Dissertations / Theses on the topic 'Multi-Photon microscope'

La microscopie multiphotonique est devenue un outil essentiel pour étudier l'activité fonctionnelle de la souris mais son application à des animaux plus grands se heurte à plusieurs obstacles. Il serait particulièrement avantageux de pouvoir l'appliquer aux macaques, car ils représentent un modèle animal de choix pour comprendre les mécanismes neuronaux des fonctions cognitives de haut niveau, telles que l'attention, la mémoire et la conscience. L'un des principaux facteurs limitant à l'imagerie chez les grands animaux est la dure-mère. Cette couche de tissu épaisse et opaque protège le cerveau, mais elle est si épaisse chez les grands animaux qu'elle entrave l'imagerie. Elle est donc couramment enlevée mais cela conduit à une préparation hautement invasive et instable. L'objectif principal de ce travail est d'étudier la possibilité d'enregistrer l'activité fonctionnelle du cortex du macaque rhésus à travers la dure-mère naturelle.Une installation de microscopie multiphotonique a été conçue avec des chemins optiques de microscopie à deux et trois photons pour pouvoir faire des enregistrements sur des primates non humains vigiles. La fréquence de répétition du laser est de 2MHz, ce qui permet une profondeur d'imagerie maximale à l'intérieur du cortex de 520µm à 960nm et 715µm à 1300nm en la présence d'une dure-mère de 120µm d'épaisseur à la surface. Le champ de vision est de 620x630µm² avec une fréquence d'acquisition de 7,8Hz en utilisant un balayage unidirectionnel. En plus de cette installation, des implants chirurgicaux ont été développés pour une imagerie stable sur le long-terme de sujets vigiles.En utilisant une étude ex vivo de la dure-mère d'un macaque rhésus, les aberrations optiques induites par celle-ci ont été étudiées en mesurant la diminution de la résolution spatiale du microscope pour une épaisseur variable de dure-mère. Ceci révèle qu'elle n'a pas d'impact significatif sur la résolution pour une épaisseur allant jusqu'à 150µm à 1300nm. La longueur d'atténuation effective de la dure-mère naturelle est estimée à 56.5±10.1µm à 960nm et 80.7±5.3µm à 1300nm. Ces valeurs sont utilisées pour modéliser la profondeur maximale d'imagerie en fonction de la fréquence de répétition du laser et de l'épaisseur de la dure-mère.Ce modèle est ajusté et validé à l'aide de données in vivo provenant de deux primates non humains. La longueur d'atténuation effective de la dure-mère naturelle et d'une repousse de tissu après une durectomie (appelée "néomembrane") sont étudiées. Des enregistrements fonctionnels ont été réalisés chez la souris et prétraités avec Suite2P. Les paramètres d'injection virale ont été testés chez trois macaques et nous avons enregistré l'activité structurelle et fonctionnelle de neurones pour l'un d'eux au moment de l'étude.Enfin, la comparaison entre l'utilisation de la microscopie à deux ou trois photons pour l'étude du primate non humain est discutée.En conclusion, nous avons mis en place et optimisé un microscope multiphotonique pour l'imagerie vigile et à long-terme du cortex de primates non-humains et montré qu'il était possible d'enregistrer le cortex jusqu'à plus de 700µm de profondeur (ce qui correspond aux couches L2/L3) tout en gardant la dure-mère naturelle en place
Multi-photon microscopy has become a standard technique to study the structural and functional activity in mice but it faces obstacles to be applied in larger animals. It would be particularly advantageous to be able to apply it to macaque monkeys, as they are the animal model of choice to understand the neural mechanisms of high-level cognitive functions such as selective attention, working memory and consciousness. One of the main limiting factors for imaging in larger animals is the dura mater. This tough and opaque layer of tissue protects the brain but is so thick in larger animals that it obstructs imaging. It is therefore commonly removed but this leads to a highly invasive and unstable preparation. The main aim of the current work is to investigate the possibility to record functional activity from the cortex of the rhesus macaque monkey through the natural dura.A multi-photon microscopy setup has been designed with a two-photon and a three-photon microscopy optical paths to record from awake macaque monkeys. The repetition rate of the laser is 2MHz which allows a maximum imaging depth inside the cortex of 520µm at 960nm and 715µm at 1300nm with an additional 120µm-thick layer of dura mater at the surface. Resonance-galvo scanning is used to allow a maximal frame rate of 15.6Hz at a field of view of 620x630µm². In addition to the setup, surgical implants have been developed for long-term and awake imaging.Using an ex vivo study of dura mater from a macaque monkey, the induced optical aberrations are studied by measuring the decrease in spatial resolution of the setup for a varying thickness of dura mater. This reveals that it has no significant impact on the spatial resolution for a thickness up to 150µm at 1300nm. The effective attenuation length of the dura mater is estimated to be 56.5±10.1µm at 960nm and 80.7±5.3µm at 1300nm. These measurements are used to model the maximum imaging depth that can be reached according to the repetition rate of the laser and the thickness of the dura.This model is adjusted and validated using in vivo data from two non-human primates. The effective attenuation length of the natural dura mater and of a regrowth of tissue following a durectomy (called a 'neomembrane') are investigated. Functional recordings have been performed in mice and preprocessed using Suite2P. Viral injection parameters have been tested in three macaque monkeys and we have so far recorded the in vivo structural and functional activity of neurons in one. Finally, the comparison between the use of two- and three-photon microscopy to study non-human primates is discussed. In conclusion, we have set up and optimized a multi-photon microscope for long-term awake imaging of the cortex of non-human primates and shown that it was possible to record down to over 700µm into the cortex (which corresponds to the layers L2/L3) while imaging through the natural dura mater or a neomembrane

Articular cartilage has been imaged using the following multi-photon modalities: Second Harmonic Generation (SHG), Two-photon Fluorescence (TPF) and Coherent Anti-Stokes Raman Scattering (CARS). A simple epi detection microscope was constructed for SHG and TPF imaging in the early stages of this research. Later the imaging was transferred to a new microscope system which allowed simultaneous forwards and epi detection and combined CARS imaging with TPF and SHG. Multiphoton spectroscopic studies were conducted on both intact tissue samples and the major components of the extracellular matrix, in order to identify sources of TPF. Fluorescence was detected from type II collagen, elastin and samples of purified collagen and elastin crosslinks. Age related glycation crosslinks of collagen may be a significant source of TPF. No fluorescence was detected from proteoglycans. In intact, unfixed healthy articular cartilage the cells were observed via CARS, surrounded in their pericellular matrix which is characterised by an increase in TPF. The collagen of the extra cellular matrix showed up clearly in the SHG images. Diseased cartilage was also imaged revealing microscopic lesion at the articular surface in early osteoarthritis and highly fibrous collagen structures and cell clusters in more advanced degeneration. In young healthy cartilage a network of elastin fibres were found lying parallel to the articular surface in the most superficial 50μm of the tissue. Regional variations in these fibres were also investigated. The fibres appeared mainly long and straight suggesting that they may be under tension, further work is needed to identify whether they have a mechanical function. The polarization sensitivity of the SHG from collagen has been investigated for both cartilage and tendon. In the most superficial tissue these measurements can be used directly to determine the collagen fibre orientation. However at increasing depths the effects of biattenuation and birefringence must be considered. Healthy cartilage has a characteristic pattern of polarization sensitivity with depth and this changes at lesions indicating a disruption of the normal collagen architecture. The methods developed in this thesis demonstrate the use of non-linear microscopy to visualise the structure of the extracellular matrix and cells in intact unstained tissue. They should also be appropriate in many areas of cell and matrix biology.

This project set out to examine the possibility that by acquiring Raman spectra and performing multi-photon imaging we can get better diagnosis and understanding of the biochemistry of an individual cancerous tumour and distinguish it from the healthy tissue. Within the frame of this study, colorectal primary and secondary cancer cells are examined with Raman spectroscopy in order to (i) study and distinguish them according to their chemical composition by applying multivariate methods and (ii) determine whether Raman spectroscopy can identify the cells which are the link between primary and secondary colorectal cancer cells, the so-called Cancer Stem Cells. The second part of this thesis is based on tissue studies. Human colorectal tissue sections are examined in a label-free manner with the use of multi-photon imaging modes (i) Two photon excitation fluorescence, (ii) stimulated Raman scattering and (iii) second harmonic generation, in order to determine whether these can provide fast and accurate diagnosis of colorectal cancer. These techniques were able to distinguish between healthy and cancerous tissue regions, based on the chemically-specific images of the tissue microenvironment and architecture. The hypothesis of Cancer stem cell is examined with the use of Raman spectroscopy shown that the CSCs have some small differences according to their tissue origin.

Motivation: Cartilage is a robust but flexible connective tissue found in most joints of the body. The collagen fibres present in the extracellular matrix of cartilage contribute to its tensile strength and stiffness. The purpose of this study is to develop and implement methods to determine the orientation and anisotropy of collagen fibres in 3-D images gen- erated with multi-photon microscopy. The motivation behind developing these techniques is to improve the foundation for further studies on understanding the characteristics of the cartilage matrix. This in turn would give a better foundation for developing artificial matrices and mechanical models, as well as improve diagnostics.Material and methods: The two methods developed in this study are based on analysing the frequency domain. One is an expansion of a previous developed method by Chaudhuri et al. [1]. This method is based on evaluating the average intensity at different directions in the frequency domain. The direction with the least average intensity is equivalent to the direction of the fibres. The other method is based on thresholding the frequency domain according to intensity followed by fitting an ellipsoid to the remaining data set. The direction of the collagen fibres is equivalent to the direction of the shortest axis of the ellipsoid. These methods are called the sector and ellipsoid method, respectively. To determine how robust these methods are a series of tests were developed. The focus of these tests was to determine if the methods are rotational invariant and if the results are influences by different preprocessing techniques. These preprocessing techniques are: median filtering, deconvolution and skeletonization of the original image containing the collagen fibres. It is also important to determine the sensitivity of the ellipsoid method according to the chosen threshold value. In addition data generated fibres and frequency domains were made to determine the accuracy of the methods.Results and conclusion: The sector method was not very robust. For most cases there is not one specific direction that has the least average intensity in the frequency domain. Instead there is a quite large minimum area. The ellipsoid method shows promising results. It managed to find the correct direction both for the data generated data sets, but also for the real images. It seems like no preprocessing nor frequency filtering, except for thresholding, is needed to still find the correct direction and its anisotropy. The only remark is that the automatically chosen threshold value was to low for one of the samples. This can probably be improved by making a slight change in the process for choosing a threshold value.

La "cryopolymérisation" permet d'obtenir des gels de polymère macroporeux ou "cryogels". Cette méthode a été utilisée pour la synthèse d'hydrogels thermosensibles à base de pNIPA. La température critique TC correspondant à la transition de volume a été déterminée par des mesures de taux de gonflement et par DSC. La macroporosité (distribution de la taille des pores et épaisseur des parois) et son évolution en fonction de T ont été étudiées par la microscopie biphotonique donnant des informations à l'échelle du µm à plusieurs dizaines de µm. La diffusion de rayons X (SAXS et WAXS) a été utilisée pour caractériser la structure multi-échelle (de quelques dixièmes à quelques dizaines de nm) du gel constituant les parois des macropores. Les courbes de diffusion ont été décrites analytiquement. L'évolution des dix paramètres contenus dans l'équation a été étudiée en fonction de T et discutée. Enfin, des expériences utilisant les phonons hyperfréquences générés par la technique des réseaux transitoires avec détection hétérodyne (HD-TG) ont été réalisées. Ces mesures ont permis de déterminer la vitesse de propagation de l'onde ultra-sonore (à 340 MHz), son atténuation, et la constante de diffusion thermique à différentes températures.

Les interactions lumière-matière dans les mileux moléculaires et bio-moléculaires peuvent mener à des processus complexes où les polarisations des champs optiques se couplent aux assemblages de dipoles de transitions moléculaires. La manipulation des polarisations des champs optiques en microscopie de uorescence peut en particulier donner accès à des modi cations nes d'arrangements moléculaires. Dans ce travail de thèse nous introduisons une méthode basée sur la variation continue d'un état de polarisation d'excitation complémentée par une analyse polarisée, appliquée à la microscopie de uorescence multi-photons. La uorescence à deux photons polarimétrique permet d'accéder à une information statique quantitative sur la forme et l'orientation de la distribution orientationnelle moléculaire dans des membranes lipidiques articielles, dans des cellules ou sur des composés molécluaires co-cristallins qui peuvent être fortement hétérogènes. La uorescence à trois photons polarimétrique apporte de plus un diagnostique de cristallinité dans des cristaux de protéines, avec une forte sensibilité à leur structure et symétrie. L'implémentation expérimentale de cette technique requiert de quantier les distortions de polarisation provenant du montage expérimental et de l'échantillon lui-même, qui sont nement analysés.

L'infiltration tissulaire des macrophages est un facteur aggravant dans de nombreuses pathologies telles que les maladies inflammatoires chroniques ou le cancer. Les macrophages qui infiltrent les tumeurs de façon continue sont appelés macrophages associés aux tumeurs (TAMs). Ils favorisent la croissance tumorale, l'angiogenèse, l'invasion tumorale et la formation de métastases. L'inhibition de l'infiltration des macrophages est donc devenue une évidence thérapeutique. Récemment, l'équipe a démontré que les macrophages utilisent le mode migratoire amiboïde (dépendant de ROCK) ou mésenchymal (dépendant des protéases) selon l'architecture de la matrice extracellulaire (MEC) en trois-dimensions (3D) qu'ils traversent. De plus, l'étude du mode migratoire mésenchymal a montré qu'il est dépendant de Hck (une tyrosine kinase spécifique des phagocytes) et de sa capacité à réorganiser les podosomes en rosettes (structures riches en actine dégradant la MEC). Mon projet de thèse s'est articulé autour de deux axes de recherche : 1) l'identification des substrats de Hck et la caractérisation de leur rôle dans l'organisation des podosomes et la migration 3D des macrophages, et 2) l'étude de la migration 3D des monocytes/macrophages primaires humains dans un modèle mimant le microenvironnement tumoral : les sphéroïdes tumoraux. Par une approche protéomique j'ai identifié des partenaires et substrats potentiels de Hck dont la Filamine A (FLNa), une protéine assurant notamment la liaison entre le cytosquelette d'actine et les intégrines. En utilisant différents outils (protéines recombinantes, anticorps, shRNA. . . ) j'ai montré que : 1) Hck phosphoryle la FLNa in vitro, 2) la FLNa est associée aux podosomes et est nécessaire à leur organisation en rosettes sous le contrôle de Hck, 3) les podosomes des cellules déficientes en Flna ont une durée de vie plus courte, et 4) l'expression de la FLNa est nécessaire à la migration mésenchymale, mais pas à la migration amiboïde des macrophages dans une MEC en 3D. Ainsi la FLNa est impliquée dans la formation et à la stabilisation des podosomes, à leur organisation en rosettes, la migration mésenchymale des macrophages et pourrait se situer dans la voie de signalisation de Hck. En parallèle, j'ai mis au point un modèle de sphéroïdes tumoraux qui m'a permis de montrer que l'infiltration des monocytes ou des macrophages, dans ce modèle tissulaire in vitro, est dépendante de ROCK et des protéases, signature de l'utilisation des deux modes migratoires. Puis en incubant ces sphéroïdes au sein de MEC, j'ai démontré que la présence de macrophages infiltrés dans les sphéroïdes est nécessaire pour déclencher le pouvoir invasif des cellules tumorales qui émigrent des sphéroïdes en suivant les macrophages et infiltrent la MEC environnante. Les macrophages Hck-/- présentant un défaut de migration mésenchymale, sont significativement moins efficaces dans la promotion de l'invasion des cellules tumorales. Ces résultats indiquent que l'activité de migration et de remodelage de la matrice exercée par les macrophages est prépondérante dans l'invasion tumorale in vitro. Ces résultats ont permis d'établir le mode migratoire des macrophages infiltrant un modèle tissulaire in-vitro et de démontrer le mécanisme d'action des macrophages dans l'invasion tumorale. Ainsi, mes travaux de thèse ont permis de progresser dans la caractérisation des mécanismes moléculaires et cellulaires de la migration 3D des macrophages humains. En effet, j'ai pu 1) identifier une protéine nécessaire à la migration mésenchymale des macrophages, 2) mettre en évidence l'utilisation par les macrophages des modes migratoires amiboïde et mésenchymal lors de leur infiltration dans un modèle de tumeur en trois-dimensions, les sphéroïdes tumoraux et 3) montrer que le remodelage de la matrice par les macrophages, lors de leur migration, joue un rôle prépondérant dans l'invasion tumorale
Tissue infiltration of macrophages is an aggravating factor in many diseases such as chronic inflammation and cancer. Macrophages that infiltrate tumors are called tumor-associated macrophages (TAMs). They promote tumor growth, angiogenesis, invasion and metastasis. Thus, inhibition of macrophage infiltration has become a therapeutic goal. Recently, the team demonstrated that macrophages use the amoeboid (depending on ROCK) or the mesenchymal (depending on proteases) migratory mode according to the extracellular matrix (ECM) architecture in three dimensions (3D). In addition, the study of the mesenchymal migration mode showed that it is dependent on Hck (a phagocyte-specific tyrosine kinase) and its ability to reorganize podosomes (ECM-degrading actin-rich structures) into rosettes. My thesis project was organized around two axes 1) the identification of substrates of Hck and the characterization of their role in the organization of podosomes and 3D migration of macrophages, and 2) the study of the 3D migration mechanisms of primary human monocytes/ macrophages within an in vitro tumor model: tumor cell spheroids. By a proteomic approach, I have identified potential partners and substrates of Hck, including the protein Filamin A (FLNa), a protein interacting with the actin cytoskeleton and integrins. Using different tools (recombinant proteins, antibodies, shRNA. . . ) I showed that: 1) Hck phosphorylates FLNa in vitro, 2) FLNa is localized to podosomes and is necessary for their organization as rosettes under the control of Hck, 3) the podosomes of FLNa-deficient cells have a shorter life span, and 4) the expression of FLNa is required for mesenchymal migration, but not for amoeboid migration of macrophages in a 3D ECM. Thus, FLNa could be a substrate of Hck necessary for the formation and stabilization of podosomes and their organization as rosettes, and is required for the mesenchymal migration of macrophages. In parallel, I developed a model of tumor cell spheroids, which allowed me to show that the infiltration of monocytes or macrophages in this in vitro tissue model of tumor is dependent on ROCK and proteases, signature of the use of the two migration modes. Then, when spheroids were embedded into ECM, I demonstrated that the presence of macrophages infiltrated into the spheroids is necessary to trigger the invasiveness of tumor cells. Indeed, macrophages infiltrate first the surrounding ECM and tumor cells follow macrophages in the matrix outside of the spheroid. Hck-/- macrophages, that are defective in mesenchymal migration, are significantly less effective in promoting the invasion of tumor cells. These results indicate that the activity of migration and matrix remodeling exerted by macrophages is prominent in tumor invasion. These results have established the migratory mode of macrophages infiltrating an in vitro tumor model and a mechanism required for tumor invasiveness promoted by macrophages. Thus during my thesis, I characterized the molecular and cellular mechanisms of 3D migration of human macrophages. Indeed, I have been able to: 1) identify a protein necessary for the mesenchymal migration of macrophages, 2) highlight the use by macrophages of the amoeboid and mesenchymal migration modes during their infiltration into an in vitro tumor model in 3D and 3) show that the matrix remodeling activity of macrophages during their migration plays a critical role in tumor cell invasion

La microscopie multiphotonique est une modalité d’imagerie de pointe offrant des opportunités d’avancées remarquables en biologie mais aussi dans le domaine médical. Afin d’en exploiter pleinement le formidable potentiel au cœur même de la pratique clinique, le développement de nombreuses sondes miniaturisées à fibre optique pour l’endomicroscopie multiphotonique (EMMP) a eu lieu depuis de nombreuses années et dans de nombreux laboratoires français et étrangers. Il s’est pour l'instant confronté à des limitations majeures comme l’impossibilité de recueillir les signaux d’auto-fluorescence des tissus qui sont intrinsèquement faibles comme ceux venant des co-enzymes métaboliques NADH et FAD. Cette limitation compromet l'utilité de l’EMMP en la restreignant à une imagerie morphologique requérant un marquage exogène des tissus. Ce manuscrit présente une architecture d’EMMP permettant de dépasser cette limitation, capable de proposer une imagerie fonctionnelle du métabolisme cellulaire en temps réel, in vivo, in situ, sans marquage. Le prototype d’EMMP proposé est une amélioration du précédent, où les Grisms en réflexions sont remplacés par des Grisms en transmission, permettant d’élargir la bande spectrale d’utilisation et la transmission du système. Ce prototype voit aussi l’adjonction d’un second laser excitateur afin d’accéder aux fluorescences du NADH et du FAD. Les résultats démontrent capable que nous sommes à même d’imager les fluorescences cellulaires intrinsèques au travers de 5 mètres de fibre optique avec une résolution subcellulaire. Parmi celles-ci nous sommes capables d’exciter et de collecter spécifiquement les fluorescences du NADH et du FAD. Enfin nous détectons assez de photons pour disposer d‘informations quantitatives et donc de proposer une image du rapport d’oxydo-réduction optique en endomicroscopie
Nonlinear microscopy is a cutting edge imaging modality leading to remarkable step forward in biology but also in the clinical field. To use it at its full potential and at the very heart of clinical practice, there has been several development of fiber-based micro-endoscope. The application for those probes is now limited by few major restrictions, such as the impossibility to collect auto-fluorescence signal from tissues theses being inherently weak such as the fluorescence from NADH or FAD. This limitation reduces the usefulness of the micro-endoscope effectively restraining it to morphological imaging modality requiring staining of the tissue. Our aim is to go beyond this limitation, showing cellular metabolism monitoring, in real time, without any staining. The experimental setup is an upgrade of our precedent one where the reflection- based Grism stretcher is replace with a new generation transmission-based Grism stretcher. Another Laser was also added in order to tune the first laser at 860nm to allow FAD imaging and the second one to 760nm for NADH. The results prove that we assess and image the level of NADH and FAD at subcellular resolution through a five-meter-long fiber. Thus we demonstrate that we are capable of measuring the optical redox ratio in a micro-endoscopic configuration

Thesis (Ph.D.)--University of Cincinnati, 2007.
Advisor: Richard J. Paul. Title from electronic thesis title page (viewed Apr. 4, 2009). Keywords: PMCA (human gene symbols; ATP2B); SERCA2 (human gene symbols; ATP2A2); NCX; bladder smooth muscle; Ca²⁺ homeostasis; gene-altered mice. Ca²⁺ waves; Ca²⁺ sparks; Fura-PE3; Fluo-4; Indo-1; multi-photon microscopy. Includes abstract. Includes bibliographical references.

碩士
國立臺灣大學
醫學工程學研究所
107
The corneal endothelium is a monolayer of hexagonal cells derived from the neural crest and is responsible for maintaining the dehydration of the cornea. Due to the limited proliferative capacity, impaired mature endothelial cells ultimately results in corneal decompensation and loss of vision. However, the cell dynamics of corneal endothelium during homeostasis and regeneration remain unclear. In this study, we characterize that the N-cadherin is the specific marker of corneal endothelial cells. Using multi-photon live imaging, the cell density and nucleus volume in corneal endothelium significantly decreases from the center to periphery. In addition, the corneal endothelial cells are quiescent, arrested at G1 phase, without migration for consecutive 3 days. We use laser ablation with precise spatiotemporal control to create a 50x50μm2 wound in corneal endothelium in vivo. Here we show that the wound healing process can be divided into three phases: latent phase, migration phase, and remodeling phase. The latent phase initiates at 6 hours and followed by the migration phase from 24 hours. The wound approaches closed at 38 hours. Interestingly, our data suggest that corneal endothelial cells detach from the Descemet’s membrane in both the latent phase and remodeling phase. This study not only develops a novel corneal endothelial injury model but also elucidates the wound healing process in vivo. Additionally, the cell loss from Descemet’s membrane during regeneration enables us to validate the new insight into ophthalmic therapy.

A thorough understanding of how neurons work is one of the greatest scientific goals in the field of experimental neuroscience. However, four fundamental technical limitations complicate any attempt to study neuronal function with sub-cellular resolution: First, neurons and neuronal processes are small, second, in realistic experimental situations they can be located deep within optically scattering tissue, third, the chemical and electrical signaling that characterizes neuronal behavior happens quickly, and fourth, neurons and neuronal processes have very three dimensional (3D) shapes. Here we develop a tool that overcomes all four listed limitations by combining the technique of multi-photon microscopy with a unique method for 3D laser beam steering. The result is an instrument capable of monitoring physiological signals at multiple locations in the volume of space occupied by a neuron, a task that is unachievable with any other available instrument.

Multi-photon fluorescence microscopy has become a primary tool for high-resolution deep tissue imaging because of its sensitivity to ballistic excitation photons in comparison to scattered excitation photons. The imaging depth of multi-photon microscopes in tissue imaging is limited primarily by background fluorescence that is generated by scattered light due to the random fluctuations in refractive index inside the media, and by reduced intensity in the ballistic focal volume due to aberrations within the tissue and at its interface. We built two multi-photon adaptive optics (AO) correction systems, one for combating scattering and aberration problems, and another for compensating interface aberrations. For scattering correction a MEMS segmented deformable mirror (SDM) was inserted at a plane conjugate to the objective back-pupil plane. The SDM can pre-compensate for light scattering by coherent combination of the scattered light to make an apparent focus even at a depths where negligible ballistic light remains (i.e. ballistic limit). This problem was approached by investigating the spatial and temporal focusing characteristics of a broad-band light source through strongly scattering media. A new model was developed for coherent focus enhancement through or inside the strongly media based on the initial speckle contrast. A layer of fluorescent beads under a mouse skull was imaged using an iterative coherent beam control method in the prototype two-photon microscope to demonstrate the technique. We also adapted an AO correction system to an existing in three-photon microscope in a collaborator lab at Cornell University. In the second AO correction approach a continuous deformable mirror (CDM) is placed at a plane conjugate to the plane of an interface aberration. We demonstrated that this “Conjugate AO” technique yields a large field-of-view (FOV) advantage in comparison to Pupil AO. Further, we showed that the extended FOV in conjugate AO is maintained over a relatively large axial misalignment of the conjugate planes of the CDM and the aberrating interface. This dissertation advances the field of microscopy by providing new models and techniques for imaging deeply within strongly scattering tissue, and by describing new adaptive optics approaches to extending imaging FOV due to sample aberrations.

碩士
國立臺灣大學
醫學工程學研究所
106
We have observed the yellow fluorescence of mesenteric ischemia, and ischemia-reperfusion rats with multi-photon fluorescence lifetime and intensity, which based on a femtosecond Ti:sapphire laser. With lifetime trace, single photon, two-photon fluorescence spectrum, and two-photon fluorescence excitation spectrum, we found out that intensity of yellow fluorescence increases during ischemia state, and recovered gradually after reperfusion. The characteristic change of the autofluorescence intensity ratio in two-photon excitation spectrum, which observed only in the ischemia state instead of normal state or reperfusion state. We compared fluorescence decay traces of preishemia, ischemia state, and ischemia-reperfusion state, the results showed these traces are almost overlapping, which indicated that there is no any new fluorescent substance producing during the whole process. From above results, the elevating intensity of ischemia state is due to the concentration of original fluorescent substances increase. We tried to identify that fluorescent substance by High-performance Liquid Chromatography-Mass Spectrophotometer (HPLC-MS), and the preliminary result is riboflavin. With our study result, we believe two-photon fluorescence excitation spectrum, a non-invasive way, is potential to serve as a tool for early diagnosis of acute mesenteric ischemia.

碩士
國立中央大學
光電科學與工程學系
103
The development of biomedical engineering has gradually improved, and molecular imaging has been one of its most important techniques. To solve the limitation of a conventional microscope that merely detects the morphology of samples, we’ve combined hyperspectral imaging with two photon fluorescence microscopy and designed the setup of two photon fluorescence hyperspectral microscopy. This neoteric technique includes the advantages of both microscopies, providing deeper depth penetration, less photo damage to sample, higher axial resolution, and the addition of receiving spectral information of the targeted sample simultaneously. With these properties, it’s more suitable for biomedical research in vivo, it also provides more information of the samples to researchers, and reduces the misjudgment probabilities of researches that are dependent on the morphology. Using the previously developed system, a non-de-scanned two-photon fluorescence hyperspectral microscope with parallel recording, experiments of cells and sectioning skin samples were carried out to test the efficiency of the system. Furthermore, the major aim is to try to actually apply this microscopy to biological researches and to overcome the limitation of multiple fluorescence labeling samples. Multispectral images might generate crosstalk and increase the difficulty of fluorescence discrimination. Therefore, we applied the applicably spectral analysis, linear unmixing, to improve the spectral discriminating ability. In the biological research, there were two parts of experiments. In the first part, cultured cell lines were labeled with multiple fluorescence dyes and the hyperspectral images of these cells were analyzed to show the ability and correctness of linear unmixing. In the second part, mouse skin tissues with hair follicles being fluorescently labeled were used as samples. According to the experimental results and the spectral analyses, the fluorescence and second harmonic generation signals was easily separated. The unexpected signal sources, second harmonic generation, can be removed to avoid influencing doctor’s diagnoses. The two-photon hyperspectral imaging combined with linear unmixing has shown its ability to biomedical fields.

碩士
國立臺灣大學
醫學工程學研究所
99
With multi-photon fluorescence spectra, multi-photon nonlinear microscopy and FLIM images, we successfully build up a bilirubin molecular imaging platform and imaged the metabolic disorders of bilirubins in HCC. By using an infrared femtosecond Cr:forsterite laser source with its wavelength away from two-photon resonance of most endogenous pigments, the distribution and amount of bilirubins can be semi-quantitively mapped over low-background autofluorescences. For non-tumor regions of liver tissues, the autofluorescences of bilirubins are strong and emitted from granular vesicles in hepatocytes. In poorly-differentiated HCC, the granular vesicles which emit fluorescence disappear and the intensity of multi-photon fluorescence is apparently reduced. If the hepatocytes have cholestasis, which is the signature of well-differentiated HCC, the fluorescence intensity were much higher than non-tumor part and the spectra showed an extra 800nm peak. These results indicate that, based on the multiphoton microscopy of autofluorescence spectra, intensities, and lifetimes, endogenous bilirubins are useful markers to indicate the presence and the staging of HCC. Without an extra labeling or chemical assays, disorder of bilirubins metabolisms in HCC can be sensitively detected.

碩士
國立臺灣大學
物理研究所
98
Liver disease represent a major health problem worldwide. Fatty liver is a common liver disease which can progress to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis. In this thesis, in order to elucidate the effects of fatty liver on hepatobiliary metabolism, we combined multi-photon microscopy with a home-built intravital hepatic imaging chamber (IHIC) to image and quantify the metabolic capacity of liver in methionine- and choline-deficient (MCD) dietary animal model. Mice were imaged at 2, 4, 6, and 8 weeks after they were first fed with MCD. Non-fluorescent 6-carboxyfluorescein diacetate (6-CFDA) which is hydrolyzed by the hepatocytes into fluorescent 6-carboxyfluorescein (6-CF) and excreted into the bile canaculi by the multidrug resistant proteins (mrps) is then used as the molecular probe in tracing the effects of fatty liver on hepatobiliary metabolism. Our data showed that the metabolic function of liver in fatty liver diseased mice is impaired in comparison with that of normal mice.

碩士
臺灣大學
物理研究所
96
The parenchyma of liver has a highly organized and densely crowded sinusoidal system. They are tiny capillary-like vessels, therefore leukocytes and other blood cells must traverse through these vessels in contact with the endothelial cells flanking the sinusoids. By the use of intravital two-photon microscopy and hepatic imaging chamber we can study the response in vivo after administration of lipopolysaccharide (LPS), which is commonly used to model inflammatory mechanism. Whereas it is a critical and apparent process of the inflammatory response that the leukocytes recruitment at the inflamed tissues, these leukocytes roll along the vascular wall until they encounter stimulus produced by the endothelium cells and macrophages. The fluorescence micrographs of the hepatic microcirculation show that leukocytes accumulate within the sinusoids, then transmigrate across endothelial layer and into the inflammatory hepatocytes. We can also observe the metabolism of 6-CFDA within hepatocytes and the subsequent excretion in bile canaliculi.

碩士
國立臺灣大學
物理研究所
99
Epithelium of the small intestine, a mono discontinuous layer which consists of many epithelial cells but few gaps, plays a critical role in supporting gut barrier function. These epithelial cells function by migrating and shedding, that is, cells are shed from villi and create gaps. Cell shedding and migration are closely related to the defect of barrier function. However, the connection between abnormality of cell shedding and serious disease such as sepsis is not clear. This study used multi-photon microscopy to understand the mechanism of septic cell shedding in vivo. We constructed a sepsis model by lipopolysaccharide (LPS) and a dangerous sepsis model by LPS puls burn. Finally, the motion of cell shedding and migration was directly observed and detail was completely presented. We found in normal model cells formed dynamic equilibrium of shedding and production. However, in dangerous sepsis model epithelium presented intense shedding and increased permeability.

In the past two decades, Fluorescence microscopy has imparted tremendous impact in Biology and Imaging. Several super-resolution Fluorescence imaging techniques (e.g. PALM, STED, STORM, 4Pi and structured illumination) have enabled diff raction-unlimited imaging. But high resolution is limited to a depth of few tens of microns. Thus, deep tissue imaging and simultaneous volume imaging have become a highly sought after feature in Fluorescence microscopy. The research work in this thesis address these issues by using spatial filtering techniques to tailor the point spread function (PSF) which uniquely characterizes the optical sys-tem. The advantage of this approach lies in the fact that intricate details about the focal region can be computed and designed with the help of well established theory and experimentation. In particular, this technique was applied to both spherical and cylindrical lenses. The former was used to generate Bessel-like, non-diffracting beams which demonstrated the ability to penetrate deep inside tissue-like media and thereby yielded an imaging depth of nearly 650μm as compared to about 200μm for a state-of-the-art confocal microscope. The latter gave rise to light-sheet and it's extended version that is ideal for planar imaging at large penetration depths. Another development is the generation of multiple light-sheet illumination pattern that can simultaneously illuminate several planes of the specimen. The proposed multiple light-sheet illumination microscopy (MLSIM) technique may enable volume imaging in Fluorescence microscopy. The first two chapters of this thesis are introductory in nature and provides a general overview of the principles of Fluorescence microscopy and three state-of-the-art Fluorescence imaging techniques; namely confocal, multi-photon and light-sheet based microscopy. Confocal microscopes are widely considered as a standard tool for biologists and this discussion shows that even though they have made signi ficant contributions in the fields of biophysics, biophotonics and nanoscale imaging, their inability to achieve better penetration depth has prevented their use in thick, scattering samples such as biological tissue. The system PSF of a confocal microscope broadens as it goes deeper in-side a scattering sample resulting in poor-resolution thereby destroying the very concept of high resolution, noise-free imaging. Additionally, confocal microscopy suffers from in-creased photo-bleaching due to o -layer (above and below the focal plane) excitation and low temporal resolution since it requires point-by-point scanning mechanism. On the other hand, multi-photon microscopy offers several advantages over confocal microscopy such as reduced photo-bleaching and inherent optical sectioning ability, however, it still lacks in providing high temporal resolution. Light-sheet based microscopy have gained popularity in recent years and promises to deliver high spatio-temporal resolution with minimized photo-bleaching. Recently, a considerable amount of research has been dedicated to further develop this promising technique for a variety of applications. The ability to look deeper inside a biological specimen has profound implications. How-ever, at depths of hundreds of microns, several effects (such as scattering, PSF distortion and noise) deteriorates the image quality and prohibits detailed study of key biological phenomenon. Chapter 3 of this thesis describes the original research work which experimentally addresses to this issue. Here, Bessel-like beam is employed in conjugation with an orthogonal detection scheme to achieve imaging at large penetration depth. Bessel beams are penetrative, non-di ffracting and have self-reconstruction properties making them a natural choice for imaging scattering prone specimens which are otherwise inaccessible by other microscopy imaging techniques such as, Widefield, CLSM, 4PI, Structural illumination microscopy and others. In this case such a Bessel-like beam is generated by masking the back-aperture of the excitation objective with a ring-like spatial filter. The proposed excitation scheme allow continuous scanning by simply translating the detection optics. Additionally, only a pencil-like region of the specimen can be illuminated at a given instance thereby reducing premature photobleaching of neighboring regions. This illumination scheme coupled with orthogonal detection shows the ability of selective imaging from a desired plane deep inside the specimen. In such a configuration, the lateral resolution of the illumination arm determines the axial resolution of the overall imaging system. Such an imaging system is a boon for obtaining depth information from any desired specimen layer that includes nano-particle tracking in thick tissue. Experiments performed by imaging the Fluorescent polymer tagged-CaCO3 particles and yeast cell in a tissue-like gel-matrix demonstrates penetration depth that extends up to 650 m. This will advance the field of fluorescence imaging microscopy and imaging. Similar to the ability to observe deep inside a sample, simultaneous 3D monitoring of whole specimens play a vital role in understanding many developmental process in Biology. At present, light-sheet based microscopy is the prime candidate amongst the various microscopy techniques, that is capable of providing high signal-to-background-ratio as far as planar imaging is concerned. Since spatial filtering technique was found to successfully give rise to novel features (such as large penetration depth) in a fluorescence microscope setup, a logical extension would be to implement a similar approach with a light-sheet based microscope setup. These implementations are discussed in Chapter 4 of this thesis where spatial filtering is employed with cylindrical lenses. For facilitating computational and experimental studies, a vectorial formalism was derived to give an explicit computable integral solution of the electric field generated at the focal region of a cylindrical lens. This representation is based on vectorial diffraction theory and further enables the computation of the point spread function of a cylindrical lens. Commonly used assumptions are made in the derivation such as no back-scattering and negligible contribution from evanescent fields. Stationary phase approximation along with the Fresnel transmission coefficients are employed for evaluating the polarization dependent electric field components. Computational studies were carried out to determine the polarization effects and calculate the system resolution. Experimental comparison of light-sheet intensity pro les show good agreement with the theoretical calculations and hence validate the model. This formalism was derived as a first step since it gives the essential understanding of tightly focused E-fields of a high N.A. cylindrical lens systems and thereby helps in further understanding the effect of spatial filtering. As the next step, generation of extended light-sheet for fluorescence microscopy is pro-posed by introducing a specially designed double-window spatial filter at the back-aperture of a cylindrical lens. The filter allows the light to pass through the periphery and center of a cylindrical lens. When illuminated with a plane wave, the proposed filter results in an extended depth-of-focus along with side-lobes which are due to other interferences in the transverse focal plane. Computational studies show a maximum extension of light-sheet by 3:38 times for single photon excitation, and 3:68 times for multi-photon excitation as compared to state-of-art single plane illumination microscopy (SPIM) system and essentially implies a larger field of view. Finally, generation of multiple light-sheet pattern is proposed and demonstrated using a different spatial filter placed at the back aperture of a cylindrical lens. A complete imaging setup consisting of multiple light-sheets for illumination and an orthogonal detection arm, is implemented for volume imaging in fluorescence microscopy. This proposed scheme is a single shot technique that enables whole volume imaging by simultaneously exciting multiple specimen layers. Experimental results confirm the generation of multiple light-sheets of thickness 6:6 m with an inter-sheet spacing of 13:4 m. Imaging of 3 5 m sized fluorescently coated Yeast cells (encaged in Agarose gel-matrix) is per-formed and conclusively demonstrates the usefulness and potential of multiple light-sheet illumination microscopy (MLSIM) for volume imaging. As part of the future scope of the research work presented in this thesis, the Bessel-beam based improved depth microscopy technique may attract applications in particle tracking deep inside tissues and optical injection apart from fluorescence imaging applications. The vectorial formalism derived for cylindrical lens can be used to predict other, complex optical setups involving cylindrical lenses. Extended light-sheet generation proposed in this work by using appropriate spatial filtering with a cylindrical lens, complements the existing and popular selective plane illumination microscopy technique and may facilitate the study of large biological specimens (such as, full-grown Zebra sh and tissue) with high spatial resolution and reduced photobleaching. Finally, the MLSIM technique presented in this thesis may accelerate the field of developmental biology, cell biology, fluorescence imaging and 3D optical data storage.

The roles of the vascular architecture and blood flow in response to neurovascular diseases are important in predicting physiological outcomes. Observing these parameters chronically with optical imaging techniques provides insight into the neurovascular recovery process. We develop and deploy optical imaging systems for monitoring the progression of vascular structure, perfusion, and functional blood response after ischemic stroke in a chronic rodent model to observe vascular dynamics of the cortex under normal and diseased pathologies. Specifically, we monitor the progression of the vascular structure and cerebral blood flow (CBF) over a chronic period in the rodent cortex after photo-thrombotic occlusion. Multi-Exposure Speckle Imaging (MESI) provides surface measurements of microvascular flow dynamics while Two-Photon Fluorescence Microscopy offers direct visualization of the microvascular structure. We observe the occurrence of vascular reorientation in the sub-surface microvascular structure over a 35 day post-occlusion period. We also correlate MESI flow estimates in the parenchyma with sub-surface microvascular volume fractions from two-photon microscopy to assess how vascular density influences the surface-integrated MESI measurements. Next, we develop and validate a MESI technique for measuring absolute changes of the functional blood flow response to forepaw stimulation in rodents, termed FA MESI. The optimal camera exposures for capturing the CBF response to forepaw stimulation are extracted from a training set of animal data and the feasibility of the technique is demonstrated in a testing animal set by comparing functional response results between new and existing techniques. We then deploy this system in a chronic study monitoring the progression of hemodynamic parameters after ischemic stroke within the functionally responding area of the cortex. The progression of the regional CBF perfusion and absolute changes in the magnitude of the functional blood flow response are monitored chronically after photo-thrombotic occlusion. We compare the differences between absolute and relative measurements of the functional blood flow responses, and validate FA MESI by comparing baseline measurements to 15-exposure MESI over the sampled flow distributions. We demonstrate the differences measured between the functional outcomes and the regional CBF perfusion over a three week post-occlusion time period.
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Indiana University-Purdue University Indianapolis (IUPUI)
The sympathetic nervous system strongly modulates the contractile and electrical function of the heart. The anatomical underpinnings that enable a spatially and temporally coordinated dissemination of sympathetic signals within the cardiac tissue are only incompletely characterized. In this work we took the first step of unraveling the in situ 3D microarchitecture of the cardiac sympathetic nervous system. Using a combination of two-photon excitation fluorescence microscopy and computer-assisted image analyses, we reconstructed the sympathetic network in a portion of the left ventricular epicardium from adult transgenic mice expressing a fluorescent reporter protein in all peripheral sympathetic neurons. The reconstruction revealed several organizational principles of the local sympathetic tree that synergize to enable a coordinated and efficient signal transfer to the target tissue. First, synaptic boutons are aligned with high density along much of axon-cell contacts. Second, axon segments are oriented parallel to the main, i.e., longitudinal, axes of their apposed cardiomyocytes, optimizing the frequency of transmitter release sites per axon/per cardiomyocyte. Third, the local network was partitioned into branched and/or looped sub-trees which extended both radially and tangentially through the image volume. Fourth, sub-trees arrange to not much overlap, giving rise to multiple annexed innervation domains of variable complexity and configuration. The sympathetic network in the epicardial border zone of a chronic myocardial infarction was observed to undergo substantive remodeling, which included almost complete loss of fibers at depths >10 µm from the surface, spatially heterogeneous gain of axons, irregularly shaped synaptic boutons, and formation of axonal plexuses composed of nested loops of variable length. In conclusion, we provide, to the best of our knowledge, the first in situ 3D reconstruction of the local cardiac sympathetic network in normal and injured mammalian myocardium. Mapping the sympathetic network connectivity will aid in elucidating its role in sympathetic signal transmisson and processing.