In this work, it was studied theoretically and experimentally biospeckle technique for analysis of rough surface with application to the investigation of biological activity. The theoretical study was conducted using light scattering theory, in particular a first order perturbation method, seeking to obtain the scattering cross-section per unit area of rough surface. Once given the cross-section, it was possible to obtain the distribution of intensities in a certain region of space as a function of time, allowing the comparison of different light distributions in different instants. The experimental investigation was made from the comparison of speckle patterns recorded (by a CCD camera) at different time instants produced from scattering of coherent light at 635 nm from a freshly harvested plant leaf. The parameter of comparison used was the Pearson correlation coefficient. The experimental result obtained from the correlation coefficient versus time showed a temporal evolution type exponential decay, indicating a degradation behavior of the leaf chemically active and regulated by equations rate. The theoretical result of the correlation function of time, obtained by simulation, also showed a behavior type exponential decay indicating a good agreement with experimental results. / Nesta dissertação, foi estudada teórica e experimentalmente a técnica de biospeckle para análise de superfície rugosa com aplicação na investigação de atividade biológica. O estudo teórico foi feito usando teoria de espalhamento da luz, em particular usando método perturbativo de primeira ordem, buscando obter a seção de choque de radiação espalhada por unidade de área da superfície rugosa. Uma vez determinada a seção de choque, foi possível obter a distribuição de intensidades numa certa região do espaço, em função do tempo, permitindo a comparação das diferentes distribuições de luz em instantes distintos. A investigação experimental foi feita a partir da comparação de padrões de speckles gravados (por uma câmera CCD) em diferentes instantes de tempo originados do espalhamento de luz coerente em 635 nm, a partir de uma folha vegetal recém colhida. O parâmetro de comparação usado foi o coeficiente de correlação de Pearson. O resultado experimental obtido do coeficiente de correlação em função do tempo mostrou uma evolução temporal do tipo exponencial decrescente, indicando um comportamento de degradação da folha quimicamente ativo e regido por equações de taxas. O resultado teórico do coeficiente de correlação em função do tempo, obtido por meio de simulação, também evidenciou o comportamento exponencial decrescente indicando uma boa concordância com o resultado experimental.

La tomographie par cohérence optique plein champ (OCT plein champ) est une technique de microscopie interférométrique basée sur l’utilisation d’une source de lumière faiblement cohérente, telle qu’une lampe halogène. Elle permet de réaliser, de façon non invasive, des images tomographiques à plusieurs centaines de micromètres de profondeur dans les tissus biologiques et avec une résolution spatiale isotrope de l’ordre de 1 µm. Ces travaux de thèse concernent le développement de plusieurs systèmes d'OCT plein champ, dans le but de proposer de nouvelles performances et de nouveaux contrastes destinés à l’imagerie en trois dimensions de tissus biologiques. Nous avons dans un premier temps exploité la large bande spectrale d’émission d’une lampe halogène, afin d’apporter une information spectroscopique et d’être capable de distinguer et de caractériser des zones d’un échantillon qui seraient sinon indiscernables. Puis nous avons optimisé la résolution spatiale d’un montage d’OCT plein champ pour atteindre une valeur record de 0,5 µm (dans l’eau) dans les trois directions de l’espace, notamment grâce à l’utilisation d’une bande spectrale adaptée à l’imagerie de tissus, tels que la peau. Un montage dont le champ de vision est élargi à 18 mm x 18 mm a ensuite été développé et appliqué à l’imagerie du signal d’amplitude ainsi qu’à la mesure quantitative du signal de phase résolu en profondeur. Enfin un système utilisant un laser à balayage spectral comme source de lumière combiné à un traitement numérique de correction de la focalisation a été mis en œuvre. Nous avons ainsi démontré la possibilité de réaliser des images en trois dimensions avec une résolution latérale relativement élevée, sans utiliser le moindre déplacement mécanique durant l’acquisition. / Full-field optical coherence tomography (FF-OCT) is an optical technology based on low-coherence interference microscopy for tomographic imaging of semitransparent samples. Non-invasive three-dimensional imaging can be performed with an isotropic spatial resolution of the order of 1 µm. During the PhD thesis, several FF-OCT systems have been reported achieving extended performances or contrast enhanced images relevant for biological tissues imaging. Firstly, a three-band, 1.9-μm axial resolution FF-OCT system has been implemented to perform spectroscopic contrast enhanced imaging of biological tissues over a 530-1700 nm wavelength range. Then, a study of the FF-OCT axial response has been carried out for maximizing the axial resolution of the system. An isotropic spatial resolution of 0.5 µm (in water) has been obtained by combining 1.2-NA microscope objectives with an optimized broad spectral band adapted to biological tissues imaging, such as skin samples. A set-up with an extended field of view of 18 mm x 18 mm has been also designed and applied to amplitude signal detection as well as depth-resolved quantitative phase signal measurement. At last, we developed a technique based on the combination of full-field swept-source optical coherence tomography (FF-SSOCT) with low spatial coherence illumination and a special numerical processing that allows for numerically focused mechanical motion-free three-dimensional imaging.

Coherence-Controlled Holographic Microscope (CCHM) and a Fluorescence Holographic Microscope (FHM) were developed particularly for quantitative phase imaging and measurement of live cell dynamics, which used to be a subject of digital holographic microscopy (DHM). CCHM and FHM in low-coherence mode extend capabilities of DHM in the study of living cells. However, this advantage following from the use of low coherence is accompanied by increased sensitivity of the system to its correct alignment. Therefore, the introduction of an automatic self-correcting system is inevitable. Accordingly, in the thesis, the theory of a suitable control system is derived and the design of an automated alignment system for both microscopes is proposed and experimentally proved. The holographic signal was identified as a significant variable for guiding the alignment procedures. On this basis the original basic realignment algorithms were proposed, which encompasses the processes for initial and advanced alignment as well as for long-term maintenance of the microscope aligned state. Automated procedures were implemented in both microscopes unique set of robotic mechanisms designed and built within the frame of the thesis work. All of the procedures described in the thesis were in real experimentally proved at real microscopes in the experimental biophotonics laboratory. In addition, the control software, which contains the needed automated procedures, was developed for FHM.

The Digital Micromirror Device (DMD) technology has been developed especially for Digital Light Processing projectors, which allow the image projection. After this succesful implementation, and thanks to the commercial availibility and low initial cost of the DMD chip, a wide range of other applications became possible. Besides, it may be used in microscopy as a spatial light modulator. For example in Coherence-Controlled Holographic Microscope (CCHM) that finds its use especially for imaging and measurement of live-cell dynamic processes. The DMD chip placed in the illumination part of CCHM allows for broadening the application possibilities. Namely it could be different illumination mode experiments or tomographic applications. The master's thesis deals with the optical design of CCHM with digital optics, i. e. DMD chip. The selection of optical elements for CCHM, the experimental verification of the imaging setup and the process of designing the illumination part are described in detail. In the end, the analysis of different designs for illumination setup with the digital optics in object arm is carried out and the results are compared.

Coherence-Controlled Holographic Microscope (CCHM) and a Fluorescence Holographic Microscope (FHM) were developed particularly for quantitative phase imaging and measurement of live cell dynamics, which used to be a subject of digital holographic microscopy (DHM). CCHM and FHM in low-coherence mode extend capabilities of DHM in the study of living cells. However, this advantage following from the use of low coherence is accompanied by increased sensitivity of the system to its correct alignment. Therefore, the introduction of an automatic self-correcting system is inevitable. Accordingly, in the thesis, the theory of a suitable control system is derived and the design of an automated alignment system for both microscopes is proposed and experimentally proved. The holographic signal was identified as a significant variable for guiding the alignment procedures. On this basis the original basic realignment algorithms were proposed, which encompasses the processes for initial and advanced alignment as well as for long-term maintenance of the microscope aligned state. Automated procedures were implemented in both microscopes unique set of robotic mechanisms designed and built within the frame of the thesis work. All of the procedures described in the thesis were in real experimentally proved at real microscopes in the experimental biophotonics laboratory. In addition, the control software, which contains the needed automated procedures, was developed for FHM.

科学研究費補助金 研究種目:一般研究(C) 課題番号:03650275 研究代表者:森 正和 研究期間:1991-1992年度