Browsing by Subject "Physical Sectioning"
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Item Physical sectioning in 3D biological microscopy(2009-05-15) Guntupalli, Jyothi SwaroopOur ability to analyze the microstructure of biological tissue in three dimensions (3D) has proven invaluable in modeling its functionality, and therefore providing a better understanding of the basic mechanisms of life. Volumetric imaging of tissue at the cellular level, using serial imaging of consecutive tissue sections, provides such ability to acquire microstructure in 3D. Three-dimensional light microscopy in biology can be broadly classified as using either optical sectioning or physical sectioning. Due to the inherent limitations on the depth resolution in optical sectioning, and the recent introduction of novel techniques, physical sectioning has become the sought-out method to obtain high-resolution volumetric tissue structure data. To meet this demand with increased processing speed in 3D biological imaging, this thesis provides an engineering study and formulation of the tissue sectioning process. The knife-edge scanning microscopy (KESM), a novel physical sectioning and imaging instrument developed in the Brain Networks Laboratory at Texas A&M University, has been used for the purpose of this study. However, the modes of characterizing chatter and its measurement are equally applicable to all current variants of 3D biological microscopy using physical sectioning. We focus on chatter in the physical sectioning process, principally characterizing it by its geometric and optical attributes. Some important nonlinear dynamical models of chatter in the sectioning process, drawn from the metal machining literature, are introduced and compared with observed measurements of chatter in the tissue cutting process. To understand the effects of the embedding polymer on tissue sectioning, we discuss methods to characterize the polymer material and present polymer measurements. Image processing techniques are introduced as a method to abate chatter artifacts in the volumetric data that has already been obtained. Ultra-precise machining techniques, using (1) free-form nanomachining and (2) an oscillating knife, are introduced as potential ways to acquire chatter-free higher-resolution volumetric data in less time. Finally, conclusions of our study and future work conclude the thesis. In this thesis, we conclude that to achieve ultrathin sectioning and high-resolution imaging, embedded plastic should be soft. To overcome the machining defects of soft plastics, we suggested free-form nanomachining and sectioning with an oscillating knife.Item Reducing Chatter in Knife-Edge Scanning Microscopy(2014-12-18) Shah, Raj SunilThe Knife-Edge Scanning Microscope (KESM) employs a novel form of physical sectioning microscopy: Imaging of tissue while sectioning. KESM was developed in the Brain Networks Lab (BNL) at Texas A&M University. The KESM has been used to section animal tissue embedded in a plastic block using a diamond knife. During each cut, the plastic block containing the tissue contacts the knife and that impact induces vibrations, known as knife chatter. These vibrations introduce noise in the image captured from the cut slice. This research is aimed at determining a metric to quantify knife chatter in the images acquired using the KESM, and to test if the use of a vibrating knife reduces knife chatter. Knife chatter appears as repeated parallel streaks in the images. A quantitative characterization of knife chatter is difficult since there is no regular pattern with which it appears. Having no regular pattern makes it very challenging to detect the chatter using automated programs. Observing the Fast Fourier Transforms of the images tells us that a narrow vertical band around the central vertical axis contains information exclusively about the chatter, while most of the information about the object in the image is outside the vertical band that represents the knife chatter. Using this information, we can quantitatively characterize knife chatter as a ratio of (1) the width of the region in the Fast Fourier Transform that corresponds to the knife chatter, and (2) the width of the region that corresponds to the object. Determining if the introduction of vibrations in the KESM diamond knife affects the amount of knife chatter present in the images was achieved by sectioning specimens of nissl-stained zebra-fish embryos embedded into araldite blocks, at different knife vibration frequencies. External sinusoidal wave vibrations were introduced in the KESM knife from a signal generator throughout the sectioning process. These electrical signals were converted to mechanical waves at the tip of the KESM knife blade. Performing such experiments at different oscillation frequencies enabled us to compare data using the metric described above. The results indicate that sectioning tissues with external vibrations does affect the amount of total data bandwidth taken up just by the chatter, and in some cases, reduces the relative width of the bandwidth taken up by the chatter.