<p>Mouse models of cancer are an invaluable tool for studying the mechanism of the disease and the effect of new therapies. Recent years have seen an explosive growth in the development of such models and consequently there is an increased need for better imaging techniques to study them. The goal of this work was to develop a technique that satisfied the requirements for preclinical cancer imaging: high spatial resolution, good soft tissue differentiation, excellent motion immunity, fast and non-invasive imaging to enable high-throughput, longitudinal studies. </p> <p>T₂-weighted and diffusion-weighted magnetic resonance imaging (MRI) has been shown to be effective for tumor characterization clinically. But translation of these techniques to the mouse is challenging. The higher spatial resolution and faster physiologic motion make conventional approaches very susceptible to phase artifacts. Additionally, at higher magnetic fields required for these studies, T<super>*</super>₂ and T₂ are significantly shorter and T₁ is longer, making in vivo imaging even harder.</p> <p>A rigorous cancer imaging protocol was developed by optimizing and integrating various components of the system, including MR hardware, animal handling, and pulse sequence design to achieve reliable, repeatable and rapid imaging. The technique presented here relies heavily on the non-Cartesian sampling strategy of PROPELLER (Periodically Rotated Overlapping ParallEL Lines with Enhanced Reconstruction) MRI. The novel data acquisition and reconstruction overcomes the adverse effects of physiological motion, allows for rapid setup and acquisition and provides excellent tissue contrast. The sequence was optimized to enable T₂-weighted and diffusion-weighted imaging in tumor-bearing mice with in-plane resolution of 117&mu;m and slice thickness of 1mm. Multi-slice datasets covering the entire thorax and abdomen were acquired in &sim;30 minutes.</p> <p>The imaging protocol developed here was applied to a high-throughput, longitudinal study in a mouse model of liver metastases. The liver is a common site of distal metastases in colon and rectal cancer, and if detected early has an improved prognosis. Unfortunately, severe respiratory motion make it hard to image. The relative merits of the proposed PROPELLER technique were analyzed with respect to the accepted gold-standard for abdominal cancer imaging, computed tomography (CT).</p> <p>The non-Cartesian MR microscopy technique proposed here is a valuable tool in the &ldquo;Cancer analysis toolkit&rdquo;. It allows for high-throughput, longitudinal experiments in free-breathing mice generating both structural and functional information with minimal artifacts and excellent spatial resolution. This work should find broad applications in various mouse models of cancer for studying the pathology of the disease, its progression as well as its response to treatment.</p> / Dissertation

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Atomic force microscopy is one of the powerful instruments used to explore the mechanical properties of nanoscale materials. It not only can produce high-resolution images and surface mechanical properties, but also can make use of its probe for surface etching. In this study, we first use atomic force microscopy to measure the Adhesion Map of insulin amyloid fibers, then conduct mechanical lithography on the surface with the probe. In the end, we discuss the effect on insulin amyloid fibrils due to exert different forces and different speeds with the probe. According to Nanoindentation theory and Hertzian model, we can derive the Young's modulus of insulin amyloid fibrils from force-indentation relations. Then we cut the Insulin amyloid fibers with probe. The results showed that when we applied 3.23 nN force by the probe, the insulin amyloid fibers began to break. When we applied 7.07 nN force, insulin amyloid fibers are cut off easily. Therefore, we can bite off insulin amyloid fibers of different lengths and sections, and arrange in the desired pattern by atomic force microscope.

Double stranded DNA bacteriophages achieve release of phage progeny by disrupting the cell envelope of the host cell. This is accomplished by two phage-encoded proteins, the holin and the endolysin. In bacteriophage lambda, the S holin is a small three TMD membrane protein that creates a lesion in the inner membrane of the host at a specific time, programmed in its primary structure. Lesion formation permits the cytoplasmic endolysin R access to the murein cell wall for degradation and cell lysis. Although it has been shown that S oligomerizes in the membrane, the structural nature of this complex has not been elucidated. In this study the S holin was purified using a mild non-ionic detergent and the structure of a ring complex formed by the holin was determined by electron microscopy and single particle analysis at a resolution of 2.6 nm. Biochemical characterization of the rings suggests that such a complex might represent the assembly formed by S in the membrane. Protein translocation in all organisms allows the export of proteins destined for localization outside the cytoplasm. In eubacteria, newly synthesized proteins are directed to the heterotrimeric membrane complex SecYEG by signals embedded in their sequence. The driving force through this complex is provided by the cytoplasmic ATPase SecA which combines ATP hydrolysis to mechanically insert proteins through the protein conducting channel. Using electron microscopy and single particle analysis we have obtained the structure of SecA from M. tuberculosis. The structure indicates that four SecA monomers assemble to form an elongated molecule with D2 symmetry. Docking of the EM map to the crystal structure of tb SecA confirms this arrangement of the subunits. This finding, that M. tuberculosis SecA forms a tetramer raises intriguing possibilities about SecA function.

The leading cause of death in patients who survive subarachnoid hemorrhage (SAH) is stroke as a result of cerebral arterial vasospasm1. Such vasospasms involve a vasoactive response, but they remain enigmatic and no clinical treatment has proven effective in prevention or reduction2. Arteries remodel in response to diverse mechanical loads and chemical factors. Following SAH, the surrounding vasculature is exposed to a radically altered chemo-mechanical environment. It is our hypothesis that chemical stimuli associated with the formation of an extravascular blood clot dominates the maladaptive growth and remodeling response early on, thus leading to important structural changes. However, it is not clear which of the many chemical factors are key players in the production of vasospasm. Before an accurate picture of the etiology of vasospasm can be produced, it is imperative to gain a better understanding of the non-pathogenic cerebral vasculature. In particular, the rabbit basilar artery is a well established model for vasospasm. However, surprisingly little is known about the mechanical properties of the rabbit basilar artery. Using an in vitro custom organ culture and mechanical testing device, acute and cultured basilar arteries from male White New Zealand specific pathogen free rabbits underwent cyclic pressurization tests at in vivo conditions and controlled levels of myogenic tone. Sections of basilar arteries were imaged for collagen fiber orientation at 0, 40 and 80 mmHg at in vivo stretch conditions using nonlinear optical microscopy. The nonlinear stress-strain curves provide baseline characteristics for acute and short-term culture basilar arteries. The active and passive testing creates a framework for interpreting the basal tone of arteries in our culture system. Nonlinear optical microscopy second harmonic generation provides unique microstructural information and allows imaging of live, intact arteries while maintaining in vivo geometries and conditions. Collagen fibers were found to be widely distributed about the axial direction in the adventitial layer and narrowly distributed about the circumferential direction in the adventitial layer. The quantified collagen fiber angles within the artery wall further support the development of accurate mathematical models.

Vulnerable plaque is a clinically silent condition of atherosclerotic plaque that leaves a large number of patients at risk of a coronary event. A method to detect vulnerable atherosclerotic plaque would greatly enhance the ability of clinicians to diagnose and treat patients at risk. Fluorescence lifetime imaging microscopy (FLIM) offers a way to extract both spatial and biochemical information from plaque by taking several wide-field images over time. The goal of this study was to determine the potential of a FLIM angioscopy system to detect and differentiate coronary atherosclerotic plaques ex-vivo into several groups including thin, fibrotic, lipid-laden, thick-cap fibroatheroma (FA), and fibrocalcified. Samples were extracted post-mortem weekly and sliced open to have their lumens imaged. For each sample, 51 time resolved wide-field images were taken over 10 nanoseconds at 390 (±40) nm, 450 (±40) nm, and 550 (±88) nm wavelengths. To analyze the samples, the intensity map and lifetime map were created at each wavelength. The intensity map was simply the wide-field images summed in time and normalized. In order to calculate lifetime at each point, a fast, model-free Laguerre deconvolution algorithm was recently developed for FLIM data analysis and was used. This allowed for fast, efficient estimations of the fluorescence decay curves at each pixel of the FLIM images and facilitated the computation of quantitative parameters describing the fluorescence emission of the tissue, specifically, the relative fluorescence intensity and lifetime at defined emission bands. Statistical analysis on these FLIM derived parameters indicated that the autofluorescence emission of the plaques allows for distinguishing relative plaque thickness: thin plaque, whose signal is dominated by elastin fluorophores, shows a marked difference between thicker plaques, such as fibrotic, fibrocalcified and thick-cap FA (who are dominated primarily by collagen). However, the ability of the current FLIM system to differentiate vulnerable plaque remains in question due to the absence of thin-cap FA samples. Further work has also been proposed; of primary concern is gathering thin-cap FA plaque samples needed to validate the system’s ability to differentiate vulnerable plaques from other common groupings.

Many Americans are afflicted with inflammation of the colon. They are also at a higher risk of developing colon cancer. Confocal microscopy of bulk epithelial tissue has the potential to provide information on tissue structural properties that may be lost in the fixation and slicing procedures required for histopathology. Optical sectioning provides images in three dimensions capturing the organizational structure of cells and colon crypts throughout the entire colon. I have constructed a custom built fluorescence and reflectance confocal microscope for imaging molecular and morphological changes associated with development of inflammation in a mouse model. A confocal microscope is a point scanning system that removes out of focus light by placing a pinhole aperture in the conjugate image plane located in front of the detector. We have two sources, 488 nm and 811 nm, for fluorescence and reflectance imaging, respectively. A polygon scanning mirror and a galvanometer scanning mirror allow for a variable scan rate between 8 and 15 fps. The lateral resolution of the system is approximately 3 μm with an axial resolution of 6 μm and 4 μm for reflectance and fluorescence mode, respectively. As colon tissue becomes inflamed, there is a distinct change in the structure and architecture of the tissue. The colon crypts are no longer uniform in size or distribution throughout the tissue. Having a large field of view of 1mm2 allows for many colon crypts to be visualized within a single frame. Histology was performed on the same tissue imaged for the inflammatory study confirming the constructed confocal microscope’s ability to characterize inflamed tissue and the potential use for guided biopsy. Mosaicing, or image tiling, is an imaging technique that stitches single frames together to produce a much larger field of view. An extended frame with 1 mm x 2 cm field of view is achieved within seconds. This extended frame would allow mosaicing of the entire mouse colon much faster than conventional methods without loss of resolution. The acquired confocal images of colon tissue demonstrate the microscope’s ability to resolve cell nuclei lining the colon crypts within a relatively large field of view.

­ì¤l¤OÅã·LÃè(AFM)¯à¦b®ð¬Û¡B¯uªÅ¡B¤Î±µªñ¥Í²z±ø¥óªº²G¬Û¤¤ª½±µ¶i¦æªí­±³æ¤À¤lªºÆ[´ú¡C¦ý¼Ë«~¤À¤l³Q©T©w«á¡A¨äµ²ºc¬O§_»P¦ÛµMª¬ºA¬ÛÃö¡A»á¥O¤H½èºÃ¡C ¥»¬ã¨s°w¹ï¶³¥À¤ùªí­±¶i¦æ¤Æ¾Ç­×¹¢¡A§Q¥ÎºëÓi(spermine)¤j¤j´£°ª¤F°òªOªí­±§lªþDNAªº¯à¤O¡C¹B¥Î»E¦X¤À¤lÃì²Î­p¤ÀªR²z½×(statistical polymer chain analysis)¡A¥H¤TºØ¤£¦Pªø«×ªº½u«¬DNA¤À¤l¡A®Ú¾ÚAFM¼v¹³¤À§O§@¤À¤l½ü¹øÁ`ªø(contour length, L)¤Î¥¼ºÝ¨âÂI¶ZÂ÷(end-to-end distance, R)ªº´ú¶q¡A¥H<R2>»PL¤§¬ÛÃö©Ê±Àª¾¼v¹³¤¤ªºDNA¤À¤lªí²{ªº¬O3D¥ßÅé®·®»ºAºc«¬(three-dimensional trapped configuration)¡A¦Ó«D¤À¤l¦b2DªÅ¶¡­«·s«Ø¥ß¥­¿Å«áªºµ²ºc¡C¥t¥~¡AÂǧïÅÜDNA¼Ë«~²Gºw¦b¶³¥À¤ù¤W°®Àꪺ®É¶¡¡A©Ò³y¦¨°òªO§lªþ¤À¤l¼Æ¥ØªºÅܤơA°t¦X¤£¥i°fÂX´²¹B°Ê¼Ò«¬±o¨ì¤F¤À¤l¥Ñ²G¬Û¨ì¹Fªí­±¿é°e¹Lµ{¤§ÂX´²«Y¼Æ¡C ¦b©Ý¾ë²§ºc酶(topoisomerase)»P¶Ê¤ÆDNA¤À¤l¤ÏÀ³¹êÅ礤¡AAFM©úÅã¿ëÃÑ¥XDNA¤À¤l¦b©Ý¾ëºc§Î¤WªºÂà¤Æ¡A¬Æ¦Üª½±µ¬Ý¨ì¸Ñ±Û¾÷¨î¤¤©Ý¾ë酶¤À¤l»PÂùªÑDNA¤¤¤@±ø³æªÑªº§@¥Î¡C

In this study, we have successfully developed an acoustic modulation microscopy that is based on a laser scanning confocal microscopy and operates in the range of a few tens of kilohertz. The induced submicron oscillation is detected through the combination of differential confocal microscopy and lock-in circuit. In this way, the mechanical properties, such as elasticity and stiffness, can be mapped in a two-dimensional way rapidly.