R-3 Repository :: Browsing by Author "Richards-Kortum, Rebecca Rae"

Browsing by Author "Richards-Kortum, Rebecca Rae"

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Array microscopy technology and its application to digital detection of Mycobacterium tuberculosis
(2013-09-16) McCall, Brian; Tkaczyk, Tomasz S.; Richards-Kortum, Rebecca Rae; Graviss, Edward A.; Baraniuk, Richard G.
Tuberculosis causes more deaths worldwide than any other curable infectious disease. This is the case despite tuberculosis appearing to be on the verge of eradication midway through the last century. Efforts at reversing the spread of tuberculosis have intensified since the early 1990s. Since then, microscopy has been the primary frontline diagnostic. In this dissertation, advances in clinical microscopy towards array microscopy for digital detection of Mycobacterium tuberculosis are presented. Digital array microscopy separates the tasks of microscope operation and pathogen detection and will reduce the specialization needed in order to operate the microscope. Distributing the work and reducing specialization will allow this technology to be deployed at the point of care, taking the front-line diagnostic for tuberculosis from the microscopy center to the community health center. By improving access to microscopy centers, hundreds of thousands of lives can be saved. For this dissertation, a lens was designed that can be manufactured as 4×6 array of microscopes. This lens design is diffraction limited, having less than 0.071 waves of aberration (root mean square) over the entire field of view. A total area imaged onto a full-frame digital image sensor is expected to be 3.94 mm2, which according to tuberculosis microscopy guidelines is more than sufficient for a sensitive diagnosis. The design is tolerant to single point diamond turning manufacturing errors, as found by tolerance analysis and by fabricating a prototype. Diamond micro-milling, a fabrication technique for lens array molds, was applied to plastic plano-concave and plano-convex lens arrays, and found to produce high quality optical surfaces. The micro-milling technique did not prove robust enough to produce bi-convex and meniscus lens arrays in a variety of lens shapes, however, and it required lengthy fabrication times. In order to rapidly prototype new lenses, a new diamond machining technique was developed called 4-axis single point diamond machining. This technique is 2-10x faster than micro-milling, depending on how advanced the micro-milling equipment is. With array microscope fabrication still in development, a single prototype of the lens designed for an array microscope was fabricated using single point diamond turning. The prototype microscope objective was validated in a pre-clinical trial. The prototype was compared with a standard clinical microscope objective in diagnostic tests. High concordance, a Fleiss’s kappa of 0.88, was found between diagnoses made using the prototype and standard microscope objectives and a reference test. With the lens designed and validated and an advanced fabrication process developed, array microscopy technology is advanced to the point where it is feasible to rapidly prototype an array microscope for detection of tuberculosis and translate array microscope from an innovative concept to a device that can save lives.
Assessment of In Vivo Microscopy for Malaria Detection
(2014-10-23) Burnett, Jennifer; Richards-Kortum, Rebecca Rae; Drezek, Rebekah; Tkaczyk, Tomasz
Half of the world’s population is at risk for malaria, yet 90% of deaths due to malaria occur in sub-Saharan Africa, with the highest mortality rates occurring in children under 5. Due to this high mortality rate, children with common flu-like symptoms are often treated for malaria presumptively. This overtreatment with anti-malarial drugs can contribute to the emergence of drug resistant species. Commercially available diagnostic tools, such as blood smear microscopy and rapid diagnostic tests, offer greater specificity than presumptive diagnosis, but require the collection of a finger prick blood sample for diagnosis, generating biohazards and requiring consumables. This thesis introduces a new diagnostic concept to detect parasitized blood cells as they circulate in vivo, avoiding the requirement for blood collection. The work presented here investigates two major components of this concept: 1) develop and characterize a needle-free malaria diagnostic system, and 2) evaluate the performance of the system in biological environments of progressively increasing complexity. Results show promising optical signatures from two biomarkers for malaria detection. This work demonstrates the feasibility for imaging circulating blood cells in vivo through a non-invasive technique, warranting future investigations in a small malaria-infected sample population.
Depth-sensitive optical spectroscopy for noninvasive diagnosis of oral neoplasia
(2010) Schwarz, Richard Alan; Richards-Kortum, Rebecca Rae
Oral cancer is the 11th most common cancer in the world. Cancers of the oral cavity and oropharynx account for more than 7,500 deaths each year in the United States alone. Major advances have been made in the management of oral cancer through the combined use of surgery, radiotherapy and chemotherapy, improving the quality of life for many patients; however, these advances have not led to a significant increase in survival rates, primarily because diagnosis often occurs at a late stage when treatment is more difficult and less successful. Accurate, objective, noninvasive methods for early diagnosis of oral neoplasia are needed. Here a method is presented to noninvasively evaluate oral lesions using depth-sensitive optical spectroscopy (DSOS). A ball lens coupled fiber-optic probe was developed to enable preferential targeting of different depth regions in the oral mucosa. Clinical studies of the diagnostic performance of DSOS in 157 subjects were carried out in collaboration with the University of Texas M. D. Anderson Cancer Center. An overall sensitivity of 90% and specificity of 89% were obtained for nonkeratinized oral tissue relative to histopathology. Based on these results a compact, portable version of the clinical DSOS device with real-time automated diagnostic capability was developed. The portable device was tested in 47 subjects and a sensitivity of 82% and specificity of 83% were obtained for nonkeratinized oral tissue. The diagnostic potential of multimodal platforms incorporating DSOS was explored through two pilot studies. A pilot study of DSOS in combination with widefield imaging was carried out in 29 oral cancer patients, resulting in a combined sensitivity of 94% and specificity of 69%. Widefield imaging and spectroscopy performed slightly better in combination than each method performed independently. A pilot study of DSOS in combination with the optical contrast agents 2-NBDG, EGF-Alexa 647, and proflavine was carried out in resected tissue specimens from 15 oral cancer patients. Improved contrast between neoplastic and healthy tissue was observed using 2-NBDG and EGF-Alexa 647.
Development and Evaluation of Approaches for Quantitative Optical Molecular Imaging of Neoplasia
(2011) Rosbach, Kelsey Jane; Richards-Kortum, Rebecca Rae
This thesis develops and evaluates three approaches for quantitative molecularly-targeted optical imaging of neoplasia. The first approach focuses on widefield imaging of biomarkers near the tissue surface for early detection applications; this approach is demonstrated in freshly resected oral tissue. Most oral cancers are not detected until the disease has spread, but topical application of targeted imaging agents allows rapid visualization of biomarker expression, giving real-time, objective information. Epidermal growth factor receptor (EGFR) expression was quantified in patient samples using fluorescent epidermal growth factor. Dysplasia (n=4) and cancer (n=13) had an average 2.3-fold and 3.8-fold increase in signal compared to normal tissue. EGFR expression was assessed along with metabolic activity using a fluorescent glucose analog, 2-NBDG, in 9 patient samples. A classification algorithm using quantitative image features resulted in an area under the curve (AUC) of 0.83, though the main advantage of this technique may be to understand spatial heterogeneity of biomarker expression and how this correlates with disease. The next approach focuses on high-resolution optical imaging through a needle to detect metastases in lymphoid tissue for clinical staging applications; this approach is demonstrated in resected lymph nodes from breast cancer patients. These patients often require removal of nodes, but an optical imaging strategy using topical application of imaging agents in vivo may classify nodes as normal or metastatic, thus reducing unnecessary removal of normal nodes and improving metastasis detection. Proflavine, a nuclear dye, was topically applied to 43 nodes. A classification algorithm developed from quantitative image features distinguished normal lymphoid tissue from metastases with an AUC of 0.84. Because optical imaging is depth limited, the final approach combines high-resolution optical imaging with magnetic resonance imaging (MRI) for multimodal evaluation of deep tissue. An imaging agent functional in both optical and MRI was developed by co-loading fluorescent EGFR antibodies and gadolinium-based contrast agents in silicon discs. These discs accumulate in tumors, resulting in localized delivery of imaging agents. The research presented here can be applied to understanding tumor biology and biomarker heterogeneity, with the future clinical goal of improving identification of disease and determination of appropriate therapy for cancer patients.
Development of Hyperspectral Imagers for Snapshot Optical Coherence Tomography
(2014-04-25) Nguyen, Thuc-Uyen; Tkaczyk, Tomasz S.; Richards-Kortum, Rebecca Rae; Baraniuk, Richard G.; Pierce, Mark C
Optical Coherence Tomography (OCT) is an established interferometry-based technique for volumetric tissue imaging with micrometer resolution, best known in many medical applications such as ophthalmologic imaging and endoscopy. Several clinically recognized examples include retinal imaging to detect glaucoma and age-related macular degeneration (AMD) or cardiovascular imaging when employed with a catheter. Scanning mechanism presenting in all current OCT technology requires moving parts, often limiting the system’s compactness, compromising light throughput and risking unwanted movement. Snapshot imaging thus allows fast and high-throughput acquisition while minimizing motion artifacts caused by instrumental vibration or samples’ transient nature. This thesis presents novel work contributing to the development of a snapshot 3-Dimensional OCT (3D-OCT) system. With theoretical and experimental evaluations, different hyperspectral imaging designs were surveyed to provide enhancements such as high throughput, dense spectral sampling, high sensitivity toward the appropriate spectrum and spatial-spectral tunability. A proof-of-concept snapshot 3D-OCT system is introduced to simultaneously collect signals of a volumetric datacube, enabling cellular visualization of scattering biological samples. This system affords diffraction-limited performance with reduced motion and requires minimal computational time.
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.
High resolution microendoscopy for quantitative diagnosis of esophageal neoplasia
(2013-09-16) Shin, Dong Suk; Richards-Kortum, Rebecca Rae; Tkaczyk, Tomasz S.; Baraniuk, Richard G.; Anandasabapathy, Sharmila
Esophageal cancer is the eighth most common cancer in the world. Cancers of the esophagus account for 3.8% of all cases of cancers, with approximately 482,300 new cases reported in 2008 worldwide. In the United States alone, it is estimated that approximately 18,000 new cases will be diagnosed in 2013, and 15,210 deaths are expected. Despite advances in surgery and chemoradiation therapy, these advances have not led to a significant increase in survival rates, primarily because diagnosis often at an advanced and incurable stage when treatment is more difficult and less successful. Accurate, objective methods for early detection of esophageal neoplasia are needed. Here, quantitative classification algorithms for high resolution miscroendoscopic images were developed to distinguish between esophageal neoplastic and non-neoplastic tissue. A clinical study in 177 patients with esophageal squamous cell carcinoma (ESCC) was performed to evaluate the diagnostic performance of the classification algorithm in collaboration with the Mount Sinai Medical Center in the United States, the First Hospital of Jilin University in China, and the Cancer Institute and Hospital, the Chinese Academy of Medical Science in China. The study reported a sensitivity and specificity of 93% and 92%, respectively, in the training set, 87% and 97%, respectively, in the test set, and 84% and 95%, respectively, in an independent validation set. Another clinical study in 31 patients with Barrett’s esophagus resulted in a sensitivity of 84% and a specificity of 85%. Finally, a compact, portable version of the high resolution microendoscopy (HRME) device using a consumer-grade camera was developed and a series of biomedical experimental studies were carried out to assess the capability of the device.
High-resolution imaging for cancer detection with a fiber bundle microendoscope
(2009) Muldoon, Timothy J.; Richards-Kortum, Rebecca Rae
Dysplasia and cancer of epithelial tissues, including the oral cavity and esophagus, typically have much higher survival rates if diagnosed at an early stage. Unfortunately, the clinical appearance of lesions in these tissues can be highly variable. To achieve a definitive diagnosis of a suspected lesion at these sites, an excisional biopsy must be examined at high-resolution. These procedures can be costly and timeconsuming, and in the case of Barrett's esophagus, surveillance biopsy strategies may not be entirely effective. Optical imaging modalities have the potential to yield qualitative and quantitative high-resolution data at low cost, enabling clinicians to improve early detection rate. This dissertation presents a low-cost high-resolution microendoscopy system based on a fiber optic bundle image guide. In combination with a topical fluorescent dye, the fiber bundle can be placed into contact with the tissue to be observed. A high-resolution image is then projected onto a CCD camera and stored on a PC. A pilot study was performed on both resected esophageal tissue containing intestinal metaplasia (a condition known as Barrett's esophagus, which can transform to esophageal adenocarcinoma) and resected oral tissue following surgical removal of cancer. Qualitative image analysis demonstrated similar features were visible in both microendoscope images and standard histology images, and quantitative image processing and analysis yielded an objective classification algorithm. The classification algorithm was developed to discriminate between neoplastic and non-neoplastic imaging sites. The performance of this algorithm was monitored by comparing the predicted results to the pathology diagnosis at each measurement site. In the oral cancer pilot study, the classifier achieved 85% sensitivity and 78% specificity with 141 independent measurement sites. In the Barrett's metaplasia pilot study, 87% sensitivity and 85% specificity were achieved with 128 independent measurement sites. The work presented in this dissertation outlines the design, testing, and initial validation of the high-resolution microendoscope system. This microendoscope system has demonstrated potential utility over a wide range of modalities, including small animal imaging, molecular-specific imaging, ex vivo and ultimately in vivo imaging.
Improvement of image reconstruction speed with GPU in cone beam CT breast imaging
(2010) Ge, Shuaiping; Richards-Kortum, Rebecca Rae
Breast cancer is the most common and the second lethal cancer among women in the United States. Our group is constructing a dedicated cone beam breast CT (CBCT) system to provide true 3D image to improve the screening and diagnostic of breast cancer. Our result shows that dedicated CBCT out-perform a lot than conventional CT when detecting micro-calcification which is essential to the detection of early stage breast cancer. I also explored the possibility of using the super parallel computing power of GPU with CUDA environment to deal with data-immense and computationally-intensive image reconstruction process of CBCT. My results show that FDK algorithm image reconstruction with GPU is over 10 times faster than that with our PC cluster system. The faster and accurate image reconstruction implies potential new applications in diagnostic and therapy technology.
Isothermal Nucleic Acid Assays Based on Nucleic Acid Sequence Based Amplification (NASBA) and Recombinase Polymerase Amplification (RPA) for HIV-1 Diagnosis and Management in Low Resource Settings
(2015-04-01) Rohrman, Brittany Ann; Richards-Kortum, Rebecca Rae; Biswal, Sibani L; McDevitt, John
Over two-thirds of the estimated 35 million people worldwide infected with HIV live in the developing world. Nucleic acid tests (NATs) are necessary for early infant diagnosis and for monitoring patients receiving therapy. However, NATs cost $50-100 USD per test and require expensive thermal cycling equipment that may be unavailable in the developing world. This thesis presents two low-cost NATs for HIV-1 diagnosis and management that are based on isothermal amplification, which eliminates the need for expensive thermal cycling equipment. In one assay, HIV-1 viral RNA is detected using nucleic acid sequence based amplification (NASBA) and a custom lateral flow test. This assay costs about \$16 USD and only requires a heat block. When coupled with NASBA, the lateral flow test detected concentrations of synthetic RNA spanning the entire clinical range. When the assay was evaluated using pediatric plasma samples, the sensitivity (61%) and limit-of-detection (10,000 HIV-1 copies/mL plasma) were lower because of the genetic diversity of the samples, and the specificity was lower (88%) due to amplicon contamination. In the other assay, HIV-1 proviral DNA is amplified using recombinase polymerase amplification (RPA). This assay, which costs about \$5 per test, was integrated into a paper and plastic microfluidic device. The device was capable of amplifying 10 copies of plasmid HIV-1 DNA to detectable levels in 15 minutes. The assay was then adapted for real-time quantification. On average, the assay predicted sample concentrations within one order of magnitude of the correct concentration. In addition, a method for incubating RPA reactions without external equipment was developed. Using human body heat for incubation, all RPA reactions with 10 copies of plasmid HIV-1 DNA and 95% of reactions with 100 copies of plasmid HIV-1 DNA tested positive. Finally, concentrations of background DNA found in whole blood were shown to prevent the amplification of target DNA by RPA. To address this problem, three sequence-specific capture methods were developed to enrich target DNA concentration relative to background DNA concentration. These methods may be enable detection of high proviral loads in 0.1 mL infant blood samples but require improvement to detect lower proviral loads.
Low cost optical imaging systems for early detection of oral cancer
(2009) Rahman, Mohammed Saidur; Richards-Kortum, Rebecca Rae
Optical imaging has the potential to improve early detection of oral cancer. Reflectance and fluorescence based optical devices have demonstrated improved sensitivity and specificity compared to conventional visual oral examination. Although these devices are increasingly used as clinical tools in developed countries, they are a less practical solution in low-resource settings as the cost of these devices is relatively high, their portability is limited, and results from them are often subjective. This dissertation focuses on development of optical imaging platforms that specifically addresses these challenges and can be used to aid in screening and detection of oral pre-cancers. The first part of this dissertation describes the construction and evaluation of a macroscopic imaging system with multi-modal imaging capability. This system can be used to screen the surface area of oral tissue at risk to detect abnormal sites. It is low-cost, portable and battery powered, which is ideal for screening and detection of oral cancer in high-risk populations in low-resource and remote settings. The system was evaluated in a clinical study in India and results from the study were used to develop a computational algorithm for objective interpretation of images from the system. In addition, this system was used to characterize optical properties of pathological conditions that are population specific. Results from this trial system are promising and show that normal oral sites can be differentiated from high-risk and cancerous sites with high sensitivity and specificity. Although results show that sites with low-risk can be differentiated from normal tissue using this system, the sample size of the low-risk measurements is relatively small and they can not be differentiated from high-risk and cancerous tissue. The second part of this dissertation involves developing a low-cost and simple microscopic system that is capable of high-resolution imaging for early detection of cancer. It was demonstrated that the 'optical-sectioning' concept of structured illumination can be integrated with optically active exogenous contrast agents for high-resolution molecular-specific imaging of pre-cancer. Finally, this dissertation also incorporates evaluation of a multi-modal miniature microscope (4M) device developed based on structured illumination. Results from the system show that the device is capable of high-resolution imaging and can be used with molecular-specific contrast agents for detection of cancer and its precursors.
Multi-Modal Imaging Techniques for Early Cancer Diagnostics
(2012-09-05) Bedard, Noah; Tkaczyk, Tomasz S.; Richards-Kortum, Rebecca Rae; Merenyi, Erzsebet; Gillenwater, Ann M.
Cancer kills more Americans under the age of 75 than any other disease. Although most cancers occur in epithelial surfaces that can be directly visualized, the majority of cases are detected at an advanced stage. Optical imaging and spectroscopy may provide a solution to the need for non-invasive and effective early detection tools. These technologies are capable of examining tissue over a wide range of spatial scales, with widefield macroscopic imaging typically spanning several square-centimeters, and high resolution in vivo microscopy techniques enabling cellular and subcellular features to be visualized. This work presents novel technologies in two important areas of optical imaging: high resolution imaging and widefield imaging. For subcellular imaging applications, new high resolution endomicroscope techniques are presented with improved lateral resolution, larger field-of-view, increased contrast, decreased background signal, and reduced cost compared to existing devices. A new widefield optical technology called multi-modal spectral imaging is also developed. This technique provides real-time in vivo spectral data over a large field-of-view, which is useful for detecting biochemical alterations associated with neoplasia. The described devices are compared to existing technologies, tested using ex vivo tissue specimens, and evaluated for diagnostic potential in a multi-patient oral cancer clinical trial.
Multimodal Optical Imaging for Detection of Cervical Neoplasia
(2013-09-16) Bubi, Tefo; Richards-Kortum, Rebecca Rae; Drezek, Rebekah A.; Baraniuk, Richard G.
Despite being the most preventable cancer, cervical cancer remains the third leading cause of cancer death worldwide. Over 85% of cervical cancer incidence and mortality occurs in low-resource countries where screening programs for early detection are either inadequate or unavailable. In the developed world, where screening programs are well organized, incidence and mortality rates are greatly reduced. Recent advances in optical imaging have the potential to enable cervical cancer screening at the point-of-care, even in the hands of less experienced providers. High performance optical imaging systems can be constructed at relatively low cost, and image analysis can be automated; thus, these technologies may provide a way to bridge the gap to cervical cancer screening for developing countries. This work focuses on the design, construction, and clinical testing of a novel multimodal optical imaging (combination of wide-field imaging and high-resolution) for early detection of cervical neoplasia. The Multimodal Digital Imager (MDI) acquires in vivo images of cervical tissue in fluorescence, narrow band reflectance, and orthogonal polarized reflectance modes using multiple illumination wavelengths. The High Resolution Microendoscope (HRME) was used to interrogate clinically suspicious areas with subcellular spatial resolution, revealing changes in nuclear to cytoplasmic area ratio. In vivo image data from the wide-field system was combined with image data from a high- resolution microendoscope (HRME) in order to test the effectiveness of the multimodal optical imaging in discriminating between cervical neoplasia and non-neoplastic. Multimodal optical imaging coupled with computer aided diagnostic achieved a sensitivity of 82% and specificity of 85% for discriminating cervical neoplastic from non-neoplastic This work has demonstrated that multimodal optical imaging; combination of wide-field and high-resolution optical imaging of the cervix can assist in the detection of cervical neoplasia and can be implemented effectively in a low-resource setting.
Multispectral optical imaging for the detection and delineation of oral neoplasia
(2009) Roblyer, Darren Michael; Richards-Kortum, Rebecca Rae
Despite the accessibility of the oral cavity to inspection, patients with oral cancer most often present at a late stage, leading to high morbidity and mortality. Multispectral widefield optical imaging has emerged as a promising technology to aid clinicians in screening and resection of oral neoplasia, but current approaches rely on subjective interpretation. This work focuses on the design, construction, and clinical testing of a novel multispectral widefield optical imaging device for objective screening and delineation of oral neoplasia. The Multispectral Digital Microscope (MDM) acquires in vivo images of oral tissue in autofluorescence, narrow band reflectance, and orthogonal polarized reflectance modes that the diagnostic value of each modality may be qualitatively and quantitatively evaluated alone and in combination. Using in vivo imaging data collected from 56 patients and 11 normal volunteers, combined with computer aided diagnostics, a sensitivity of 100% and a specificity of 91.4% was achieved for discriminating oral dysplasia and cancer from normal tissue in an independent validation set. A single feature calculated from the autofluorescence images at 405 nm excitation was used to achieve this performance. Disease probability maps were constructed using this feature to help identify areas with a high probability of abnormality. Autofluorescence imaging at 405 nm excitation also provided the greatest image contrast which was significantly higher than that using standard white-light illumination. Features extracted from other imaging types did not appear to aid in diagnosis. Ex vivo image data from the MDM was combined with image data from a high-resolution microendoscope (HRME) in order to determine if a synergistic relationship existed between these devices. The ability to objectively diagnose oral lesions substantially increased when using both devices in combination compared to using either alone. This combination of devices provides a practical means of screening the entire mucosal surface for suspicious regions, using the MDM, and then using the HRME for confirmation of diagnosis. This work has demonstrated that widefield autofluorescence imaging at 405 nm excitation can be highly effective for the objective discrimination of oral lesions.
Nanosystems: From their design to characterization as advanced MRI contrast agents
(2013-07-29) Sethi, Richa; Wilson, Lon J.; McDevitt, John T.; Richards-Kortum, Rebecca Rae; Decuzzi, Paolo
Ultra-short single-walled carbon nanotubes (US-tubes) have been previously shown to be efficient carriers of imaging agents. In particular, gadonanotubes (GNTs) synthesized by loading and nanoscale confinement of Gd3+ ions within US-tubes have been established as high-performance MRI contrast agents (CAs) with efficiencies 40 to 90 times greater than the current clinical CAs. Using nuclear magnetic resonance dispersion (NMRD) and electron spin resonance (ESR) techniques, this work discusses the origin of the magnetic and proton relaxation behavior in MRI of the GNTs and related structures at low magnetic fields. The likely causes for the observed paramagnetism for these materials are explored and their effect on water proton relaxation is discussed. In addition, Gd3+ chelates, which are currently approved for clinical MRI use, provide relaxivities (or contrast enhancement) well below their theoretical limit, and they also lack tissue speciﬁcity. In this dissertation, using vascularly injectable mesoporous silicon nanoparticles (SiMPs), general methods for increasing the efficiency of Gd3+-based MRI CAs are described. Two different strategies have been successfully tested where Gd3+ chelates are either geometrically confined within the pores of SiMPs or covalently attached to the surface of SiMPs. For both the approaches, SiMPs with different pore sizes have been used to generate a dominant role in the resulting relaxivity. The nanoconstructs designed using these approaches have been shown to produce relaxivities that are many-fold greater than the free CAs in solution. This enhancement is attributed to the optimization of the molecular parameters that govern relaxivity. Co-loading the pores with a Gd3+-based CA and a fluorescently-labeled antibody has shown the potential of SiMP nanoconstructs as multimodal agents. The strategies outlined in this dissertation are general and can be successfully applied to any imaging agent and porous nanosystem. In summary, this work highlights two key outcomes. First, it provides a better understanding of the magnetic and MRI behavior of the GNTs. Second, it demonstrates that geometrical confinement of CAs and covalent functionalization of nanoparticles are universal strategies for enhancing the performance of Gd3+-based CAs.
Optical biopsy systems using ultra-slim objectives for the diagnosis of breast cancer
(2013-09-16) Kyrish, Matthew; Tkaczyk, Tomasz S.; Richards-Kortum, Rebecca Rae; Dick, Andrew J.; Huang, Huey W.
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 remo
Optical Contrast Agents to Distinguish Benign Inflammation from Neoplasia in Epithelial Tissues
(2016-01-29) Hellebust, Anne E; Richards-Kortum, Rebecca Rae; Farach-Carson, Mary; Gillenwater, Ann; Sikora, Andrew; Tkaczyk, Tomasz
A minimally-invasive, optical strategy to detect and discriminate between inflammation and neoplasia could improve early cancer detection by reducing the number of false positive exams due to benign inflammation. This thesis describes research to optimize optical molecular contrast agents to observe architectural, metabolic, and biomolecular changes from inflammation and cancer in the gastrointestinal tract. My goal was to: 1) understand the limitations of autofluorescence imaging for cancer detection, 2) image exogenous fluorescent contrast agents specific to inflammation and neoplasia in rodent models, and 3) topically deliver a contrast agent cocktail in vivo in a mouse model. Wide field autofluorescence imaging of oral tissue utilizes endogenous tissue contrast to discriminate neoplastic from normal tissue; clinical studies of this technique show good sensitivity but poor specificity. I conducted a confocal microscopy study of 47 biopsies from 20 patients; results showed a similar decrease in autofluorescence in the stroma of inflamed and neoplastic tissue. This finding helps explain the low specificity of wide field autofluorescence imaging. Topically applied exogenous contrast agents could be used to improve discrimination between neoplasia and inflammation. I tested individual fluorescent contrast agents and contrast agent cocktails in chemically induced rodent models of inflammation and neoplasia. The first model used autofluorescence imaging with fluorescence imaging of proflavine to highlight cell nuclei and 2-NBDG to assess metabolic activity for oral cancer detection. A classification algorithm based on proflavine and 2-NBDG staining separated neoplastic from non-neoplastic areas on the tongue with 91% sensitivity and specificity. In the second model, a contrast agent cocktail composed of proflavine, a fluorescently labelled CD45-targeted antibody to identify inflammatory cells, and permeation enhancers was evaluated for topical in vivo delivery to image ulcerative colitis. The antibody identified the presence of inflammation and established topical delivery of antibody sized agents in vivo. These results provide evidence that topically applied contrast agent cocktails could improve discrimination between inflammation and neoplasia when endogenous contrast is insufficient. An optical-based strategy utilizing contrast agent cocktails to observe architectural, metabolic, and biomolecular changes associated with inflammation and cancer could improve early cancer detection by reducing the number of false positives from inflammation.
Optical Imaging Techniques for the Detection of Esophageal Neoplasia in Barrett’s Esophagus
(2013-09-16) Thekkek, Nadhi; Richards-Kortum, Rebecca Rae; Baraniuk, Richard G.; Anandasabapathy, Sharmila; McDevitt, John T.
The main objective of this research was to develop a two-stage optical imaging platform to improve detection of cancer in Barrett’s esophagus (BE). BE caused by chronic reflux and patients with BE are at a higher risk for developing esophageal adenocarcinoma (EAC). However, neoplasia in BE is often unidentifiable under standard endoscopy, and studies have shown nearly half of early cancers can go unidentified by this method. Widefield imaging (resolves ~100 microns) allows efficient surveillance of large BE segments. Two widefield imaging techniques were identified to improve contrast between benign and abnormal lesions during an ex vivo 15 patient feasibility study. Cross-polarized imaging (CPI) reduced specular reflection and improved vascular contrast. Vital-dye fluorescence imaging (VFI) using topically-applied proflavine improved visualization of glandular pattern. Moreover, relevant pathologic features visible during VFI were seen in corresponding histology slides as well as high resolution images of the same sites. Based on these results, a cap-based Multispectral Digital Endoscope (MDE) was designed and built. The MDE can image in three different imaging modes: white light imaging, CPI, and VFI. Modifications to a Pentax EPK-i video processor and a Pentax endoscope were made to incorporate these imaging modes into one system. A 21 patient in vivo pilot study with 65 pathologically correlated sites demonstrated the feasibility of using this system in vivo; image criteria were developed to classify neoplasia with a sensitivity and specificity of 100% and 76% respectively. High resolution imaging (resolves ~2-5 micron) may verify the disease presence in suspicious areas identified using widefield techniques. 2-NBDG, a fluorescent metabolic marker, was used as to identify neoplastic biopsies. In a study with 21 patients yielding 38 pathologically correlated biopsies and 158 image sites, 2-NBDG imaging allowed classification of cancerous biopsies with a sensitivity of 96% and specificity of 90%. The unique contributions of these results is the development of a multimodal cap-based endoscopic system to identify suspicious areas in BE, and using a metabolic marker to verify the presence of disease. This application extends beyond esophageal cancer detection and may be explored for cancer detection in other organ sites characterized by columnar epithelium.
Optimization of Microfluidic, Point of Care, Flow-Through, Bead-Based Microarrays: Towards Affordable Healthcare
(2012-10-12) Chou, Jie; McDevitt, John T.; Richards-Kortum, Rebecca Rae; Biswal, Sibani Lisa
Recently, there has been much interest on the development of affordable, portable diagnostic devices for the detection of a wide range of analytes. Advancements in microfluidics and miniaturization bring promise for their use at the point of care over traditional, and for the most part laboratory-confined approaches. The integration of porous beads with microfluidics has demonstrated potential as highly sensitive sensing elements with the capability to detect multiple biological and chemical agents simultaneously. When used in a flow through microarray platform known as the Programmable Bio-Nano-Chip (p-BNC), these beads have demonstrated opportunities for detection of low volumes of sample under short analysis times. However, limitations in traditional microfluidic materials such as silicon and inefficient fractional capture of analytes by porous beads hinder the translation of the p-BNC into broad global and clinical adoption where tests are single use with short analysis times to detect low concentrations of sample. This dissertation aims to optimize the p-BNC through engineering design choices to enhance the performance and reduce the costs associated with the p-BNC. The development of a computational tool to model the porous bead-based system is described herein and used to lead in the design optimization of the system. This tool provides insights into the transport and capture of analytes within the bead array with capture performance as a function of flow rate, porosity, capture distances, molecular affinities, and binding densities. To transition away from a single use and expensive silicon-based microarray, a thermoplastics-based microarray, fabricated through the hot embossing of polyethylene from replicated molds from silicon, is developed and described. Further, to transition towards point of care conditions where sample volume is low and analysis times are short, the geometry of the bead microwell design is optimized to improve the fractional capture efficiency of analytes by the beads in flow through microcontainers. Finally, to improve the imprecision performance in bead-to-bead signal variation within the microarray, exploration of a split design and use of smaller beads reveal a decrease in imprecision.
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.

Browsing by Author "Richards-Kortum, Rebecca Rae"

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