Tesis sobre el tema "ULTRAFAST WIDE FIELD IMAGING"

The light collecting apertures of space telescopes are currently limited in part by the size and weight restrictions of launch vehicles, ultimately limiting the spatial resolution that can be achieved by the observatory. A technique that can overcome these limitations and provide superior spatial resolution is interferometric imaging, whereby multiple small telescopes can be combined to produce a spatial resolution comparable to a much larger monolithic telescope. In astronomy, the spectrum of the sources in the scene are crucial to understanding the material composition of the sources. So, the ultimate goal is to have high-spatial-resolution imagery and obtain sufficient spectral resolution for all points in the scene. This goal can be accomplished through spatio-spectral interferometric imaging, which combines the aperture synthesis aspects of a Michelson stellar interferometer with the spectral capabilities of Fourier transform spectroscopy.

Spatio-spectral interferometric imaging can be extended to a wide-field imaging modality, which increases the collecting efficiency of the technique. This is the basis for NASA’s Wide-field Imaging Interferometry Testbed (WIIT). For such an interferometer, there are two light collecting apertures separated by a variable distance known as the baseline length. The optical path in one of the arms of the interferometer is variable, while the other path delay is fixed. The beams from both apertures are subsequently combined and imaged onto a detector. For a fixed baseline length, the result is many low-spatial-resolution images at a slew of optical path differences, and the process is repeated for many different baseline lengths and orientations. Image processing and synthesis techniques are required to reduce the large dataset into a single high-spatial-resolution hyperspectral image.

Our contributions to spatio-spectral interferometry include various aspects of theory, simulation, image synthesis, and processing of experimental data, with the end goal of better understanding the nature of the technique. We present the theory behind the measurement model for spatio-spectral interferometry, as well as the direct approach to image synthesis. We have developed a pipeline to preprocess experimental data to remove unwanted signatures in the data and register all image measurements to a single orientation, which leverages information about the optical system’s point spread function. In an experimental setup, such as WIIT, the reference frame for the path difference measured for each baseline is unknown and must be accounted for. To overcome this obstacle, we created a phase referencing technique that leverages point sources within the scene of known separation in order to recover unknown information regarding the measurements in a laboratory setting. We also provide a method that allows for the measurement of spatially and spectrally complicated scenes with WIIT by decomposing them prior to scene projection.

This dissertation explores computational optical imaging methods to circumvent the physical limitations of classical sensing. An ideal imaging system would maximize resolution in time, spectral bandwidth, three-dimensional object space, and polarization. Practically, increasing any one parameter will correspondingly decrease the others.

Spectrometers strive to measure the power spectral density of the object scene. Traditional pushbroom spectral imagers acquire high resolution spectral and spatial resolution at the expense of acquisition time. Multiplexed spectral imagers acquire spectral and spatial information at each instant of time. Using a coded aperture and dispersive element, the coded aperture snapshot spectral imagers (CASSI) here described leverage correlations between voxels in the spatial-spectral data cube to compressively sample the power spectral density with minimal loss in spatial-spectral resolution while maintaining high temporal resolution.

Photography is limited by similar physical constraints. Low f/# systems are required for high spatial resolution to circumvent diffraction limits and allow for more photon transfer to the film plain, but require larger optical volumes and more optical elements. Wide field systems similarly suffer from increasing complexity and optical volume. Incorporating a multi-scale optical system, the f/#, resolving power, optical volume and wide field of view become much less coupled. This system uses a single objective lens that images onto a curved spherical focal plane which is relayed by small micro-optics to discrete focal planes. Using this design methodology allows for gigapixel designs at low f/# that are only a few pounds and smaller than a one-foot hemisphere.

Computational imaging systems add the necessary step of forward modeling and calibration. Since the mapping from object space to image space is no longer directly readable, post-processing is required to display the required data. The CASSI system uses an undersampled measurement matrix that requires inversion while the multi-scale camera requires image stitching and compositing methods for billions of pixels in the image. Calibration methods and a testbed are demonstrated that were developed specifically for these computational imaging systems.

Breast conserving surgery (BCS) is a common treatment option for breast cancer patients. The goal of BCS is to remove the entire tumor from the breast while preserving as much normal tissue as possible for a better cosmetic outcome after surgery. Specifically, the excised specimen must have at least 2 mm of normal tissue surrounding the diseased mass. Unfortunately, a staggering 20-70% of patients undergoing BCS require repeated surgeries due to the incomplete removal of the tumor diagnosed post-operatively. Due to these high re-excision rates as well as limited post-operative histopathological sampling of the tumor specimen, there is an unmet clinical need for margin assessment. Quantitative diffuse reflectance spectral imaging has previously been explored as a promising, method for providing real-time visual maps of tissue composition to help surgeons determine breast tumor margins to ensure the complete removal of the disease during breast conserving surgery. We have leveraged the underlying sources of contrast in breast tissue, specifically total hemoglobin content, beta-carotene content, and tissue scattering, and developed various fiber optics based spectral imaging systems for this clinical application. Combined with a fast inverse Monte Carlo model of reflectance, previous studies have shown that this technology may be able to decrease re-excision rates for BCS. However, these systems, which all consist of a broadband source, fiber optics probes, an imaging spectrograph and a CCD, have severe limitations in system footprint, tumor area coverage, and speed for acquisition and analysis. The fiber based spectral imaging systems are not scalable to smaller designs that cover a large surveillance area at a very fast speed, which ultimately makes them impractical for use in the clinical environment. The objective of this dissertation was to design, develop, test, and show clinical feasibility of a novel wide field spectral imaging system that utilizes the same scientific principles of previously developed fiber optics based imaging systems, but improves upon the technical issues, such as size, complexity, and speed,to meet the demands of the intra-operative setting.

First, our simple re-design of the system completely eliminated the need for an imaging spectrograph and CCD by replacing them with an array of custom annular photodiodes. The geometry of the photodiodes were designed with the goal of minimizing optical crosstalk, maximizing SNR, and achieving the appropriate tissue sensing depth of up to 2 mm for tumor margin assessment. Without the imaging spectrograph and CCD, the system requires discrete wavelengths of light to launch into the tissue sample. A wavelength selection method that combines an inverse Monte Carlo model and a genetic algorithm was developed in order to optimize the wavelength choices specifically for the underlying breast tissue optical contrast. The final system design consisted of a broadband source with an 8-slot filter wheel containing the optimized set of wavelength choices, an optical light guide and quartz light delivery tube to send the 8 wavelengths of light in free space through the back apertures of each annular photodiode in the imaging array, an 8-channel integrating transimpedance amplifier circuit with a switch box and data acquisition card to collect the reflectance signal, and a laptop computer that controls all the components and analyzes the data.

This newly designed wide field spectral imaging system was tested in tissue-mimicking liquid phantoms and achieved comparable performance to previous clinically-validated fiber optics based systems in its ability to extract optical properties with high accuracy. The system was also tested in various biological samples, including a murine tumor model, porcine tissue, and human breast tissue, for the direct comparison with its fiber optics based counterparts. The photodiode based imaging system achieved comparable or better SNR, comparable extractions of optical properties extractions for all tissue types, and feasible improvements in speed and coverage for future iterations. We show proof of concept in performing fast, wide field spectral imaging with a simple, inexpensive design. With a reduction in size, cost, number of wavelengths used, and overall complexity, the system described by this dissertation allows for a more seamless scaling to higher pixel number and density in future iterations of the technology, which will help make this a clinically translatable tool for breast tumor margin assessment.

Wide field-of-view (FOV) microscopy is of high importance to biological research and clinical diagnosis where a high-throughput screening of samples is needed. This thesis presents the development of several novel wide FOV imaging technologies and demonstrates their capabilities in longitudinal imaging of living organisms, on the scale of viral plaques to live cells and tissues.

The ePetri Dish is a wide FOV on-chip bright-field microscope. Here we applied an ePetri platform for plaque analysis of murine norovirus 1 (MNV-1). The ePetri offers the ability to dynamically track plaques at the individual cell death event level over a wide FOV of 6 mm × 4 mm at 30 min intervals. A density-based clustering algorithm is used to analyze the spatial-temporal distribution of cell death events to identify plaques at their earliest stages. We also demonstrate the capabilities of the ePetri in viral titer count and dynamically monitoring plaque formation, growth, and the influence of antiviral drugs.

We developed another wide FOV imaging technique, the Talbot microscope, for the fluorescence imaging of live cells. The Talbot microscope takes advantage of the Talbot effect and can generate a focal spot array to scan the fluorescence samples directly on-chip. It has a resolution of 1.2 μm and a FOV of ~13 mm². We further upgraded the Talbot microscope for the long-term time-lapse fluorescence imaging of live cell cultures, and analyzed the cells’ dynamic response to an anticancer drug.

We present two wide FOV endoscopes for tissue imaging, named the AnCam and the PanCam. The AnCam is based on the contact image sensor (CIS) technology, and can scan the whole anal canal within 10 seconds with a resolution of 89 μm, a maximum FOV of 100 mm × 120 mm, and a depth-of-field (DOF) of 0.65 mm. We also demonstrate the performance of the AnCam in whole anal canal imaging in both animal models and real patients. In addition to this, the PanCam is based on a smartphone platform integrated with a panoramic annular lens (PAL), and can capture a FOV of 18 mm × 120 mm in a single shot with a resolution of 100─140 μm. In this work we demonstrate the PanCam’s performance in imaging a stained tissue sample.

Optical microscopy has become an indispensable tool for biological researches since its invention, mostly owing to its sub-cellular spatial resolutions, non-invasiveness, instrumental simplicity, and the intuitive observations it provides. Nonetheless, obtaining reliable, quantitative spatial information from conventional wide-field optical microscopy is not always intuitive as it appears to be. This is because in the acquired images of optical microscopy the information about out-of-focus regions is spatially blurred and mixed with in-focus information. In other words, conventional wide-field optical microscopy transforms the three-dimensional spatial information, or volumetric information about the objects into a two-dimensional form in each acquired image, and therefore distorts the spatial information about the object. Several fluorescence holography-based methods have demonstrated the ability to obtain three-dimensional information about the objects, but these methods generally rely on decomposing stereoscopic visualizations to extract volumetric information and are unable to resolve complex 3-dimensional structures such as a multi-layer sphere.

The concept of optical-sectioning techniques, on the other hand, is to detect only two-dimensional information about an object at each acquisition. Specifically, each image obtained by optical-sectioning techniques contains mainly the information about an optically thin layer inside the object, as if only a thin histological section is being observed at a time. Using such a methodology, obtaining undistorted volumetric information about the object simply requires taking images of the object at sequential depths.

Among existing methods of obtaining volumetric information, the practicability of optical sectioning has made it the most commonly used and most powerful one in biological science. However, when applied to imaging living biological systems, conventional single-point-scanning optical-sectioning techniques often result in certain degrees of photo-damages because of the high focal intensity at the scanning point. In order to overcome such an issue, several wide-field optical-sectioning techniques have been proposed and demonstrated, although not without introducing new limitations and compromises such as low signal-to-background ratios and reduced axial resolutions. As a result, single-point-scanning optical-sectioning techniques remain the most widely used instrumentations for volumetric imaging of living biological systems to date.

In order to develop wide-field optical-sectioning techniques that has equivalent optical performance as single-point-scanning ones, this thesis first introduces the mechanisms and limitations of existing wide-field optical-sectioning techniques, and then brings in our innovations that aim to overcome these limitations. We demonstrate, theoretically and experimentally, that our proposed wide-field optical-sectioning techniques can achieve diffraction-limited optical sectioning, low out-of-focus excitation and high-frame-rate imaging in living biological systems. In addition to such imaging capabilities, our proposed techniques can be instrumentally simple and economic, and are straightforward for implementation on conventional wide-field microscopes. These advantages together show the potential of our innovations to be widely used for high-speed, volumetric fluorescence imaging of living biological systems.