenzanregung verbessert werden kann. Die ExPAN (excitation polarization angle narrowing) genannte Technik nutzt stimulierte Emission, um den Effekt der Photoselektion zu vergrößern. ExPAN lässt sich potentiell für verschiedene Methoden einsetzen, in denen fluoreszenzmarkierte Proben untersucht werden und ist insbesondere im Kontext von Fluoreszenzanisotropie-Messungen oder der Bestimmung von molekularen Orientierungen von Interesse. Solche Methoden finden in den Biowissenschaften breite Anwendung und werden z.B. zum Studium von Rezeptor-Liganden-Interaktionen oder der Proteindynamik eingesetzt. Im Rahmen der Arbeit wird ExPAN in Kombination mit einem neuen Ansatz in der Weitfeldmikroskopie untersucht, bei der die Orientierung von Farbstoffmolekülen als Kontrastmechanismus genutzt wird. Dabei wird die Polarisationsrichtung des Anregungslichts rotiert, um Informationen über die molekulare Orientierung zu gewinnen. Aufgrund der Photoselektion weist das Fluoreszenzsignal von Molekülen mit bevorzugter Ausrichtung dadurch eine periodische Modulation auf. Es wird gezeigt, dass diese Information zur Unterscheidung von Molekülen mit abweichender Orientierung genutzt werden kann, selbst wenn sich deren Signale räumlich überlagern. Für die Versuche wurde ein modifiziertes Weitfeld-Mikroskop konstruiert und die Methode zum einen experimentell an Einzelmolekülen und zum anderen mittels Simulationen erprobt. Dabei konnten Signale von Farbstoffmolekülen mit einem Abstand von bis zu 80 nm separiert werden. Darüber hinaus wurde ein moduliertes Fluoreszenzsignal bei oberflächenmarkierten Mikropartikeln in wässriger Lösung sowie bei fixierten biologischen Proben beobachtet. Eine Verbesserung der Photoselektion durch ExPAN wird experimentell nachgewiesen und gezeigt, dass mit ExPAN auch ähnlich orientierte Moleküle unterschieden werden können. Im zweiten Teil der Arbeit wird eine Methode zur Verbesserung der Auflösung von konfokalen Laser-Scanning-Mikroskopen vorgestellt, die als Multidetektor-Scanning (MDS) bezeichnet wird und auf dem Prinzip der Image-Scanning-Mikroskopie (ISM) beruht. Mit ISM lässt sich die Auflösung von Fluoreszenzmikroskopen theoretisch verdoppeln. Da ISM einen Flächendetektor voraussetzt, wurden in der Vergangenheit hauptsächlich CCD oder CMOS Kameras als Detektoren eingesetzt. In dieser Arbeit werden anstelle einer Kamera mehrere Einzelphotonendetektoren verwendet und über ein Glasfaserbündel zu einem Flächendetektor kombiniert. Dadurch ist es erstmals möglich, die Methode in Verbindung mit Fluoreszenzlebensdauer-Mikroskopie (FLIM) einzusetzen. FLIM hat sich in den Biowissenschaften als wichtige Mikroskopie-Technik etabliert und wird unter anderem bei Protein-Protein-Interaktionsstudien oder zur Untersuchung des NADH-Metabolismus eingesetzt. Die Verbesserung der räumlichen Auflösung von FLIM mit MDS ist somit für eine Reihe von biologischen Fragestellungen von potentiellem Interesse. Im Rahmen der Arbeit wurde ein Multidetektor-Scanning-Mikroskop konstruiert und durch die Vermessung von fluoreszierenden Mikropartikeln charakterisiert. Eine Verbesserung der Auflösung durch MDS wird an fixierten biologischen Proben demonstriert. Dabei wurde eine Auflösung von 168 nm mit MDS sowie 146 nm mit MDS und Dekonvolution erreicht. Schließlich wird die Kombination der Methode mit Fluoreszenzlebensdauer-Mikroskopie demonstriert.

Atomic Force Microscopy (AFM) is a powerful technique that has evolved from being a purely imaging tool to a one capable of providing multifunctional information, offering exciting new possibilities for nano-biotechnology. The project focuses on the use of the AFM in order to morphologically and mechanically characterise cells and biomaterials demonstrating how versatile this instrument can be. The project is divided in the following parts:Part 1: establishment of AFM protocols for the nano-scale morphological and mechanical characterisation of soft and hard macroscopic substrates and of objects such as adsorbed nanoparticles. In particular, these techniques were tested on:Hyaluronic acid (HA)/poly(ethylene glycol) (PEG)-based hydrogels, which provide an artificial model for the mechanical behaviour of some biological tissues and organs. The elastic modulus, measured via AFM nanoindentation, of these hydrogels increased by decreasing the concentration and the molecular weight (MW) of HA in the hydrogels. We have then verified a clear relation between the mechanical properties of the hydrogels and the proliferation of cells cultured on them. Chitosan nanoparticle (popular carriers for the delivery of negatively charged macromolecular payloads, e.g. nucleic acids) cross-linked with triphosphate (TPP) and then coated with HA. We focussed on the influence of chitosan molecular weight (Mw) on nanoparticle properties. HA was able to penetrate into the more porous nanoparticles (high MW chitosan), whereas it formed a corona around the more cross-linked ones (low MW chitosan). AFM imaging was used to highlight the presence of this corona and also to estimate its apparent thickness to about 20-30 nm (in dry state).Silicone substrates modified with amphiphilic triblock copolymer (Sil-GMMA) layers. Extensive AFM (imaging and nanoindentation) provided evidence that silicone substrates are prevalently coated with Sil-GMMA thin layers that exhibit negligible hydrophobic recovery during drying and change the surface from more to less cell-adhesive. Part 2: AFM mechanical characterisation of fibroblast-to-myofibroblast differentiation process. Fibroblasts were stimulated to differentiate into myofibroblasts by Transforming Grow Factor β1 (TGFβ1) on hard substrate. AFM force maps performed both on fibroblasts (untreated cells) and myofibroblasts (TGFβ1-treated cells) revealed a significant increase in the elastic modulus in treated cells. Part 3: preparation and AFM characterisation of poly(ethylene glycol) diacrylate/acrylate (PEGDA/A) hydrogels. Since the mechanical properties of the substrate plays a pivotal role in fibroblast-to-myofibroblast differentiation process, hydrogels were prepared and characterised at the macro/nanoscale with AFM indentation, providing us with cell-adhesive substrates that cover a wide range of elastic modulus. These substrates are optimal candidates for future investigations to better understand and possibly control the differentiation process.

This thesis is a systematic description of normal and abnormal dog spermatozoa by means of bright field light and transmission electron microscopy, and an investigation into the effect that abnormal sperm have on canine fertility. A total of 101 ejaculates were collected from 88 dogs, of 34 different breeds. Sperm samples were examined macroscopically for volume, colour, consistency, and pH. Microscopic evaluation of sperm motility was conducted on all samples. Morphological evaluation using light microscopy was conducted on 71 of the samples. Samples from 10 of the dogs were examined ultrastructurally. A novel classification for abnormal dog sperm is presented. Abnormal sperm were classified into one of the following groups: Acrosomal defects, head defects, midpiece defects, tail defects and other abnormalities. Abnormalities were further sub-divided into major and minor defects. The most common abnormalities encountered were major sperm head defects. The abnormalities are compared with those described for other species, in particular the bull and man. The association between the percentage abnormal sperm in the ejaculate and the fertility of the dog was statistically evaluated. On this basis, the dogs were divided into normal and sub-normal groups. The percentage normal morphology below which fertility was adversely affected was found to be sixty percent. The fertility of dogs with greater than or equal to 60 percent normal morphology was 61 percent, while the fertility of dogs with · less than 60 percent normal morphology was 13 percent. There was no statistical difference between the ages of the dogs in the two groups; from this it was concluded that sub-fertility may affect a dog at any age. A means of evaluating dogs for reproductive potential is discussed.

Indiana University-Purdue University Indianapolis (IUPUI) / Secondary caries and the replacement of existing restorations account for 50 to 70 percent of operative dentistry today. Quantitative Light Fluorescence (QLF) has been shown to be effective at diagnosing very early tooth demineralization on smooth surfaces (less than 50 μ in depth); however, QLF has never been utilized to evaluate secondary caries in dentin. The objective of this study was to validate the accuracy of QLF in diagnosing early secondary caries and then verify the results using confocal microscopy and polarized light microscopy. Seventy-five mandibular molar teeth were prepared with Class V amalgam preparations on the mesial surface. A fluoridated varnish and 1.23- percent acidulated phosphate fluoride (APF) were introduced to this evaluation system, two agents known to effectively inhibit tooth demineralization. The artificial caries system utilized was adjusted to ensure that secondary caries would occur at restoration/tooth surface interfaces. The teeth were exposed to this artificial caries challenge for five days and following lesion formation, QLF was used to determine if incipient demineralization could be detected. The results of the QLF analysis were then compared with the data gathered using confocal microscopy and polarized light microscopy. Our results demonstrate that QLF detected 100 percent of the lesions seen with confocal microscopy and polarized light microscopy; however, no sound specimens were analyzed with any of the three techniques. There were no consistent significant differences between the fluoridated varnish and APF (p < 0.05) with any of the three methods utilized. We conclude that QLF can be used in early caries diagnosis and that emphasis should now be focused on treatment of the early lesion.

In this work, digital holography (DH) is extensively utilized to characterize microparticles. Here, “characterization” refers to the determination of a particle’s shape, size, and, in some cases, its surface structure. A variety of microparticles, such as environmental dust, pollen, volcanic ash, clay, and biological samples, are thoroughly analyzed. In this technique, the microscopically fine interference pattern generated by the coherent superposition of an object and a reference wave fields is digitally recorded using an optoelectronic sensor, in the form of a hologram, and the desired particle property is then computationally extracted by performing a numerical reconstruction to form an image of the particle. The objective of this work is to explore, develop, and demonstrate the feasibility of different experimental arrangements to reconstruct the image of various arbitrary-shaped particles. Both forward- and backward-scattering experimental arrangements are constructed and calibrated to quantify the size of several micron-sized particles. The performance and implications of the technique are validated using the National Institute of Standards and Technology (NIST)-traceable borosilicate glass microspheres of various diameters and a Thorlabs resolution plate. After successful validation and calibration of the system, the resolution limit of the experimental setup is estimated, which is ~10 microns. Particles smaller than 10 microns in size could not be imaged well enough to ensure that what appeared like a single particle was not in fact a cluster. The forward- and backward-scattering holograms of different samples are recorded simultaneously and images of the particles are then computationally reconstructed from these recorded holograms. Our results show that the forward- and backward-scattering images yield different information on the particle surface structure and edge roughness, and thus, reveal more information about a particle profile. This suggests that the two image perspectives reveal aspects of the particle structure not available from a more commonly used forward-scattering based image alone. The results of this work could be supportive to insight more on the particles’ morphology and subsequently important for the advancement of contactree particle characterization technique.

No description available.

No description available.