Browsing by Author "Spann, Claire"
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Item Expansion Microscopy for Super-Resolution Imaging of the Rodent Brain(2024-03-21) Ampofo, Hannah; Berry, Raymond; Spann, Claire; Liu, Ran; Yang, ShaohuaExpansion Microscopy for Super-Resolution Imaging of the Rodent Brain Hannah Ampofo, Raymond Berry, Claire Spann, Ran Liu, Shaohua Yang Pharmacology and Neuroscience Department, School of Biomedical Sciences, University of North Texas Health Science Center, Texas, United States. Purpose-To establish Expansion microscopy (ExM) for super-resolution imaging of the rodent brain. Background- ExM is a remarkable imaging technology that enables nanoscale resolution in three-dimensional (3-D) imaging of preserved cells and tissues. ExM, which was invented in 2015, physically expands specimens using a hydrogel, allowing high-resolution imaging to be done with conventional diffraction-limited microscopes. The basic idea is to attach anchors to biomolecules or labels chemically and link them to a hydrogel that is uniformly distributed throughout the material. This polymerization technique separates biomolecules while maintaining their spatial organization by enabling isotropic expansion. The procedure is similar to sketching an outline on an inflating object and blowing it up: the ink particles will move apart, but their relative organization remains the same. Traditional optical imaging is unable to resolve nanoscale structures with dimensions smaller than 200–300 nm due to the fundamental physical limitations imposed by diffraction. ExM offers faster imaging speeds as compared to super-resolution methods, and enhanced antibody efficiency due to the decrowding effect generated by expanding biomolecules. The original ExM could resolve the specimen at 70 nm, however, new variants such as iterative ExM, 10X ExM microscopy, and nine-fold microscopy can resolve down to 15 to 30 nm, comparable to super-resolution microscopes. Method- The Paper-MAP version of expansion microscopy, a modified MAP method that allows immunostaining and expansion within two days was employed. The procedure involved staining floating mouse brain sections and incubating with a Paper-MAP cocktail (consisting of TEMED and sodium acrylate) and ammonium persulfate solution. The hydrogel matrix was created in situ through the crosslinking of sodium acrylate and bisacrylamide, forming a dense polyelectrolyte hydrogel. A denaturing solution was used to mechanically homogenize the sample and then expanded using deionized water. The pre and post-expanded sections were imaged using a Zeiss LSM 510 confocal microscope. Results- Following the addition of the monomer solution, the expansion procedure produced a 2 fold increase in size. This was followed by an evident 4 to 5 fold increase after the expansion was completed. We compared the pre-expansion image to the post-expansion image and observed intricate and detailed structures with significantly enhanced resolution that were previously indistinguishable in the pre-expansion section using confocal microscope. Conclusion- Expansion microscopy is a versatile and accessible imaging technique that resulted in significant improvements in imaging the microscopic configuration of the mouse brain. Its broad application offers a powerful tool for biological research in diverse organisms.Item Tractography as a method for mapping brain connectivity(2024-03-21) Spann, Claire; Yang, Shaohua; Liu, Ran; Berry, Raymond; Ampofo, Hannah; Colon-Perez, LuisPurpose. Mapping the brain and its complex connectivity has proved a challenging feat for neuroscience, though with the development of diffusion tensor imaging and tractography, we are one step closer to understanding brain anatomical connections. This method utilizes diffusion-weighted magnetic resonance imaging, which takes advantage of the Brownian motion of water molecules, to produce a diffusion tensor. In the white matter of the brain, diffusion varies in direction due to cellular membranes and myelin, and the diffusion tensor measures this anisotropic diffusivity to indicate possible tissue orientation. The generalized q-sampling imaging tractography method, developed by Frank Yeh in 2010, uses the diffusion tensor to approximate the course of white matter tracts and can be used to determine the exact location and termination of white matter bundles to assess connectivity between and within different brain regions. Despite limitations that decrease the accuracy of white matter tracking, tractography remains the only method to visualize white matter trajectories in vivo and non-invasively. Though commonly used for human diffusion-weighted images, here we verify tractography as a method to visualize and measure white matter trajectories in the rat brain. Methods. A male 3-month Sprague Dawley rat was used to acquire DWI images that were analyzed using DSI Studio. The DWI was superimposed with the corresponding T2W image and regions of interest (ROIs) were drawn in the corpus callosum and were applied via the built-in Waxholm Space rat atlas. Fiber tracking was seeded from the ROI, and fractional anisotropy, quantitative anisotropy, isotropy, mean diffusivity, axial diffusivity, and radial diffusivity was calculated at each ROI by DSI software. Results. Tractography of the corpus callosum was easily visualized using both drawn and atlas-applied ROIs. Fiber tractography from both ROIs included fibers from the internal and external capsules to ensure the integrity of all corpus callosum fibers. Diffusion metrics were not drastically different between the two seeding methods. Conclusion. This study presents tractography as a tool for visualizing white matter tracts and quantifying different diffusion metrics. Using both hand-drawn regions and regions from the rat atlas, white matter tracts in diseased brains can be compared to controls to measure several aspects of pathology, such as edema, axonal integrity, and axonal density. One application includes the imaging and quantification of both acute and chronic stroke, which exhibit different pathologies that can be visualized and measured with diffusion metrics, allowing for more precise targets of therapy. The use of tractography in adjunct with other established methods can improve the understanding of disease and assist in the development of better treatment.