Investigations into high resolution imaging and contouring for diagnostic bio-applications

Cancer is a leading cause of deaths in the world, with lung, breast and colorectal cancers being the top killers. As early detection is important to prevent deaths due to cancer, current emphasis is on periodic screening for persons over the age of 50. Depending on the organ being assessed, a variety of diagnostics tools are presently available to detect abnormal state of the tissue, with each technique having its own set of advantages and disadvantages. Of these, currently, only endoscopy provides a possibility of abnormality detection based on direct tissue imaging and visual differentiation in body cavities such as colon. However, the effectiveness of endoscopy is limited in that polyps are missed as they fail to catch the attention of the endoscopist during the visual examination. They are missed out in blind biopsies or are not well delineated as abnormalities, especially as in the case of flat dysplasia. If abnormal regions are well identified, targeted biopsies can be carried out minimising the pain and risk of tissue perforations to the patient. Research is in progress around the world for finding methods which are minimally invasive and which can provide accurate results with minimum patient discomfort. The hallmarks of cancer are changes in the tissue’s biochemical makeup, biological activity level, vasculature, blood perfusion and cellular structure. In this context, this research thesis focuses on coming up with such methodologies using optical concepts and imaging modalities for diagnostic medical imaging in a probe based scheme. Complementary optical methodologies, which can enable easy identification and diagnosis of potential abnormal growth sites that represent cancerous state of tissue, were investigated in this research, as it is envisioned that information from one modality can be used to strengthen judgment from another independent modality. For the modalities in whole field imaging mode, a target size of 5 mm and below was set as detection capability for the abnormalities for early diagnosis, as polyp sizes up to 5 mm were deemed as low risk and are only marked for observation in subsequent screening sessions. An enlargement of the size of cell nucleus from about 5-10 pm to about 20 pm being an indication of pre-cancerous state, a resolution capability of less than 5 pm was set as a target for high resolution imaging mode. A novel RGB grayscale value based spectral shift approach was successfully developed for easy identification of abnormality based on biochemical signature differences that can be identified optically, namely fluorescence. A CCD camera based image analysis scheme was conceptualised and configured using spectral shift calculations. The Euclidian distances for the change in Red, Green and Blue gray values between the illuminating wavelengths and the points in the image were used to reconstruct a pseudo-image which shows the differences between normal and abnormal areas in the tissues. The proposed algorithm was validated with phantom tissues as well as real mouse colon tissues and mouse with tumour to distinguish abnormal regions, with abnormality sizes ranging from 2 mm to about 12 mm. The proposed algorithm and developed methodology apply pixel level processing, and hence, processing of both whole field and high resolution images can be carried out.Contouring of abnormality based on bioactivity differences that can be identified optically, namely dynamic speckle based methodology, was the second target area of the research. A novel thermal perturbation based approach was proposed and researched to enable identification and contouring of abnormalities by way of differences in speckle pattern changes arising out of tissue mass and perfusion differences. Thermal perturbation of the target tissue with infrared beam was used to effect the required change in the bio-speckle pattern. Subsequent contouring of the abnormal region in the tissue could be delineated due to the speckle pattem differences that evolved over. It was found, using phantom tissue as test samples, that a time lapse of about 1 second between imaging frames captured with a CCD camera could enable identification and contouring of sub-surface abnormalities. An image reconstruction scheme using variation of intensity at corresponding pixel locations was also investigated. The effects of perfusion and simulated mucus layer were investigated. Experimental verification was carried out on silicone phantoms and to a limited extent, with animal model, with abnormality sizes ranging from about 5 mm to 8 mm. A fiber based high resolution imaging methodology was investigated that is capable of producing microscopic images of the targeted tissues. A quasi- confocal imaging approach with an imaging fiber bundle was developed to image and contour microscale objects. Non-scanning (fixed frame) as well as scanning (lD as well as 2D) based approaches were investigated. For the nonscan based approach, with axial and lateral resolutions of 15.98 ± 0.94 pm and 3.47 pm, respectively, the system capability was demonstrated with glassbubbles, polystyrene beads, cells and micro-fluidic flow channels. The scan based methodology developed, with the probe in proximity with the target tissue, could overcome loss of visual information due to the fiberlet interspaces and achieved a significant improvement in lateral resolution to 2.19 pm with the same optical elements as with the non-scan based scheme. With the fiber bundle enabling multiple scan points, thus saving scan time, and scanning steps designed to match the imaging pixel size for best resolution, a scheme to reconstruct high resolution images from the whole-field scan, at any region of interest was worked out. It was found that the central pixel in each of the fiberlet image are the optimal scan points as it gave the best contrast and the least offset in the scan image compared with other pixels within the fiberlet. The capability of the system was demonstrated using micron-sized fluorescent polystyrene beads and cells mounted on a microscope slide. Besides providing imaging capability, the imaging fibre can double up as a light conduit to illuminate the target tissue, which would be in synergy with the first two modalities, namely, the RGB and the dynamic speckle analyses. The research carried out in this thesis involved exploring and laying the foundations for a synergistic combination of optical methods which could potentially be integrated in future into a single probe for endoscopic applications. It is capable of identifying early stage cancerous regions of less than 5 mm in size from a distance and also micro-level imaging of structures less than 5 pm at proximity of the targeted area. As a future work direction, an actual probe system could be engineered integrating a micro-CCD camera and an imaging fiber bundle by applying the proposed multiple optical bubbles, polystyrene beads, cells and micro-fluidic flow channels. The scan based methodology developed, with the probe in proximity with the target tissue, could overcome loss of visual information due to the fiberlet interspaces and achieved a significant improvement in lateral resolution to 2.19 pm with the same optical elements as with the non-scan based scheme. With the fiber bundle enabling multiple scan points, thus saving scan time, and scanning steps designed to match the imaging pixel size for best resolution, a scheme to reconstruct high resolution images from the whole-field scan, at any region of interest was worked out. It was found that the central pixel in each of the fiberlet image are the optimal scan points as it gave the best contrast and the least offset in the scan image compared with other pixels within the fiberlet. The capability of the system was demonstrated using micron-sized fluorescent polystyrene beads and cells mounted on a microscope slide. Besides providing imaging capability, the imaging fibre can double up as a light conduit to illuminate the target tissue, which would be in synergy with the first two modalities, namely, the RGB and the dynamic speckle analyses. The research carried out in this thesis involved exploring and laying the foundations for a synergistic combination of optical methods which could potentially be integrated in future into a single probe for endoscopic applications. It is capable of identifying early stage cancerous regions of less than 5 mm in size from a distance and also micro-level imaging of structures less than 5 pm at proximity of the targeted area. As a future work direction, an actual probe system could be engineered integrating a micro-CCD camera and an imaging fiber bundle by applying the proposed multiple optical bubbles, polystyrene beads, cells and micro-fluidic flow channels. The scan based methodology developed, with the probe in proximity with the target tissue, could overcome loss of visual information due to the fiberlet interspaces and achieved a significant improvement in lateral resolution to 2.19 pm with the same optical elements as with the non-scan based scheme. With the fiber bundle enabling multiple scan points, thus saving scan time, and scanning steps designed to match the imaging pixel size for best resolution, a scheme to reconstruct high resolution images from the whole-field scan, at any region of interest was worked out. It was found that the central pixel in each of the fiberlet image are the optimal scan points as it gave the best contrast and the least offset in the scan image compared with other pixels within the fiberlet. The capability of the system was demonstrated using micron-sized fluorescent polystyrene beads and cells mounted on a microscope slide. Besides providing imaging capability, the imaging fibre can double up as a light conduit to illuminate the target tissue, which would be in synergy with the first two modalities, namely, the RGB and the dynamic speckle analyses. The research carried out in this thesis involved exploring and laying the foundations for a synergistic combination of optical methods which could potentially be integrated in future into a single probe for endoscopic applications. It is capable of identifying early stage cancerous regions of less than 5 mm in size from a distance and also micro-level imaging of structures less than 5 pm at proximity of the targeted area. As a future work direction, an actual probe system could be engineered integrating a micro-CCD camera and an imaging fiber bundle by applying the proposed multiple optical methodologies for effective identification, contouring and confirmation of abnormalities that can lead to early diagnosis of disease, such as cancer, in body cavities such as colon. These findings are expected to contribute to future research towards realising an in vivo optical biopsy probe.Doctor of Philosophy (MAE

Red, green, and blue gray-value shift-based approach to whole-field imaging for tissue diagnostics

Identification of abnormal pathology in situ remains one of the challenges of medicine. The interpretation of tissue conditions relies mainly on optical assessment, which can be difficult due to inadequate visual differences or poor color delineation. We propose a methodology to identify regions of abnormal tissue in a targeted area based on red, green, blue (RGB) shift analysis employing a simple CCD color camera and light-emitting diode illumination in a whole-field-imaging scheme. The concept involves analysis of RGB components in an image with respect to a reference set of RGB values under different illumination wavelengths. The magnitude of the gray value shift is estimated by calculating the Euclidean distance between their normalized RGB coordinates. The shift values obtained using these concepts are thereafter used to construct pseudo-colored images with high contrast, enabling easy identification of abnormal areas in the tissue. Images processed from experiments conducted with excised Wistar rat colon sample (lightly doped with Alexafluor 488) and with simulated tumor (cancer cell pellet placed on colon) showed clear localization of tumor region. This proposed approach and methodology is expected to find potential applications for the in vivo diagnosis of disease.Published versio

Effect of fiber layer formation on mechanical and wear properties of natural fiber filled epoxy hybrid composites

Natural fiber-reinforced polymer matrix composites are gathering significance in future trend applications such as automotive, aerospace, sport, and other engineering applications due to their superior enhanced mechanical, wear, and thermal properties. Compared to synthetic fiber, natural fiber is low adhesive and flexural strength properties. The research aims to synthesize the epoxy hybrid composites by utilizing the silane (pH = 4) treated Kenaf (KF) and sisal fiber (SF) as layering by uni, bi, and multi-unidirectional via hand layup techniques. Thirteen composite samples have been prepared by three-layer formation adopted with different weight ratios of E/KF/SF such as 100E/0KF/0SF, 70E/30KF/0SF, 70E/0KF/30SF, 70E/20KF/10SF, and 70E/10KF/20SF respectively. The effect of layer formation on the tensile, flexural, and impact strength of composites is studied by ASTM D638, D790, and D256 standards. The unidirectional fiber layer formed (sample 5) 70E/10KF/20SF composite is found maximum tensile and flexural strength of 57.9 ± 1.2 MPa and 78.65 ± 1.8 MPa. This composite is subjected to wear studies by pin-on-disc wear apparatus configured with a hardened grey cast-iron plate under an applied load of 10, 20, 30, and 40 N at different sliding velocities of 0.1, 0.3, 0.5, and 0.7 m/s. The wear rate of the sample progressively increases with increasing load and sliding speed of the composite. The minimum wear rate of 0.012 mg/min (sample 4) is found on 7.6 N frictional force at 0.1 m/s sliding speed. Moreover, sample 4 at a high velocity of 0.7 m/s with a low load (10 N) shows a wear rate of 0.034 mg/min. The wear-worn surface is examined and found adhesive and abrasive wear on a high frictional force of 18.54 N at 0.7 m/s. The enhanced mechanical and wear behavior of sample 5 is recommended for automotive seat frame applications

Cytokines and growth factors in the developing intestine and during necrotizing enterocolitis.

Cytokines and growth factors play diverse roles in the uninflamed fetal/neonatal intestinal mucosa and in the development of inflammatory bowel injury during necrotizing enterocolitis (NEC). During gestational development and the early neonatal period, the fetal/premature intestine is exposed to high levels of many inflammatory cytokines and growth factors, first via swallowed amniotic fluid in utero and then, after birth, in colostrum and mother\u27s milk. This article reviews the dual, seemingly counter-intuitive roles of cytokines, where these agents play a trophic role and promote maturation of the uninflamed mucosa, but can also cause inflammation and promote intestinal injury during NEC

All-Trans Retinoic Acid Induces TGF-β2 in Intestinal Epithelial Cells via RhoA- and p38α MAPK-Mediated Activation of the Transcription Factor ATF2.

We have shown previously that preterm infants are at risk of necrotizing enterocolitis (NEC), an inflammatory bowel necrosis typically seen in infants born prior to 32 weeks' gestation, because of the developmental deficiency of transforming growth factor (TGF)-β2 in the intestine. The present study was designed to investigate all-trans retinoic acid (atRA) as an inducer of TGF-β2 in intestinal epithelial cells (IECs) and to elucidate the involved signaling mechanisms.AtRA effects on intestinal epithelium were investigated using IEC6 cells. TGF-β2 expression was measured using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Western blots. Signaling pathways were investigated using Western blots, transiently-transfected/transduced cells, kinase arrays, chromatin immunoprecipitation, and selective small molecule inhibitors.AtRA-treatment of IEC6 cells selectively increased TGF-β2 mRNA and protein expression in a time- and dose-dependent fashion, and increased the activity of the TGF-β2 promoter. AtRA effects were mediated via RhoA GTPase, Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), p38α MAPK, and activating transcription factor (ATF)-2. AtRA increased phospho-ATF2 binding to the TGF-β2 promoter and increased histone H2B acetylation in the TGF-β2 nucleosome, which is typically associated with transcriptional activation.AtRA induces TGF-β2 expression in IECs via RhoA- and p38α MAPK-mediated activation of the transcription factor ATF2. Further studies are needed to investigate the role of atRA as a protective/therapeutic agent in gut mucosal inflammation

AtRA-induced TGF-β₂ expression in IECs is mediated via RhoA GTPase and ROCK1.

<p><i>A</i>. Representative Western blots show increased expression of activated RhoA (RhoA-GTP) in IEC6 cells treated with atRA × 4h. Activated RhoA was pulled down from cell lysates using Rhotekin-agarose beads. Bar-diagram (means ± SE) summarizes densitometric data. <i>Inset</i>: Left panel: ATRA also increased the expression of total RhoA in IECs. Right panel: AtRA-treatment did not increase Rac1-GTP in IEC6 cells. <i>B</i>. Western blots show that atRA-induced TGF-β₂ expression in IEC6 cells was reproduced by over-expression of the constitutively-active GL4V mutant of RhoA. <i>C</i>. Cells expressing the TN19 dominant-negative RhoA mutant did not show atRA-induced TGF-β₂ expression. <i>D</i>. Western blots show cleaved ROCK1 in IEC6 cells, depicted as a function of the duration of atRA treatment. Bar-diagram (means ± SE) summarizes densitometric data. <i>Inset</i>: Fluorescence photomicrographs (magnification 630x) show nuclear localization of ROCK1 (green) in IEC6 cells treated with atRA × 2h. Nuclear staining (blue) was obtained with DAPI (blue). <i>E</i>. Pharmacological inhibition of ROCK1 by Y-27632 blocked atRA-induced TGF-β₂ expression in IEC6 cells. Western blots show TGF-β₂ and cleaved ROCK1 expression. Bar-diagram (means ± SE) summarizes densitometric data, normalized against β-actin. Data represent 3 separate experiments; * <i>p</i><0.05 compared to cells cultured in media alone; # indicates <i>p</i><0.05 compared to atRA-treated cells.</p

Transforming Growth Gactor-β\u3csub\u3e2\u3c/sub\u3e is Sequestered in Preterm Human Milk by Chondroitin Sulfate Proteoglycans

Human milk contains biologically important amounts of transforming growth factor-β2 isoform (TGF-β2), which is presumed to protect against inflammatory gut mucosal injury in the neonate. In preclinical models, enterally administered TGF-β2 can protect against experimental necrotizing enterocolitis, an inflammatory bowel necrosis of premature infants. In this study, we investigated whether TGF-β bioactivity in human preterm milk could be enhanced for therapeutic purposes by adding recombinant TGF-β2 (rTGF-β2) to milk prior to feeding. Milk-borne TGF-β bioactivity was measured by established luciferase reporter assays. Molecular interactions of TGF-β2 were investigated by nondenaturing gel electrophoresis and immunoblots, computational molecular modeling, and affinity capillary electrophoresis. Addition of rTGF-β2 (20–40 nM) to human preterm milk samples failed to increase TGF-β bioactivity in milk. Milk-borne TGF-β2 was bound to chondroitin sulfate (CS) containing proteoglycan(s) such as biglycan, which are expressed in high concentrations in milk. Chondroitinase treatment of milk increased the bioactivity of both endogenous and rTGF-β2, and consequently, enhanced the ability of preterm milk to suppress LPS-induced NF-κB activation in macrophages. These findings provide a mechanism for the normally low bioavailability of milk-borne TGF-β2 and identify chondroitinase digestion of milk as a potential therapeutic strategy to enhance the anti-inflammatory effects of preterm milk

Transforming Growth Gactor-β\u3csub\u3e2\u3c/sub\u3e is Sequestered in Preterm Human Milk by Chondroitin Sulfate Proteoglycans

Human milk contains biologically important amounts of transforming growth factor-β2 isoform (TGF-β2), which is presumed to protect against inflammatory gut mucosal injury in the neonate. In preclinical models, enterally administered TGF-β2 can protect against experimental necrotizing enterocolitis, an inflammatory bowel necrosis of premature infants. In this study, we investigated whether TGF-β bioactivity in human preterm milk could be enhanced for therapeutic purposes by adding recombinant TGF-β2 (rTGF-β2) to milk prior to feeding. Milk-borne TGF-β bioactivity was measured by established luciferase reporter assays. Molecular interactions of TGF-β2 were investigated by nondenaturing gel electrophoresis and immunoblots, computational molecular modeling, and affinity capillary electrophoresis. Addition of rTGF-β2 (20–40 nM) to human preterm milk samples failed to increase TGF-β bioactivity in milk. Milk-borne TGF-β2 was bound to chondroitin sulfate (CS) containing proteoglycan(s) such as biglycan, which are expressed in high concentrations in milk. Chondroitinase treatment of milk increased the bioactivity of both endogenous and rTGF-β2, and consequently, enhanced the ability of preterm milk to suppress LPS-induced NF-κB activation in macrophages. These findings provide a mechanism for the normally low bioavailability of milk-borne TGF-β2 and identify chondroitinase digestion of milk as a potential therapeutic strategy to enhance the anti-inflammatory effects of preterm milk