Browsing by Author "Tkaczyk, Tomasz S"

Now showing 1 - 7 of 7
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    Development of Fluorescence-based Optical Detection Techniques for Accessible and Efficient Point-of-Care Diagnostics
    (2019-04-12) Wong, Cynthia; Tkaczyk, Tomasz S
    Diagnostics performed at the point of care need to provide portable, rapid, inexpensive, and accurate results while overcoming challenges not typically seen in a central laboratory setting. Fluorescence microscopy gives a unique opportunity to address these needs. Since fluorescence looks for the presence of a target rather than morphological features in a sample, optical performance requirements may be reduced and medical device designs may be simplified. In this dissertation, the development of two classes of devices are presented. The first application targets white blood cell (WBC) differential counting, which can be used to determine bacterial or viral infections, evaluate allergic conditions, diagnose and monitor malignant diseases such as leukemia, and stage HIV infections. Two devices were developed to perform WBC differential counting: (1) a tunable fluorescence microscope using electrowetted lenses and (2) a fluorescence microscope using an ultraviolet (UV) LED as an excitation source. The electrowetted lenses incorporated in the tunable microscope were used to sharply focus on a specific wavelength at a time, simplifying the optical design as chromatic aberrations did not need to be corrected. The use of UV in the second system allowed for the removal of excitation, emission, and dichroic filters, as UV is absorbed by glass components, detectors are typically not sensitive to UV, and many fluorescent dyes are excitable by UV. Thus, this microscope could be fabricated out of commercial optics and use commercially available, low cost sample cartridges. The second application in the dissertation targets patient monitoring for tuberculosis (TB) treatment. Mycobacteria have an intrinsic molecule (thought to be used during metabolism) that is autofluorescent. This molecule had been previously shown in methanobacteria to have different photobleaching rates based on whether the molecule resided in live or dead organisms. Tracking of changes in autofluorescence decay may be used to augment current TB patient monitoring methods that cannot determine viability of organisms and may additionally reduce the time to determining viability from weeks to minutes.
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    Development of low-cost optical detection systems for point-of-care infectious disease detection and monitoring in low-resource settings
    (2016-04-22) Forcucci, Alessandra; Tkaczyk, Tomasz S
    Nearly half the world’s population lives below the global poverty line of $2.50 per day. This population is disproportionately affected by infectious diseases due to a lack of regular screenings and early detection and intervention. Optical diagnostics provide a highly sensitive and specific, low-cost method by which to improve access to disease screening and monitoring at the point of care in low-resource settings. Point of care settings range from a patient’s bedside to a hospital, and there is a need for instrumentation that may be used across the point of care setting spectrum. This work describes a platform of miniature fluorescence microscopes designed for use in resource-limited point of care settings ranging from rural communities to hospital laboratories. The prototype units of the platform described here are three miniature microscopes: 1) A monochromatic infinity-corrected 0.25 NA microscope that was used for optical readout of multiplexed bead-based bioassays. This prototype was validated with a commercially available assay reader (Luminex’s MAGPIX); 2) An achromatic 0.35 NA finite conjugate objective designed for three-part white blood cell differential counts in rural areas. This unit was validated against a commercially available benchtop hematology analyzer (Beckman Coulter Ac T diff2) and is currently in use in clinical trials at Lyndon B. Johnson General Hospital; and 3) A tunable 0.25 NA infinity-corrected fluorescence microscope designed for use with a variety multiplexed biological samples. The tunable system does not require manual adjustment by a trained user between emission wavelengths due to the addition of two low-cost, commercially available electrowetted lenses. It was validated by isolating green and red emission signal of blood stained with acridine orange. Each microscope prototype costs at least one order of magnitude less than its commercially available instrumentation counterpart. Additionally, the per-test cost for all devices is <$1.00 since the sample platform used for all three modules is a standard microscope slide with low cost reagents, not a specialized cartridge or cuvette. With the platform of low-cost, high-performance microscopes described in this work, it is possible for diagnostic tests to be performed without expensive equipment or highly-trained clinicians, thereby delivering testing access to low-resources settings.
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    Development of Recombinase Polymerase Amplification (RPA) Assays to Diagnose Infectious Diseases
    (2015-06-24) Crannell, Zachary; Richards-Kortum, Rebecca Rae; Bennett, George; Tkaczyk, Tomasz S; White, Arthur C
    This thesis describes the development of Recombinase Polymerase Amplification (RPA) assays that can be used to improve access to diarrheal diagnostics and thereby reduce the number of preventable deaths that occur each year due to persistent diarrhea. In low-resource settings (LRS), where the majority of the almost 1.5 million annual diarrheal deaths occur, a major obstacle to receiving life-saving treatment is the inability to identify the specific cause of diarrhea. Diagnosis in LRS is usually done via stool smear microscopy, which fails to identify the cause of diarrhea up to half of the time. The widely considered gold standard diagnostic method is Polymerase Chain Reaction (PCR), which detects trace amounts of pathogen DNA from stool samples. While highly sensitive, PCR requires highly trained technicians and access to expensive thermal cycling equipment, restricting its use to centralized reference laboratories. The RPA diagnostics presented here amplifies trace amounts of pathogen DNA (much like PCR), but unlike PCR do not require the use of expensive thermal cycling equipment and can function at low temperatures, alleviating the need for any external heating equipment. RPA-based diagnostics and sample preparation protocols that are appropriate for low resource settings were developed to detect Cryptosporidium, Giardia, and Entamoeba, three of the leading causes of diarrhea. The three diagnostic assays were individually characterized on the benchtop where they demonstrated limits-of-detection and specificities comparable to the gold standard of PCR. The assays were further characterized in field studies using clinical samples where they demonstrated sensitivity and specificity nearly equivalent to that of the gold standard PCR. The three individual assays were then integrated into a multiplexed test designed to simultaneously amplify and detect DNA from Cryptosporidium, Giardia, and Entamoeba. This test was also characterized on the benchtop and in pre-clinical studies. All of the assays presented here are read using lateral flow strips that can easily be used in the field. This work demonstrates for the first time that multiplex RPA results can be read with lateral flow strips. By modifying the DNA primers, this diagnostic platform could be adapted to diagnose a broad variety of infectious diseases.
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    Light-guide snapshot imaging spectrometer for remote sensing and environmental imaging applications
    (2019-04-12) Wang, Ye; Tkaczyk, Tomasz S
    Optical fibers provide a high degree of design freedom and enable the building of imaging spectrometers with optimized compactness. Moreover, the ability to arbitrarily reformat the fibers’ input/output configurations allows the tuning between spatial and spectral sampling to meet specific application requirements. However, currently the system’s spatial sampling is limited by the basic structure design of the fiber bundle, which confined its application in various scenarios. In this thesis, I aim to advance the current fiber-based snapshot imaging spectrometers by developing a compact system with >30000 spatial samplings. Specific aims include the system design, the fabrication of a compact light-guiding fiber bundle by both semi-manual assembling and fully automatic 3D printing, the development of a rapid calibration method, and the system performance assessment for environmental applications. As a result, a fiber-based snapshot imaging spectrometer was developed with a maximum of 31853 (~ 188 x 170) spatial sampling and 61 spectral channels in the 450nm-750nm range. A compact fiber bundle was fabricated with semi-manual assembling to sample the object image at the input and create void spaces between rows at the output for dispersion. 3D printing using custom controlled fused deposition modeling (FDM) is also explored as an alternative fabrication technique, with an air-clad fiber optic faceplate fabricated as a proof of concept. To calibrate the >30000 spatial samples of the system, a rapid spatial calibration method was developed based on Phase-Shifting Interferometry (PSI). Preliminary hyperspectral imaging results of the Rice University campus landscape is presented to demonstrate the system’s spectral imaging capability for distant scenes. The spectrum of different plant species with different health conditions were in accordance with reference instrument measurements. The Houston city traffic was also imaged to demonstrate the system’s snapshot hyperspectral imaging capability on dynamic scenes. Potential applications of the system include terrestrial monitoring land use, air pollution, water resources, and lightning spectroscopy and so on.
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    Multimodal Foveated Endomicroscope for the Early Detection of Oral and Esophageal Cancer
    (2016-04-11) Shadfan, Adam Harbi; Tkaczyk, Tomasz S
    Digestive tract cancers will be responsible for nearly 30% of all cancer deaths in the United States in 2016. Oral and esophageal cancers alone will result in over 25,000 deaths, with an estimated 65,000 new cases. Most of these deaths can be attributed to the late detection of cancers, when treatment options become more limited. This late detection is often due to the limitations of current standard screening procedures, which often struggle with rapid and reliable recognition of precancerous warning signs. Optical imaging methods have the potential to become powerful, non-invasive early diagnostic tools. However, most systems are often limited by several factors including insufficient optical resolution, limited field of view, or a lack diagnostically relevant data, leading to devices with either low specificity or low sensitivity. This work presents the design and development of several technologies with the goal of creating a multimodal endomicroscope that overcomes the limits of current diagnostic techniques for the early detection of oral and esophageal cancer with high sensitivity and specificity. The first stage in the development of the endomicroscope is the design, fabrication and validation of a miniature foveated objective, which provides both widefield and high resolution imaging in a compact form. The objective accomplishes this task by introducing distortion into the optical system in order to nominally mimic the variable resolution regions in the fovea of the human eye. Two image relay techniques were developed to integrate the objective with (1) a snapshot image spectrometer with the ability to capture spatial and spectral data simultaneously in order to rapidly locate suspicious areas of interest and (2) a custom confocal microscope capable of high resolution imaging to observe morphological changes in the tissue. The performance of the integrated device was evaluated through the imaging of mouse and human cancer samples.
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    Snapshot Hyperspectral Imaging for Complete Fundus Oximetry
    (2017-04-21) Dwight, Jason G; Tkaczyk, Tomasz S
    In this work, a snapshot hyperspectral imager capable of tuning its average spectral resolution from 22.7 nm to 13.9 nm in a single integrated form is presented. The principle of this system will enable future snapshot systems to dynamically adapt to a wide range of imaging situations. Additionally, the system overcomes datacube size limitations imposed by detector array size limits. The work done in this thesis also advances oximetry of the retina using data collected by the Image Mapping spectrometer (IMS), a snapshot spectrometer. Hyperspectral images of the retina are acquired, and oximetry of individual vessels in four diseased eyes is presented. Further, oximetry of the entire fundus is performed using a novel algorithm with data collected with the IMS. We present oxyhemoglobin concentration maps of the eye and demonstrate oxygen sensitivity of the maps by comparing normal and diseased eyes. The aim of this work is to advance the general capabilities of snapshot hyperspectral imagers and to advance the integration of retinal oximetry into the standard ophthalmology instrument repertoire.
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    Structured Illumination in a Fiber-Optic Microendoscope to Image Nuclear Morphometry in Columnar Epithelium
    (2015-03-23) Keahey, Pelham; Richards-Kortum, Rebecca Rae; Tkaczyk, Tomasz S; Natelson, Douglas; Drezek, Rebekah A
    Fiber-optic microendoscopes have shown promise to image the changes in nuclear morphometry that accompany the development of precancerous lesions in tissue with squamous epithelium such as in the oral mucosa and cervix. However, fiber-optic microendoscopy image contrast is limited by out-of-focus light generated by scattering within tissue. The scattering coefficient of tissues with columnar epithelium can be greater than that of squamous epithelium resulting in decreased image quality. To address this challenge, I present a small and portable microendoscope system capable of performing optical sectioning using structured illumination (SI) in real-time. Several optical phantoms were developed and used to quantify the sectioning capabilities of the system. Columnar epithelium from cervical tissue specimens was then imaged ex vivo, and I demonstrate that the addition of SI achieves higher image contrast, enabling visualization of nuclear morphology.