Guntupalli, Jyothi Swaroop (2007-12). Physical sectioning in 3D biological microscopy. Master's Thesis. Thesis uri icon

abstract

  • Our 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.
  • Our 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.

publication date

  • December 2007