Subcellular Light Activation, Control And Manipulation Using A Dynamically Reconfigurable Nanopatterning System

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Subcellular Light Activation, Control And Manipulation Using A Dynamically Reconfigurable Nanopatterning System

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Title: Subcellular Light Activation, Control And Manipulation Using A Dynamically Reconfigurable Nanopatterning System
Author: Joshi, Amruta Rajiv
Abstract: Cellular communication is the basis of all cellular activities and functions. Cell signaling is crucial in the processes of tissue maintenance, tissue development and homeostasis. Errors in transmission or interpretation of these signals can result in the onset of diseases like cancer. Efforts have been made to probe into the communication between cells, and the extracellular signals that cells transmit to each other. There is however, a necessity to understand cell communication within the cellular components themselves and to be able to perform a nanoscale analysis of the cell organelle behavior. Many cellular mechanisms involve processes at the subcellular level, and many disorders too, are organelle specific. Cell interaction with submicron structures in the cell has not been well understood due to a lack of methods to achieve precise submicron resolution cell patterning, control and probing. Therefore an organelle level perturbation apparatus will be a boon to the investigation of how organelle specific disorders occur. We constructed a nanopatterning system, as an application of nanotechnology to biology, born out of the need to study nanoscale subcellular events and manipulate individual cell organelles. This system is a photoactivated perturbation apparatus that allows for submicron control of the cell machinery. The Digital Micromirror Device from Texas Instruments coupled with an epifluorescence microscope, form the major components of this nanopatterning system. Ultraviolet light was directed to fall on the DMD surface, which generated digital light masks to specifically illuminate the cell organelles while not illuminating others and vice versa. This system thus enables photoactivated subcellular and localized control of the cell machinery. This thesis work presents the design, optimization and characterization of the nanopatterning array system used to demonstrate the varying sensitivity of individual cell components to ultraviolet light. The desired environmental conditions, optimum dye concentrations used and the system resolution are discussed. Experiments investigating the severity of damage caused within a human fibroblast cell in response to local cytoplasm and local nucleus UVA irradiation, and a relation between these damage sites are then described. Our data demonstrates that the cell cytoplasm is about three times more sensitive to UVA damage than the cell nucleus. Experiments involving UVA light illumination of only the cytoplasmic membrane cause the fibroblast cells to sense and avoid the UV illumination by retracting their body from the site of illumination. The average cytoplasmic membrane retraction velocity ranges from about 2.6 µm/min to 9.3 µm/min for a UVA intensity of 3.6mW/cm2. Our nanopatterning system provides new avenues for light activated organelle specific treatments for cases that call for localized targets. This technique can aid research in the treatment of diseases, in which the functions of an intracellular organelle are impaired and also those disorders in which interorganelle communication is defective.
Date: 2008-09-17

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