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Item Development of an active/adaptive laser scanning microscope(Montana State University - Bozeman, College of Engineering, 2018) Archer-Zhang, Christian Chunzi; Chairperson, Graduate Committee: David L. DickensheetsLaser scanning techniques such as confocal microscopy and two-photon excitation fluorescence microscopy (TPM) are powerful tools for imaging biological samples with high resolution, offering three-dimensional (3D) visualization of the behavior of cells in their natural environment. Traditionally, the 3D images are acquired from 2D image stacks with focusing depth controlled through mechanical movement of the specimen relative to the objective lens. The slow mechanical movement (~<20Hz) does not allow the spot of light to be scanned axially sufficiently fast to monitor cell:cell and cell:environment interactions in real time over hundreds of microns in all three dimensions. A fast focus control mirror supports agile scan patterns such as vertical or oblique planes or even arbitrary surfaces, minimizing the time and photo damage required to monitor features of interest within the 3D volume. Because aberrations cause image quality to decrease as the focal point of the beam penetrates deeper into the sample, adaptive optics can enhance resolution and contrast at depth for confocal microscopy and TPM. Combining a fast focus control mirror with a fast aberration correcting mirror leads to a flexible platform called the active/adaptive laser scanning microscope, capable of aberration-corrected beam scanning throughout a 3D volume of tissue. This opens up the possibility of fully corrected, variable-depth imaging along oblique sections or more complex user-defined surfaces within a single image frame.Item Dynamic performance of microelectromechanical systems deformable mirrors for use in an active/adaptive two-photon microscope(2016-12) Archer-Zhang, Christian Chunzi; Foster, Warren B.; Downey, Ryan D.; Arrasmith, Christopher L.; Dickensheets, David L.Active optics such as deformable mirrors can be used to control both focal depth and aberrations during scanning laser microscopy. If the focal depth can be changed dynamically during scanning, then imaging of oblique surfaces becomes possible. If aberrations can be corrected dynamically during scanning, an image can be optimized throughout the field of view. Here, we characterize the speed and dynamic precision of a Boston Micromachines Corporation Multi-DM 140 element aberration correction mirror and a Revibro Optics 4-zone focus control mirror to assess suitability for use in an active and adaptive two-photon microscope. Tests for the multi-DM include both step response and sinusoidal frequency sweeps of specific Zernike modes (defocus, spherical aberration, coma, astigmatism, and trefoil). We find wavefront error settling times for mode amplitude steps as large as 400 nm to be less than 52 mu s, with 3 dB frequencies ranging from 6.5 to 10 kHz. The Revibro Optics mirror was tested for step response only, with wavefront error settling time less than 80 mu s for defocus steps up to 3000 nm, and less than 45 mu s for spherical aberration steps up to 600 nm. These response speeds are sufficient for intrascan correction at scan rates typical of two-photon microscopy. (C) The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License.