Wavelength swept spectrally encoded confocal microscopy for biological and clinical applications

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2007.Includes bibliographical references (p. 157-168).Spectrally encoded confocal microscopy (SECM) is a technique that facilitates the incorporation of confocal microscopy into small, portable clinical instruments. This would allow in vivo evaluation of cellular and sub-cellular features in a non-destructive, minimally invasive manner. Prior studies have demonstrated the potential of the techniques as well as highlighted the need for faster acquisition rates and higher sensitivity. In this thesis, new laser sources, optical fiber arrangements and probe designs are explored to ultimately evaluate SECM's relevance as a clinical tool. Clinical imaging at cellular scales requires imaging rates on the order of tens of frames per second to reduce motion artifacts from unavoidable patient movements. Rapid SECM imaging was achieved through the development of a novel wavelength swept laser which simultaneously provided high output power (> 10mrW), narrow linewidth (10GHz), broad wavelength tuning (80 nm centered at 1310 nm) and fast repetition rates (up to 16,000 Hz), while being compact and environmentally stable. Imaging with a wavelength swept SECM system was characterized by coupling the laser to a tabletop imaging arm comprising a high density holographic grating, a galvanometer mounted mirror and a 0.9 NA water immersion microscope objective.(cont.) Rapid SECM imaging is performed at a transverse resolution of 1.4 microns, axial resolution of 6 microns over a field of view of 440x440 microns and allows subcellular imaging ex vivo (excised specimens) and in vivo (human skin). A study on 40 excised head and neck specimens showed that SECM has the potential to perform tissue identification, but also revealed the presence of speckle noise due to the coherent nature of the illumination and collection schemes through a single mode optical fiber. A partially coherent system based on single mode fiber for illumination and multimode fiber for detection was simulated, implemented and tested to find adequate balance between attenuation of speckle noise and conservation of resolution. A coupling of 20 modes was found to reduce speckle by a factor 4.5 with a minimal sectioning penalty of 0.25, while allowing a signal increase of 8dB. This improvement in sensitivity allowed SECM table top system to be used for investigations in developmental biology where Dual clad fibers (DCF) were previously shown to allow partially coherent endoscopic imaging, using the single mode core for illumination and inner clad for multimodal collection.(cont.) Commercially available DCF's which propagate thousands of modes are ill suited for confocal endoscopes as collecting such a number of modes would destroy the axial resolution. Based on results from the previous section and through modal analysis, a DCF was designed, drawn - via a collaboration with Boston University Photonics Center -, and tested for use with SECM. The prototype DCF yielded promising results (3 fold speckle attenuation, optical sectioning degradation of 0.85), and showed the need for implementation of better coupling mechanisms to take advantage of increased signal collection. Finally, a portable SECM system was built for in vivo evaluation of pediatric vocal fold. A preliminary study on porcine and cadaveric tissue showed that SECM can distinguish between epithelium, superior and intermediate layers of the lamina propria, which could help elucidate the development mechanism of the voice apparatus if performed in vivo. The handheld instrument comprises a custom grating scanner imaging the scanning pivot onto the back pupil of a high NA microscope objective. The imaging tube can easily be interchanged to accommodate geometrical constraints imposed by different age groups.(cont.) The probe, currently under review by the biomedical engineering committee, revealed cellular and sub cellular details of human skin in vivo at depth and acquisition rates sufficient to capture blood cells flowing through capillaries. Through major improvements in acquisition speeds, sensitivity, and speckle appearance, this work established SECM as a potent clinical and biological imaging tool. Ultimate confirmation will be revealed through in vivo studies to come, but limitations are likely to be of engineering nature rather than from physical considerations. Future work should explore the possibility to combine SECM with other contrast mechanisms to provide imaging with increased specificity.by Caroline Boudoux.Ph.D

3D imaging and quantitative analysis of intact tissues and organs

Embryonic development and tumor growth are highly complex and dynamic processes that exist in both time and space. To fully understand the molecular mechanisms that control these processes, it is crucial to study RNA expression and protein translation with single-cell spatiotemporal resolution. This is feasible by microscopic imaging that enables multidimensional assessments of cells, tissues, and organs. Here, a time-lapse calcium imaging and three-dimensional imaging was used to study physiological development of the brain or pathological development of cancer, respectively. In Paper I, spatiotemporal calcium imaging revealed a new mechanism of neurogenesis during brain development. In Paper II, a new clearing method of clinically stored specimens, DIPCO (diagnosing immunolabeled paraffin-embedded cleared organs), was developed that allows better characterization and staging of intact human tumors. In Paper III, the DIPCO method was applied to determine tumor stage and characterize the microlymphatic system in bladder cancer. In Paper IV, a novel method for RNA labeling of volumetric specimens, DIIFCO (diagnosing in situ and immunofluorescence-labeled cleared onco-sample) was developed to study RNAs expression and localization in intact tumors. Overall, the aim of the thesis was to demonstrate that multidimensional imaging extends the understanding of both physiological and pathological biological developmental processes

Computational methods to create and analyze a digital gene expression atlas of embryo development from microscopy images

Abstract The creation of atlases, or digital models where information from different subjects can be combined, is a field of increasing interest in biomedical imaging. When a single image does not contain enough information to appropriately describe the organism under study, it is then necessary to acquire images of several individuals, each of them containing complementary data with respect to the rest of the components in the cohort. This approach allows creating digital prototypes, ranging from anatomical atlases of human patients and organs, obtained for instance from Magnetic Resonance Imaging, to gene expression cartographies of embryo development, typically achieved from Light Microscopy. Within such context, in this PhD Thesis we propose, develop and validate new dedicated image processing methodologies that, based on image registration techniques, bring information from multiple individuals into alignment within a single digital atlas model. We also elaborate a dedicated software visualization platform to explore the resulting wealth of multi-dimensional data and novel analysis algo-rithms to automatically mine the generated resource in search of bio¬logical insights. In particular, this work focuses on gene expression data from developing zebrafish embryos imaged at the cellular resolution level with Two-Photon Laser Scanning Microscopy. Disposing of quantitative measurements relating multiple gene expressions to cell position and their evolution in time is a fundamental prerequisite to understand embryogenesis multi-scale processes. However, the number of gene expressions that can be simultaneously stained in one acquisition is limited due to optical and labeling constraints. These limitations motivate the implementation of atlasing strategies that can recreate a virtual gene expression multiplex. The developed computational tools have been tested in two different scenarios. The first one is the early zebrafish embryogenesis where the resulting atlas constitutes a link between the phenotype and the genotype at the cellular level. The second one is the late zebrafish brain where the resulting atlas allows studies relating gene expression to brain regionalization and neurogenesis. The proposed computational frameworks have been adapted to the requirements of both scenarios, such as the integration of partial views of the embryo into a whole embryo model with cellular resolution or the registration of anatom¬ical traits with deformable transformation models non-dependent on any specific labeling. The software implementation of the atlas generation tool (Match-IT) and the visualization platform (Atlas-IT) together with the gene expression atlas resources developed in this Thesis are to be made freely available to the scientific community. Lastly, a novel proof-of-concept experiment integrates for the first time 3D gene expression atlas resources with cell lineages extracted from live embryos, opening up the door to correlate genetic and cellular spatio-temporal dynamics. La creación de atlas, o modelos digitales, donde la información de distintos sujetos puede ser combinada, es un campo de creciente interés en imagen biomédica. Cuando una sola imagen no contiene suficientes datos como para describir apropiadamente el organismo objeto de estudio, se hace necesario adquirir imágenes de varios individuos, cada una de las cuales contiene información complementaria respecto al resto de componentes del grupo. De este modo, es posible crear prototipos digitales, que pueden ir desde atlas anatómicos de órganos y pacientes humanos, adquiridos por ejemplo mediante Resonancia Magnética, hasta cartografías de la expresión genética del desarrollo de embrionario, típicamente adquiridas mediante Microscopía Optica. Dentro de este contexto, en esta Tesis Doctoral se introducen, desarrollan y validan nuevos métodos de procesado de imagen que, basándose en técnicas de registro de imagen, son capaces de alinear imágenes y datos provenientes de múltiples individuos en un solo atlas digital. Además, se ha elaborado una plataforma de visualization específicamente diseñada para explorar la gran cantidad de datos, caracterizados por su multi-dimensionalidad, que resulta de estos métodos. Asimismo, se han propuesto novedosos algoritmos de análisis y minería de datos que permiten inspeccionar automáticamente los atlas generados en busca de conclusiones biológicas significativas. En particular, este trabajo se centra en datos de expresión genética del desarrollo embrionario del pez cebra, adquiridos mediante Microscopía dos fotones con resolución celular. Disponer de medidas cuantitativas que relacionen estas expresiones genéticas con las posiciones celulares y su evolución en el tiempo es un prerrequisito fundamental para comprender los procesos multi-escala característicos de la morfogénesis. Sin embargo, el número de expresiones genéticos que pueden ser simultáneamente etiquetados en una sola adquisición es reducido debido a limitaciones tanto ópticas como del etiquetado. Estas limitaciones requieren la implementación de estrategias de creación de atlas que puedan recrear un multiplexado virtual de expresiones genéticas. Las herramientas computacionales desarrolladas han sido validadas en dos escenarios distintos. El primer escenario es el desarrollo embrionario temprano del pez cebra, donde el atlas resultante permite constituir un vínculo, a nivel celular, entre el fenotipo y el genotipo de este organismo modelo. El segundo escenario corresponde a estadios tardíos del desarrollo del cerebro del pez cebra, donde el atlas resultante permite relacionar expresiones genéticas con la regionalización del cerebro y la formación de neuronas. La plataforma computacional desarrollada ha sido adaptada a los requisitos y retos planteados en ambos escenarios, como la integración, a resolución celular, de vistas parciales dentro de un modelo consistente en un embrión completo, o el alineamiento entre estructuras de referencia anatómica equivalentes, logrado mediante el uso de modelos de transformación deformables que no requieren ningún marcador específico. Está previsto poner a disposición de la comunidad científica tanto la herramienta de generación de atlas (Match-IT), como su plataforma de visualización (Atlas-IT), así como las bases de datos de expresión genética creadas a partir de estas herramientas. Por último, dentro de la presente Tesis Doctoral, se ha incluido una prueba conceptual innovadora que permite integrar los mencionados atlas de expresión genética tridimensionales dentro del linaje celular extraído de una adquisición in vivo de un embrión. Esta prueba conceptual abre la puerta a la posibilidad de correlar, por primera vez, las dinámicas espacio-temporales de genes y células

Complexity in Developmental Systems: Toward an Integrated Understanding of Organ Formation

During animal development, embryonic cells assemble into intricately structured organs by working together in organized groups capable of implementing tightly coordinated collective behaviors, including patterning, morphogenesis and migration. Although many of the molecular components and basic mechanisms underlying such collective phenomena are known, the complexity emerging from their interplay still represents a major challenge for developmental biology. Here, we first clarify the nature of this challenge and outline three key strategies for addressing it: precision perturbation, synthetic developmental biology, and data-driven inference. We then present the results of our effort to develop a set of tools rooted in two of these strategies and to apply them to uncover new mechanisms and principles underlying the coordination of collective cell behaviors during organogenesis, using the zebrafish posterior lateral line primordium as a model system. To enable precision perturbation of migration and morphogenesis, we sought to adapt optogenetic tools to control chemokine and actin signaling. This endeavor proved far from trivial and we were ultimately unable to derive functional optogenetic constructs. However, our work toward this goal led to a useful new way of perturbing cortical contractility, which in turn revealed a potential role for cell surface tension in lateral line organogenesis. Independently, we hypothesized that the lateral line primordium might employ plithotaxis to coordinate organ formation with collective migration. We tested this hypothesis using a novel optical tool that allows targeted arrest of cell migration, finding that contrary to previous assumptions plithotaxis does not substantially contribute to primordium guidance. Finally, we developed a computational framework for automated single-cell segmentation, latent feature extraction and quantitative analysis of cellular architecture. We identified the key factors defining shape heterogeneity across primordium cells and went on to use this shape space as a reference for mapping the results of multiple experiments into a quantitative atlas of primordium cell architecture. We also propose a number of data-driven approaches to help bridge the gap from big data to mechanistic models. Overall, this study presents several conceptual and methodological advances toward an integrated understanding of complex multi-cellular systems

Development of High-speed Optical Coherence Tomography for Time-lapse Non-destructive Characterization of Samples

Optical coherence tomography (OCT) is an established optical imaging modality which can obtain label-free, non-destructive 3D images of samples with micron-scale resolution and millimeter penetration. OCT has been widely adopted for biomedical researches

Microsphere Traction Force Microscopy

This work is a culmination of our efforts in understanding cellular mechanics at the scale of single cells and small tissues. We developed methods to quantify cell-generated traction forces using cell-sized, synthetic, functionalized hydrogel microspheres. Cell-sized solid microspheres can provide information regarding cell-generated normal and shear forces while allowing natural cell-cell interactions and facilitating a convex cell-hydrogel interface. Therefore, they are a better mimic than the current methods for understanding the natural cell-cell interactions in a physiologically relevant geometry. In the analysis of the microsphere deformations, we use a boundary spectral method based on spherical harmonics decomposition of the traction field on the spherical gel surface. Using the techniques developed here, we measure the boundary traction profiles that mammalian cells exert on the synthetic microspherical hydrogel bodies. In this report, we briefly review the state of the art in cellular force quantification methods and discuss the contributions of our work to the field and its strengths as well as its limitations

Development of optical methods for real-time whole-brain functional imaging of zebrafish neuronal activity

Each one of us in his life has, at least once, smelled the scent of roses, read one canto of Dante’s Commedia or listened to the sound of the sea from a shell. All of this is possible thanks to the astonishing capabilities of an organ, such as the brain, that allows us to collect and organize perceptions coming from sensory organs and to produce behavioural responses accordingly. Studying an operating brain in a non-invasive way is extremely difficult in mammals, and particularly in humans. In the last decade, a small teleost fish, zebrafish (Danio rerio), has been making its way into the field of neurosciences. The brain of a larval zebrafish is made up of 'only' 100000 neurons and it’s completely transparent, making it possible to optically access it. Here, taking advantage of the best of currently available technology, we devised optical solutions to investigate the dynamics of neuronal activity throughout the entire brain of zebrafish larvae

Assessing the safety of engineered nanoparticles designed for therapeutic use

In 1959, physicist and Nobel laureate-to-be Richard Feynman held his famous speech “There’s Plenty of Room at the Bottom”. Herein he prophesized the upcoming of a new research field dedicated entirely to the nanometer scale. Six decades later we find ourselves amid the vision of Feynman where nanomaterials are omnipresent in daily life. Especially engineered nanoparticles (ENP) have gained enormous interest in medicine showing promising potentials as novel drug carrier systems, drug entities, and contrast agents. However, due to their size and physico-chemical properties, ENP inherit a unique toxicological profile that remains poorly understood. Moreover, the lack of generally accepted guidelines designed specifically for ENP prevents their proper safety regulation and risk assessment. In collaboration with the Swiss Center for Applied Human Toxicology (SCAHT) and the Horizion2020 NanoReg2 consortium, we focused our research on establishing ENP-specific characterization strategies and assay toolboxes that should provide robust information on the safety of ENP-organism interactions. Our results have shown that ENP toxicity is mediated by the chemical identity of a particle along with specific physico-chemical properties but also depends on the surrounding biological system. In vitro, in vivo, and in situ analyses ranging from simple viability studies to complex signaling pathway analysis have helped to link adverse effects and immune responses to different ENP types or specific properties thereof. Furthermore, we established the first protocol for the 3D-visualization of non-labeled ENP in vivo by synchrotron-radiation phase contrast x-ray micro-computer-tomography in therapeutically relevant dose-ranges. This novel method will aid to better understand the behavior of ENP after intravenous injection and may serve as a novel platform for the monitoring of ENP biodistribution and targeting. Conclusively, the combined results obtained during this thesis have led to the proposal of a novel safety assessment strategy which specifically targets ENPs designed for therapeutic use. This proposal highlights the importance and the benefits of application-specific decision-making with a strong emphasis on physico-chemical characterization and in vitro hazard assessment. This strategy is meant to contribute to the ongoing establishment of much needed nano-specific safety guidelines and was developed to be in line with the interests of regulatory authorities and researchers alike