Snapshot hyperspectral imaging : near-infrared image replicating imaging spectrometer and achromatisation of Wollaston prisms

Conventional hyperspectral imaging (HSI) techniques are time-sequential and rely on temporal scanning to capture hyperspectral images. This temporal constraint can limit the application of HSI to static scenes and platforms, where transient and dynamic events are not expected during data capture. The Near-Infrared Image Replicating Imaging Spectrometer (N-IRIS) sensor described in this thesis enables snapshot HSI in the short-wave infrared (SWIR), without the requirement for scanning and operates without rejection in polarised light. It operates in eight wavebands from 1.1μm to 1.7μm with a 2.0° diagonal field-of-view. N-IRIS produces spectral images directly, without the need for prior topographic or image reconstruction. Additional benefits include compactness, robustness, static operation, lower processing overheads, higher signal-to-noise ratio and higher optical throughput with respect to other HSI snapshot sensors generally. This thesis covers the IRIS design process from theoretical concepts to quantitative modelling, culminating in the N-IRIS prototype designed for SWIR imaging. This effort formed the logical step in advancing from peer efforts, which focussed upon the visible wavelengths. After acceptance testing to verify optical parameters, empirical laboratory trials were carried out. This testing focussed on discriminating between common materials within a controlled environment as proof-of-concept. Significance tests were used to provide an initial test of N-IRIS capability in distinguishing materials with respect to using a conventional SWIR broadband sensor. Motivated by the design and assembly of a cost-effective visible IRIS, an innovative solution was developed for the problem of chromatic variation in the splitting angle (CVSA) of Wollaston prisms. CVSA introduces spectral blurring of images. Analytical theory is presented and is illustrated with an example N-IRIS application where a sixfold reduction in dispersion is achieved for wavelengths in the region 400nm to 1.7μm, although the principle is applicable from ultraviolet to thermal-IR wavelengths. Experimental proof of concept is demonstrated and the spectral smearing of an achromatised N-IRIS is shown to be reduced by an order of magnitude. These achromatised prisms can provide benefits to areas beyond hyperspectral imaging, such as microscopy, laser pulse control and spectrometry

A review of intrinsic optical imaging serial blockface histology (ICI-SBH) for whole rodent brain imaging

In recent years, multiple serial histology techniques were developed to enable whole rodent brain imaging in 3-D. The main driving forces behind the emergence of these imaging techniques were the genome-wide atlas of gene expression in the mouse brain, the pursuit of the mouse brain connectome, and the BigBrain project. These projects rely on the use of optical imaging to target neuronal structures with histological stains or fluorescent dyes that are either expressed by transgenic mice or injected at specific locations in the brain. Efforts to adapt the serial histology acquisition scheme to use intrinsic contrast imaging (ICI) were also put forward, thus leveraging the natural contrast of neuronal tissue. This review focuses on these efforts. First, the origin of optical contrast in brain tissue is discussed with emphasis on the various imaging modalities exploiting these contrast mechanisms. Serial blockface histology (SBH) systems using ICI modalities are then reported, followed by a review of some of their applications. These include validation studies and the creation of multimodal brain atlases at a micrometer resolution. The paper concludes with a perspective of future developments, calling for a consolidation of the SBH research and development efforts around the world. The goal would be to offer the neuroscience community a single standardized open-source SBH solution, including optical design, acquisition automation, reconstruction algorithms, and analysis pipelines

Defect and thickness inspection system for cast thin films using machine vision and full-field transmission densitometry

Quick mass production of homogeneous thin film material is required in paper, plastic, fabric, and thin film industries. Due to the high feed rates and small thicknesses, machine vision and other nondestructive evaluation techniques are used to ensure consistent, defect-free material by continuously assessing post-production quality. One of the fastest growing inspection areas is for 0.5-500 micrometer thick thin films, which are used for semiconductor wafers, amorphous photovoltaics, optical films, plastics, and organic and inorganic membranes. As a demonstration application, a prototype roll-feed imaging system has been designed to inspect high-temperature polymer electrolyte membrane (PEM), used for fuel cells, after being die cast onto a moving transparent substrate. The inspection system continuously detects thin film defects and classifies them with a neural network into categories of holes, bubbles, thinning, and gels, with a 1.2% false alarm rate, 7.1% escape rate, and classification accuracy of 96.1%. In slot die casting processes, defect types are indicative of a misbalance in the mass flow rate and web speed; so, based on the classified defects, the inspection system informs the operator of corrective adjustments to these manufacturing parameters. Thickness uniformity is also critical to membrane functionality, so a real-time, full-field transmission densitometer has been created to measure the bi-directional thickness profile of the semi-transparent PEM between 25-400 micrometers. The local thickness of the 75 mm x 100 mm imaged area is determined by converting the optical density of the sample to thickness with the Beer-Lambert law. The PEM extinction coefficient is determined to be 1.4 D/mm and the average thickness error is found to be 4.7%. Finally, the defect inspection and thickness profilometry systems are compiled into a specially-designed graphical user interface for intuitive real-time operation and visualization.M.S.Committee Chair: Tequila Harris; Committee Member: Levent Degertekin; Committee Member: Wayne Dale

Sizing individual dielectric nanoparticles with quantitative differential interference contrast microscopy

We report a method to measure the size of single dielectric nanoparticles with high accuracy and precision using quantitative differential interference contrast (DIC) microscopy. Dielectric nanoparticles are detected optically by the conversion of the optical phase change into an intensity change using DIC. Phase images of individual nanoparticles were retrieved from DIC by Wiener filtering, and a quantitative methodology to extract nanoparticle sizes was developed. Using polystyrene beads of 100 nm radius as size standard, we show that the method determines this radius within a few nm accuracy. The smallest detectable polystyrene bead is limited by background and shot-noise, which depend on acquisition and analysis parameters, including the objective numerical aperture, the DIC phase offset, and the refractive index contrast between particles and their surrounding. A sensitivity limit potentially reaching down to 1.8 nm radius was inferred. As application example, individual nanodiamonds with nominal sizes below 50 nm were measured, and were found to have a nearly exponential size distribution with 28 nm mean value. Considering the importance of dielectric nanoparticles in many fields, from naturally occurring virions to polluting nanoplastics, the proposed method could offer a powerful quantitative tool for nanoparticle analysis, combining accuracy, sensitivity and high-throughput with widely available and easy-to-use DIC microscop

Multiscale imaging of the mouse cortex using two-photon microscopy and wide-field illumination

The mouse brain can be studied over vast spatial scales ranging from microscopic imaging of single neurons to macroscopic measurements of hemodynamics acquired over the majority of the mouse cortex. However, most neuroimaging modalities are limited by a fundamental trade-off between the spatial resolution and the field-of-view (FOV) over which the brain can be imaged, making it difficult to fully understand the functional and structural architecture of the healthy mouse brain and its disruption in disease. My dissertation has focused on developing multiscale optical systems capable of imaging the mouse brain at both microscopic and mesoscopic spatial scales, specifically addressing the difference in spatial scales imaged with two-photon microscopy (TPM) and optical intrinsic signal imaging (OISI). Central to this work has been the formulation of a principled design strategy for extending the FOV of the two-photon microscope. Using this design approach, we constructed a TPM system with subcellular resolution and a FOV area 100 times greater than a conventional two-photon microscope. To image the ellipsoidal shape of the mouse cortex, we also developed the microscope to image arbitrary surfaces within a single frame using an electrically tunable lens. Finally, to address the speed limitations of the TPM systems developed during my dissertation, I also conducted research in large-scale neural phenomena occurring in the mouse brain imaged with high-speed OISI. The work conducted during my dissertation addresses some of the fundamental principles in designing and applying optical systems for multiscale imaging of the mouse brain

Modeling and applications of the focus cue in conventional digital cameras

El enfoque en cámaras digitales juega un papel fundamental tanto en la calidad de la imagen como en la percepción del entorno. Esta tesis estudia el enfoque en cámaras digitales convencionales, tales como cámaras de móviles, fotográficas, webcams y similares. Una revisión rigurosa de los conceptos teóricos detras del enfoque en cámaras convencionales muestra que, a pasar de su utilidad, el modelo clásico del thin lens presenta muchas limitaciones para aplicación en diferentes problemas relacionados con el foco. En esta tesis, el focus profile es propuesto como una alternativa a conceptos clásicos como la profundidad de campo. Los nuevos conceptos introducidos en esta tesis son aplicados a diferentes problemas relacionados con el foco, tales como la adquisición eficiente de imágenes, estimación de profundidad, integración de elementos perceptuales y fusión de imágenes. Los resultados experimentales muestran la aplicación exitosa de los modelos propuestos.The focus of digital cameras plays a fundamental role in both the quality of the acquired images and the perception of the imaged scene. This thesis studies the focus cue in conventional cameras with focus control, such as cellphone cameras, photography cameras, webcams and the like. A deep review of the theoretical concepts behind focus in conventional cameras reveals that, despite its usefulness, the widely known thin lens model has several limitations for solving different focus-related problems in computer vision. In order to overcome these limitations, the focus profile model is introduced as an alternative to classic concepts, such as the near and far limits of the depth-of-field. The new concepts introduced in this dissertation are exploited for solving diverse focus-related problems, such as efficient image capture, depth estimation, visual cue integration and image fusion. The results obtained through an exhaustive experimental validation demonstrate the applicability of the proposed models

Efficient and Accurate Disparity Estimation from MLA-Based Plenoptic Cameras

This manuscript focuses on the processing images from microlens-array based plenoptic cameras. These cameras enable the capturing of the light field in a single shot, recording a greater amount of information with respect to conventional cameras, allowing to develop a whole new set of applications. However, the enhanced information introduces additional challenges and results in higher computational effort. For one, the image is composed of thousand of micro-lens images, making it an unusual case for standard image processing algorithms. Secondly, the disparity information has to be estimated from those micro-images to create a conventional image and a three-dimensional representation. Therefore, the work in thesis is devoted to analyse and propose methodologies to deal with plenoptic images. A full framework for plenoptic cameras has been built, including the contributions described in this thesis. A blur-aware calibration method to model a plenoptic camera, an optimization method to accurately select the best microlenses combination, an overview of the different types of plenoptic cameras and their representation. Datasets consisting of both real and synthetic images have been used to create a benchmark for different disparity estimation algorithm and to inspect the behaviour of disparity under different compression rates. A robust depth estimation approach has been developed for light field microscopy and image of biological samples

Content-aware approach for improving biomedical image analysis: an interdisciplinary study series

Biomedicine is a highly interdisciplinary research area at the interface of sciences, anatomy, physiology, and medicine. In the last decade, biomedical studies have been greatly enhanced by the introduction of new technologies and techniques for automated quantitative imaging, thus considerably advancing the possibility to investigate biological phenomena through image analysis. However, the effectiveness of this interdisciplinary approach is bounded by the limited knowledge that a biologist and a computer scientist, by professional training, have of each other’s fields. The possible solution to make up for both these lacks lies in training biologists to make them interdisciplinary researchers able to develop dedicated image processing and analysis tools by exploiting a content-aware approach. The aim of this Thesis is to show the effectiveness of a content-aware approach to automated quantitative imaging, by its application to different biomedical studies, with the secondary desirable purpose of motivating researchers to invest in interdisciplinarity. Such content-aware approach has been applied firstly to the phenomization of tumour cell response to stress by confocal fluorescent imaging, and secondly, to the texture analysis of trabecular bone microarchitecture in micro-CT scans. Third, this approach served the characterization of new 3-D multicellular spheroids of human stem cells, and the investigation of the role of the Nogo-A protein in tooth innervation. Finally, the content-aware approach also prompted to the development of two novel methods for local image analysis and colocalization quantification. In conclusion, the content-aware approach has proved its benefit through building new approaches that have improved the quality of image analysis, strengthening the statistical significance to allow unveiling biological phenomena. Hopefully, this Thesis will contribute to inspire researchers to striving hard for pursuing interdisciplinarity

Multi-Aperture Fourier Ptychographic Microscopy: development of a high-speed gigapixel coherent computational microscope

Medical research and clinical diagnostics require imaging of large sample areas with sub-cellular resolution. Conventional imaging techniques can provide either high-resolution or wide field-of-view (FoV) but not both. This compromise is conventionally defeated by using a high NA objective with a small FoV and then mechanically scan the sample in order to acquire separate images of its different regions. By stitching these images together, a larger effective FoV is then obtained. This procedure, however, requires precise and expensive scanning stages and prolongs the acquisition time, thus rendering the observation of fast processes/phenomena impossible. A novel imaging configuration termed Multi-Aperture Fourier Ptychographic Microscopy (MA-FPM) is proposed here based on Fourier ptychography (FP), a technique to achieve wide-FoV and high-resolution using time-sequential synthesis of a high-NA coherent illumination. MA-FPM configuration utilises an array of objective lenses coupled with detectors to increase the bandwidth of the object spatial-frequencies captured in a single snapshot. This provides high-speed data-acquisition with wide FoV, high-resolution, long working distance and extended depth-of-field. In this work, a new reconstruction method based on Fresnel diffraction forward model was developed to extend FP reconstruction to the proposed MA-FPM technique. MA-FPM was validated experimentally by synthesis of a 3x3 lens array system from a translating objective-detector system. Additionally, a calibration procedure was also developed to register dissimilar images from multiple cameras and successfully implemented on the experimental data. A nine-fold improvement in captured data-bandwidth was demonstrated. Another experimental configuration was proposed using the Scheimpflug condition to correct for the aberrations present in the off-axis imaging systems. An experimental setup was built for this new configuration using 3D printed parts to minimise the cost. The design of this setup is discussed along with robustness analysis of the low-cost detectors used in this setup. A reconstruction model for the Scheimpflug configuration FP was developed and applied to the experimental data. Preliminary experimental results were found to be in agreement with this reconstruction model. Some artefacts were observed in these results due to the calibration errors in the experiment. These can be corrected by using the self-calibration algorithm proposed in the literature, which is left as a future work. Extensions to this work can include implementing multiplexed illumination for further increasing the data acquisition speed and diffraction tomography for imaging thick samples