Cryoimaging-Microscopy Implementation for 3D Optical Imaging

The structures and biochemistry properties of biological tissues are mostly affected by diseases. The visualization of organ structure and biochemistry helps in early detection and progression monitoring of diseases. Although, 2D imaging has traditionally been used to gain information from the tissue, it does not accurately represent many of the structures and functions. There currently exists a need for sensitive and specific methods to show detailed information about the structure of the tissue with high resolution and in 3D. The potential advantage of the high resolution 3D images is the ability to accurately probe structural and biochemical properties of the tissue. Not only the changes in structure, but also the changes in temporal physiological responses affected by oxidative stress (OS) at cellular levels. Thus, it would be valuable to detect the cellular metabolic states, which play a key role in understanding the pathogenesis of the disease, and to develop instruments to detect high resolution 3D images of the tissue. The objective of this research is to develop a second generation fluorescence optical imaging instrument to image the cellular redox state in 3D, in control and diseases conditions. I have improved upon one of optical instrument, called cryoimager software and hardware wise to enable higher resolution images. This higher resolution imaging resembles the microscopy capability in cryo temperatures for high resolution 3D imaging. In conclusion, high resolution optical instrumentation combined with signal and image processing tools provide quantitative physiological and structural information of diseased tissue

Measuring Corneal Topography Using Wavefront Analysis Technique

Refractive surgery has emphasized the need of accurate and precise methods for measuring the power of the human cornea. Available techniques for measuring corneal topography include keratometry, videokeratoscopy, and scanning slit imaging. A new type of instrument, a corneal topographer based on a Hartmann-Shack wavefront sensor for measuring corneal elevation over a spherical surface, is presented in this thesis. The performed tests of the topography system showed a high accuracy and reproducibility of the measurements on spherical as well as on toric test sample surfaces that approximate the curvature of the central human cornea. A comparison between the data obtained by the topographer and those provided by a videokeratoscope showed that the topographer is as precise as standard instruments used in clinical praxis. The measurements on human corneas demonstrated the importance of a Z-tracker module for the correct placement of the corneal surface. To enhance the accuracy of the topography system, a better tracking of the corneal position is necessary to compensate for the axial eye movements during the examination that strongly affect the measurements. Additionally, more clinical studies are necessary to test the topographer on human cornea and to give an evidence for the clinical acceptance of the instrument

Compact microscopy systems with non-conventional optical techniques

This work has been motivated by global efforts to decentralize high performance imaging systems through frugal engineering and expansion of 3D fabrication technologies. Typically, high resolution imaging systems are confined in clinical or laboratory environment due to the limited means of producing optical lenses on the demand. The use of lenses is an essential mean to achieve high resolution imaging, but conventional optical lenses are made using either polished glass or molded plastics. Both are suited for highly skilled craftsmen or factory level production. In the first part of this work, alternative low-cost lens-making process for generating high quality optical lenses with minimal operator training have been discussed. We evoked the use of liquid droplets to make lenses. This unconventional method relies on interfacial forces to generate curved droplets that if solidified can become convex-shaped lenses. To achieve this, we studied the droplet behaviour (Rayleigh-Plateau phenomenon) before creating a set of 3D printed tools to generate droplets. We measured and characterized the fabrication techniques to ensure reliability in lens fabrication on- demand at high throughput. Compact imaging requires a compact optical system and computing unit. So, in the next part of this work, we engineered a deconstructed microscope system for field-portable imaging. Still a core limitation of all optical lenses is the physical size of lens aperture – which limits their resolution performance, and optical aberrations – that limit their imaging quality performance. In the next part of this work, we investigated use of computational optics-based optimization approaches to conduct in situ characterization of aberrations that can be digitally removed. The computational approach we have used in this work is known as Fourier Ptychography (FP). It is an emerging computational microscopic technique that combines the use of synthetic aperture and iterative optimization algorithms, offering increased resolution, at full field-of-view (FOV) and aberration-removal. In using FP techniques, we have shown measurements of optical distortions from different lenses made from droplets only. We also, investigated the limitations of FP in aberration recovery on moldless lenses. In conclusion, this work presents new opportunities to engineer high resolution imaging system using modern 3D printing approaches. Our successful demonstration of FP techniques on moldless lenses will usher new additional applications in digital pathology or low-cost mobile health

Single-photon counting lidar for long-range three-dimensional imaging

Single-photon time-of-flight (ToF) distance ranging lidar is a candidate technology for high-resolution depth imaging for use, for example, from airborne platforms. This approach enables low average power pulsed laser sources to be used while allowing imaging from significantly longer target ranges compared to analogue imaging. The recent availability of Geiger-mode (Gm) arrays has revolutionised photon-counting lidar as they provide single-photon full-frame data in short acquisition times. This thesis presents work on the opto-mechanical design, tolerance analysis and performance evaluation of a re-configurable single-photon counting lidar which can accommodate either a single-element single-photon avalanche photodiode (SPAD) or a 32 × 32 Gm-array. By incorporating an inter-changeable lens, the two configurations were designed to provide identical pixel resolution for both the single-pixel system and the Gm-array configurations in order to permit a performance comparison to be conducted. This is the first time that such a comparison has been reported and the lidar is one of the earliest to assess the performance of a short-wave infra-red (SWIR) Gm-array. Both detection configurations used InGaAs/InP SPAD detectors and operated at a wavelength of 1550 nm. The main benefits of operating within the SWIR band include reduced solar background, lower atmospheric loss, improved covertness, as well as improved laser eye-safety thresholds. The system estimates target range by measuring the ToF using time-correlated single-photon counting (TCSPC) and was used to produce high-resolution three-dimensional images of targets at between 800 m and 10.5 km range. The single-element system has the potential to provide improved depth resolution over the array due to a smaller timing jitter but requires longer acquisition times due to the need for two-dimensional scanning. The acquisition time of the array configuration can be up to three orders of magnitude faster than the single-element configuration but requires significantly higher average laser power levels. The Gm-array provided a simultaneous estimation of angle-of-arrival and intensity fluctuations from which a comparable strength of atmospheric turbulence could be measured. This demonstrated that Gm-arrays provide a new way of high-speed turbulence measurement with time intervals much shorter than those offered by existing scintillometers

Interferometric Metrology Using Reprogrammable Binary Holograms

Interferometric methods for surface metrology have been widely used for many years due to their speed, accuracy and versatility. It is frequently necessary however to produce a known comparison reference surface to minimise the optical path difference and hence enhance the dynamic range. An alternative to this is to use a computer generated hologram to act as the reference wave, or to correct a spherical reference wave to match a highly aspheric optic in order to achieve a null test. This thesis shall present a novel method of producing such holograms through the use of a binary ferroelectric liquid crystal on silicon spatial light modulator (FLCOS SLM) rather than using the more common lithographically produced plates. One of the primary advantages this could introduce is the ability for arbitrarily reprogrammable holograms to be created upon demand rather than needing to produce a series of holographic plates, saving both time and money in the testing of surfaces. We present results characterising the ability of a FLCOS SLM to produce increasingly large Zernike aberrations as well as quantifying the resulting errors, before using the device to reduce interferometric fringe density allowing us to measure aberrated optics and reveal low amplitude surface variations on the scale of 0.045 waves RMS

Optical biopsy systems using ultra-slim objectives for the diagnosis of breast cancer

One in eight women in America will develop breast cancer at some point in their lives. Breast cancer is the second deadliest form of cancer for women in the United States. When a suspicious region of the breast is detected, the tissue is diagnosed by rem

Fabrication and current optical performance of a large diamond-machined ZnSe immersion grating

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New technologies for optical coherence microscopy

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2008.Includes bibliographical references.According to the American Cancer Society, gastrointestinal (GI) cancers are among the most common forms of malignancies suffered today, affecting -200,000 people and causing -80,000 deaths in the United States every year. The prognosis depends heavily on the detection of early-stage lesions. The process of endoscopic surveillance, excisional biopsy, and histologic examination is the current gold standard for screening and diagnosis of many GI cancers. This process, however, is invasive, time-consuming, and can suffer from unacceptable false negative rates. Optical imaging technology that provides real-time, high-resolution imaging of human tissue in vivo with resolution at or near that of histopathology may significantly improve clinicians' capabilities to identify malignancies at curable stages. The ability to assess histologic hallmarks of GI cancer at the tissue architectural and cellular levels without excisional biopsy would be a major advance in GI cancer management. Development of techniques to reliably image cellular and subcellular structure through endoscopic devices is one of the most outstanding challenges in biomedical imaging today and holds tremendous promise for surgical applications and for early diagnostic screening and staging of epithelial malignancies. Optical coherence microscopy (OCM) is an in vivo cellular imaging technique that combines OCT with confocal microscopy. Due to the unique feature of using two distinct optical sectioning techniques, OCM can provide superior imaging depth in highly scattered tissues and can overcome important imaging probe design limitations that hinder confocal microscopy. Two novel designs for OCM systems are proposed and developed for high resolution cellular imaging. The first uses Fourier domain optical coherence detection, and the second implements line-field illumination and detection. Differences in performance from the standard time-domain optical coherence microscopy systems will be studied.by Shu-Wei Huang.S.M

Determining the Influence of Environment and Minimizing Residual Roughness in Laser Corneal Refractive Surgery

Aims: This dissertation deals with multiple topics, with a global aim of determining the influence of environment and minimizing residual roughness in laser corneal refractive surgery. The multiple topics under consideration are listed below: •TOPIC A: To analyze the effect of seasonal changes in PMMA Performance using the SCHWIND AMARIS laser system •TOPIC B: To analyze impact of various humidity and temperature settings on excimer laser ablation of PET, PMMA and porcine corneal tissue •TOPIC C: To analyze the impact of residual roughness after corneal ablation in perception and vision •TOPIC D: To outline a rigorous simulation model for simulating shot-by-shot ablation process. Furthermore, to simulate the impact of laser beam characteristics like super Gaussian order, truncation radius, spot geometry, spot overlap and lattice geometry on ablation smoothness. •TOPIC E: To test the impact of laser beam truncation, dithering, and jitter on residual roughness after PMMA ablations, using a close-to-Gaussian beam profile. Methods: TOPIC A: By analyzing PMMA and PET ablation performance by a large series of AMARIS laser systems (Schwind eye-tech solutions, Germany) inside a climate controlled environment, the influence purely coming from the seasonal changes was investigated in a large scale retrospective cross sectional review. Seasonal outcomes were evaluated in terms of PMMA and PET Performance stratified for every month in a year, as well as stratified for each season in a year. TOPIC B: A Study was conducted using AMARIS system placed inside a climate chamber. Ablations were performed on PET, PMMA and porcine cornea. Impact of wide range of temperature (~18°C to ~30°C) and relative humidity (~25% to ~80%) on laser ablation outcomes was tested using nine climate test settings. Multiple linear regression was performed using least square method with predictive factors: Temperature, Relative Humidity, Time stamp. Influence of climate settings was modelled for Pulse Energy, Pulse Fluence, ablation efficiency on PMMA and porcine cornea tissue. TOPIC C: The Indiana Retinal Image Simulator (IRIS) was used to simulate the polychromatic retinal image. Using patient-specific Zernike coefficients and pupil diameter, the impact of different levels of chromatic aberrations was calculated. Corneal roughness was modeled via both random and filtered noise, using distinct pre-calculated higher order Zernike coefficient terms. The outcome measures for the simulation were simulated retinal image, Strehl Ratio and Visual Strehl Ratio computed in frequency domain. The impact of varying degree of roughness, spatial frequency of the roughness, and pupil dilation was analyzed on these outcome measures. TOPIC D: Given the super Gaussian order, the theoretical beam profile was determined following Lambert-Beer model. The intensity beam profile originating from an excimer laser was measured with a beam profiler camera. For both, the measured and theoretical, beam profiles, two spot geometries (round and square spots) were considered, and two types of lattices (reticular and triangular) were simulated with varying spot overlaps and ablated material (cornea or PMMA). The roughness in ablation was determined by the root-mean-square per square root of layer depth. TOPIC E: A study was conducted using a modified AMARIS system. For the PMMA ablations, two configurations (with a 0.7mm pinhole and 0.75mJ and without pinhole and 0.9mJ (for fluences of 329mJ/cm2 and 317mJ/cm2 and corneal spot volumes of 174pl and 188pl)) were considered, along with two types of lattices (with and without ordered dithering to select the optimum pulse positions), and two types of spot placement (with and without jitter). Real ablations on PMMA (ranging from -12D to +6D with and without astigmatism) completed the study setup. The effect of the 2x2x2 different configurations was analyzed based on the roughness in ablation estimated from the root mean square error in ablation. Results: TOPIC A: The seasons winter and summer showed statistical significant variations with respect to the global values for all the tested parameters except the nominal number of laser pulses for high and low fluence setting. The metric technical performance of the analyzed systems showed a stronger PMMA ablation performance in summer time compared to a weaker performance in the winter time, with the maximum seasonal deviation of 6%. The results were consistently confirmed in seasonal as well as monthly analyses. TOPIC B: Temperature changes did not affect laser pulse energy, pulse fluence (PET), and ablation efficiency (on PMMA or porcine corneal tissue) significantly. Changes in relative humidity were more critical and significantly affected laser pulse energy, high fluence and low fluence. Opposite trend was observed between the ablation performance on PMMA and porcine cornea. TOPIC C: In case of a constant roughness term, reducing the pupil size resulted in improved outcome measures and simulated retinal image. The calculated image quality metrics deteriorated dramatically with increasing roughness. Clear distinction was observed in outcome measures for corneal roughness simulated as random noise compared to filtered noise, further influenced by the spatial frequency of filtered noise. TOPIC D: Truncating the beam profile increased the roughness in ablation, Gaussian profiles theoretically resulted in smoother ablations, round spot geometries produced lower roughness in ablation compared to square geometry, triangular lattices theoretically produced lower roughness in ablation compared to the reticular lattice, theoretically modelled beam profiles showed lower roughness in ablation compared to the measured beam profile, and the simulated roughness in ablation on PMMA tend to be lower than on human cornea. For given input parameters, proper optimum parameters for minimizing the roughness has been found. TOPIC E: Truncation of the beam was negatively associated to a higher level of residual roughness; ordered dithering to select the optimum pulse positions was positively associated to a lower level of residual roughness; jitter was negatively associated to a higher level of residual roughness. The effect of dithering was the largest, followed by truncation, and jitter had the lowest impact on results. Conclusions: The large scale retrospective cross sectional study presented in this work, demonstrated a cyclic winter-summer variation in PMMA ablation using the AMARIS lasers. These seasonal variations were further substantiated with the experiments conducted in the climate chamber, over a wide range of temperature and humidity. Temperature changes did not affect laser pulse energy, pulse fluence, and ablation efficiency (on PMMA or porcine corneal tissue) significantly. However, changes in relative humidity were more critical and significantly affected laser pulse energy, high fluence and low fluence. The proposed well-fitting multi-linear model can be utilized for compensation of temperature and humidity changes on ablation efficiency. The relationship between calibration materials like PMMA and corneal tissue shall be analyzed cautiously before designing the calibration routine, in order to obtain optimum outcomes with minimum deviations. Despite its limitations, the simple and robust method proposed here for quantifying the influence of post-ablation roughness on vision and perception, can be utilized in different applications. From the simulations of the shot-by-shot ablation process, a theoretical proper optimum configuration was found for minimizing the roughness in ablation for defined input parameters. The PMMA experiments confirmed the theoretical proper optimum settings in real world conditions. The results and improvements derived out of this work can be directly applied to the laser systems for corneal refractive surgery, to help reduce the complications and occurrence of adverse events during and after refractive surgery, and improve the short term and long term postoperative clinical outcomes