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Item Widefield micro-camera integrated into the objective lens of a reflectance confocal microscope for concurrent image registration(Montana State University - Bozeman, College of Engineering, 2023) Aist, Joseph Nicholas; Chairperson, Graduate Committee: David L. DickensheetsWith millions of new skin disease cases reported annually, non-invasive imaging methods have been developed to diagnose skin disease accurately. Reflectance confocal microscopes (RCM) have led these new technologies with high sensitivity and specificity. However, current methods use multiple devices: a digital camera, a dermoscope, and an RCM, which are not co-registered. Therefore, locating small, microscopic RCM fields-of-view (0.5x0.5 mm) at specific suspicion sites within the larger dermoscopic field-of-view (10x10 mm) is extremely difficult. This 'blind' RCM imaging results in lower and more variable diagnostic accuracy, particularly sensitivity, where positive and negative predictive values can drop by up to 30%. Our team has designed a new objective lens with an integrated micro-camera to deliver a concurrent widefield image of the skin surface surrounding the location of microscopic RCM imaging. The widefield image can be used directly to provide context for RCM or can be registered to a previously stored high-resolution clinical image to show where RCM imaging is occurring. In this thesis, the micro-camera is characterized and tested in laboratory and clinical settings. In addition, this thesis investigates a co- and cross-polarized micro-camera and LED system. It compares them to the non-polarized system to explore whether the cross-polarized version enhances feature contrast and enables better dermoscopic imaging. Non-polarized, co-polarized, and cross-polarized mock-up probes of the objective lens with a micro-camera were designed and built for testing. Images of resolution targets, color charts, and skin were taken to obtain modulation transfer function (MTF) measurements, color analysis data, and representative skin images. The results showed improvement in the MTF for the cross- polarized probe when compared to the co- and non-polarized probes. It was also found that the polarization of the imaging system did not significantly affect the color quality of the images. When tested by scientists at Memorial Sloan Kettering Cancer Center, sub-surface features not seen with the co- and non-polarized probes were observed with the cross-polarized probe. The cross-polarized probe suppressed the surface reflections, allowing for sub-surface information to be captured.Item MEMS 3-dimensional scanner with SU-8 flexures for a handheld confocal microscope(Montana State University - Bozeman, College of Engineering, 2018) Liu, Tianbo; Chairperson, Graduate Committee: David DickensheetsThe conventional method for diagnosing skin cancer is to perform a biopsy followed by pathology. However not only are biopsies invasive and likely to leave permanent scarring, they also sample the body sparsely. Fortunately, a non-invasive method of imaging called confocal laser scanning microscopy has shown great potential to replacing invasive biopsies. Confocal microscopy can use light to achieve high-resolution imaging of cells that lie underneath the surface of the skin. However, the large size of current confocal microscopes limits their application to all but the most accessible sites. In this dissertation, I address the miniaturization of confocal microscopy through the development of a new microelectromechanical systems scan mirror that can scan a focused beam in three dimensions. The scanner has a 4 mm aperture, and has the capability to replace all of the bulky beam scanners and focus mechanisms that contribute to the large size of current confocal microscopes. The fabrication of the scanner explores the use of the polymer SU-8 for its mechanical structures. The gimbal mirror has demonstrated scan angles in excess of plus or minus 3° mechanical for lateral scanning, and its deformable surface provided controllable deflection up to 10 microns for focus control. This newly developed scanner was integrated into a confocal system to test its imaging capabilities. The device demonstrated high-resolution scanning with simultaneous focus adjustment suitable for the next generation of miniaturized confocal laser scanning microscopes.Item Development of an active/adaptive laser scanning microscope(Montana State University - Bozeman, College of Engineering, 2018) Archer-Zhang, Christian Chunzi; Chairperson, Graduate Committee: David L. DickensheetsLaser scanning techniques such as confocal microscopy and two-photon excitation fluorescence microscopy (TPM) are powerful tools for imaging biological samples with high resolution, offering three-dimensional (3D) visualization of the behavior of cells in their natural environment. Traditionally, the 3D images are acquired from 2D image stacks with focusing depth controlled through mechanical movement of the specimen relative to the objective lens. The slow mechanical movement (~<20Hz) does not allow the spot of light to be scanned axially sufficiently fast to monitor cell:cell and cell:environment interactions in real time over hundreds of microns in all three dimensions. A fast focus control mirror supports agile scan patterns such as vertical or oblique planes or even arbitrary surfaces, minimizing the time and photo damage required to monitor features of interest within the 3D volume. Because aberrations cause image quality to decrease as the focal point of the beam penetrates deeper into the sample, adaptive optics can enhance resolution and contrast at depth for confocal microscopy and TPM. Combining a fast focus control mirror with a fast aberration correcting mirror leads to a flexible platform called the active/adaptive laser scanning microscope, capable of aberration-corrected beam scanning throughout a 3D volume of tissue. This opens up the possibility of fully corrected, variable-depth imaging along oblique sections or more complex user-defined surfaces within a single image frame.Item Adhesion and subsequent biofilm formation of Candida albicans on chemically different surfaces as investigated using confocal scanning laser microscopy(Montana State University - Bozeman, College of Engineering, 2002) Wesenberg, Karen EmmaItem A Confocal Microscope and Raman Spectroscopy Probe for Mars exploration(Montana State University - Bozeman, College of Engineering, 2002) Crowder, Dawn MichelleItem Mems 3-D scan mirror for an endoscopic confocal microscope(Montana State University - Bozeman, College of Engineering, 2005) Shao, Yuhe; Chairperson, Graduate Committee: David L. Dickensheets.Optical MEMS is at a very exciting stage and has become an enabling technology for a variety of applications in telecommunications and high-resolution display and imaging. Among many novel MEMS devices, MEMS scanners and deformable membrane mirrors are especially useful for scanned-beam imaging systems. MEMS scanners are usually used for laser beam scanning. Deformable mirrors provide an approach to modify the optical wavefront adaptively. In optical microsystems, packaging is often a critical part of the system design. Combining functionalities at a single device can reduce complexity of the system. Biaxial beam scanning and focus control are combined together in the MEMS 3-D scan mirror. The mirror surface is made of a goldcoated silicon nitride deformable membrane and works as a positive lens of variable focal length. The membrane sits on a torsional plate that can scan about two orthogonal axes. This architecture is able to move the focus of a laser beam throughout a threedimensional space with a single optical surface. The overall size of the 3-D mirror is 1.5 mm with a usable optical aperture of 0.7 mm. Both the inner and outer scanning axes achieved more than 5o zero-to-peak mechanical scan angles; the deformable membrane achieved a maximum center displacement of more than 3.7 æm, corresponding to an adjustable focal length from infinity to approximately 8 mm. For a confocal laser scanning microscope with illumination wavelength at 500 nm, they provide Nx = Ny = 488 resolvable spots for lateral resolution, and Nz = 32 depth-of-focus distances for depth resolution. To drive the 3-D mirror, the inner axis is operated near resonance (~ 650 Hz); and the outer axis quasi-statically. Operating the outer axis at 2 Hz provides a line resolution of 325 lines/frame at a refresh rate of 2 frames/second. The performance of the 3-D mirror is well matched to the intended application of endoscopic confocal microscopy, and similar devices could prove useful in a variety of optical microsystems needing beam scanning and focus control. This dissertation describes the design, fabrication, characterization, imaging experiment and target applications of the MEMS 3- D scan mirror.Item Spatial and temporal patterns of antimicrobial action against Staphylococcus Epidermidis biofilms(Montana State University - Bozeman, College of Engineering, 2008) Davison, William Marshall; Chairperson, Graduate Committee: Philip S. Stewart; Joseph D. Seymour (co-chair)This study investigated the spatio-temporal patterns of antimicrobial action against Staphylococcus epidermidis planktonic and biofilm bacteria. Bacteria were stained with a fluorogenic esterase substrate, Calcein-AM, which allowed for the visualization of cells that possessed intact cell membranes. Four different antimicrobial agents were tested for their effect upon cell viability as associated with membrane integrity. The four biocides were Barquat®, glutaraldehyde, chlorine, and nisin. Planktonic bacteria were analyzed with flow cytometry, observing fluorescence loss during 1 h antimicrobial treatment. Treatment with Barquat resulted in initial fluorescence loss, which increased during the treatment period to levels which were present prior to the introduction of biocide, along with a decrease in cell density. Treatments with glutaraldehyde and chlorine resulted in increased average fluorescence intensity for the cell population, accompanied by decreased cell density for chlorine and increased cell density for glutaraldehyde. Nisin treatment resulted in a decrease in CAM fluorescence with an increase in cell density. Viable cell plate counts showed average log reductions in CFU/mL of 3.61, 3.83, 4.12, 4.26, and 4.67 for Barquat, glutaraldehyde, high and low concentrations of chlorine, and nisin treatments, respectively. There was no apparent correlation between plate counts and flow cytometry data. Biofilm bacteria were analyzed with time-lapse confocal scanning laser microscopy, observing fluorescence loss during biocide treatment. Biofilms treated with Barquat lost an average of 91.5% of their initial fluorescence, and clusters decreased in areal coverage by 9%. Fluorescence loss during Barquat treatment suggested the presence of a tolerant subpopulation of bacteria in the interior regions of the biofilm. Glutaraldehyde treatment reduced the average fluorescence by 16%, and cluster area did not change. There was CTC staining after glutaraldehyde treatment only. The high and low concentrations of chlorine treatment showed averages of 100% and 79% reductions in CAM staining, with liquefaction of biomass causing erosion events which reduced areal coverage by 90% and 43%, respectively. Nisin treatment reduced CAM staining by an average of 100%, while shrinking the cluster area by 8%. Corner biofilms showed qualitative differences during treatment than isolated clusters. Mathematically-predicted biocide diffusion times were much faster than experimentally observed fluorescence loss in biofilms.Item A quantitative description at multiple scales of observation of accumulation and displacement patterns in single and dual-species biofilms(Montana State University - Bozeman, College of Engineering, 2007) Klayman, Benjamin Joseph; Chairperson, Graduate Committee: Anne CamperThis research represents a novel approach for describing biofilm accumulation at multiple scales of observation in both single and dual-species biofilms. Pseudomonas aeruginosa PAO1 and Escherichia coli O157:H7 were grown as single and dual-species biofilms in 1 mm glass capillary flow cells and monitored over time using confocal microscopy. Colonization and biofilm development patterns were associated with the fluid flow regime as evaluated using the finite volume analysis program CFX (ANSYS Europe, Ltd). The shear stress was shown to vary along the surface from a minimum near the edges to a maximum in the center of the flow path. Initial colonization by both species occurred at the outer edges of the flow path (low shear). P. aeruginosa was subsequently observed to migrate perpendicular to the flow direction towards the center of the flow path (high shear), but E. coli was never observed outside of the 200 micron outer edge. E. coli was unable to persist in the flow cell unless P. aeruginosa was present as a colonizing partner. Bio-volumes of each species were calculated using the Metamorph (Molecular Devices) image analysis program and are reported over time.Item Vascular shutdown as an effect of using photodynamic therapy to treat cancer(Montana State University - Bozeman, College of Letters & Science, 2008) Pascucci, Elizabeth Mary; Chairperson, Graduate Committee: Jean Starkey; Sandra Halonen (co-chair)Photodynamic therapy (PDT) is a treatment that uses the combination of a photosensitizing drug and light to selectively kill cancer cells. PDT has many potential advantages such as minimal side effects, excellent cosmetic results, and no cellular resistance burdening traditional cancer treatments such as chemotherapy and radiation. Currently used in the clinic, a limitation is depth of light penetration; therefore, PDT can only be used to treat superficial disease. Our novel PDT agent utilizes two-photon laser technology, which increases the depth of light penetration, greatly increasing the potential uses. Our PDT is able to kill selective cells because the PDT drug has a targeting agent so the drug only goes to cancer cells that overexpress the Somatostatin receptor 2 (SSTr2) on their cell surface. Laser light irradiates the cancer cells causing cytotoxic singlet oxygen to be produced damaging the cells. It was observed, through in vivo imaging of the tumors before and after treatment, that vascular flow was diminished as a result of PDT. It is well established that angiogenesis must occur when a tumor reaches a certain size in order for the cells to remain viable and the tumor to continue to grow; therefore, vascular shutdown could be an important mechanism of how PDT works. A series of experiments on the tumor vasculature using in vivo imaging, immunohistochemistry and confocal microscopy has taken place since this observation to determine what may be happening. Results of these studies have shown that tumor vessels do express the SSTr2 and therefore effected by treatment. Experiments using a somatostatin analog, octreoate, detected by a fluor has shown that the endothelial cells do take the drug up. To ensure that it is blood vessels that were being studied, tumor tissue was stained for both SSTr2 and vonWillebrand Factor (vWF), a recognized endothelial cell marker. Staining patterns for both antibodies were similar. To strengthen the argument, SSTr2- negative tumor cells also showed a positive staining pattern of their vessels, demonstrating that even though the tumor cells are SSTr2- negative, the tumor vessels can be SSTr2-positive and thus responsive to PDT.Item Heterodyne detection fiber confocal microscope for in vivo skin imaging(Montana State University - Bozeman, College of Engineering, 2011) Xue, Xiaohu; Chairperson, Graduate Committee: David L. DickensheetsConfocal microscopy has been demonstrated to be a very effective tool for imaging in-vivo samples. The confocal imaging geometry provides a dramatic optical advantage for microscopy by discriminating against out-of-focus background with minimal loss of image-forming signal. Because of these advantages, the confocal laser scanning microscope (CLSM) can image a thin layer clearly from a thick sample without biopsy. However, current usage of CLSM is limited by the signal-to-noise ratio using conventional optical detection. In order to achieve deeper penetration into the skin in a clinical setting, a technique called heterodyne detection is incorporated into the CLSM system. This thesis describes the optical, mechanical and electrical design of the system, evaluates system noise and imaging performance, and provides initial skin images collected by the heterodyne system, comparing the results with direct detection. The heterodyne detection system is proved to have deeper penetration than the direct detection system, but the image quality is degraded.