Three-dimensional imaging of cells and sub cellular structures has enormous potential in studies on cell physiology and medicine, since it enables the observation of cell behavior in a variety of environments. Many three-dimensional imaging techniques to date require cells to be stained or dyed to produce images of structures that are normally not observable. We propose to develop a three-dimensional optical coherence phase-contrast microscope (OCPM) based on Spectral Domain Optical Coherence Tomography (SD-OCT) technology that can reveal three-dimensional structures and dynamics of cells in their natural state by measuring depth resolved phase differences with high sensitivity. The OCPM will be integrated with Two Photon Microscopy (TPM) into a standard inverted microscope. Phase sensitivity, lateral resolution, and structural imaging capability will be evaluated on calibration objects and cells, where nanometer axial and sub-micron lateral resolution are targeted. Interpretation of OCPM images will be investigated by simultaneous two-photon fluorescence imaging of the same specimen. The potential of OCPM will be studied by imaging cell responses to external stimuli. The ability for deep tissue imaging (250 ?m) and the exploitation of phase sensitivity will be investigated by monitoring action potentials in axons embedded in larger nerve bundles. Neural activity has been demonstrated to induce rapid changes in nerves (swelling) on the order of 1-3 nanometers. The spatio-temporal resolution of OCPM is capable of detecting these rapid changes during action potential propagation. OCPM will serve as a powerful imaging tool for processes in cell biology and will enhance the understanding of the structure, function, and the behavior of living cells. The overall goal of this research is A) to develop an OCPM system for 3 dimensional high resolution phase contrast imaging of cells, B) to determine the phase resolution and sectioning capability of OCPM, C) to investigate the potential of OCPM for the study of cell dynamics in response to external stimuli, D) to non- invasively measure action potentials of axons within nerve bundles.
Three-dimensional imaging of cells and sub cellular structures has enormous potential in studies on cell physiology and medicine, since it enables the observation of cell behavior in a variety of environments. Many three-dimensional imaging techniques to date require cells to be stained or dyed to produce images of structures that are normally not observable. We will develop a new form of microscopy (OCPM) that is able to detect minute changes in cells on the order of nanometers. OCPM will serve as a powerful imaging tool for processes in cell biology and will enhance the understanding of the structure, function, and the behavior of living cells.
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