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Differential Polarization Nonlinear Optical Microscopy with Adaptive Optics Controlled Multiplexed Beams
Department of Physics and Institute for Optical Sciences, University of Toronto, 60 St. George Street, Toronto, ON M5S 1A7, Canada
Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, ON L5L 1C6, Canada
These authors contributed equally to this work.
* Author to whom correspondence should be addressed.
Received: 16 July 2013; in revised form: 21 August 2013 / Accepted: 30 August 2013 / Published: 9 September 2013
Abstract: Differential polarization nonlinear optical microscopy has the potential to become an indispensable tool for structural investigations of ordered biological assemblies and microcrystalline aggregates. Their microscopic organization can be probed through fast and sensitive measurements of nonlinear optical signal anisotropy, which can be achieved with microscopic spatial resolution by using time-multiplexed pulsed laser beams with perpendicular polarization orientations and photon-counting detection electronics for signal demultiplexing. In addition, deformable membrane mirrors can be used to correct for optical aberrations in the microscope and simultaneously optimize beam overlap using a genetic algorithm. The beam overlap can be achieved with better accuracy than diffraction limited point-spread function, which allows to perform polarization-resolved measurements on the pixel-by-pixel basis. We describe a newly developed differential polarization microscope and present applications of the differential microscopy technique for structural studies of collagen and cellulose. Both, second harmonic generation, and fluorescence-detected nonlinear absorption anisotropy are used in these investigations. It is shown that the orientation and structural properties of the fibers in biological tissue can be deduced and that the orientation of fluorescent molecules (Congo Red), which label the fibers, can be determined. Differential polarization microscopy sidesteps common issues such as photobleaching and sample movement. Due to tens of megahertz alternating polarization of excitation pulses fast data acquisition can be conveniently applied to measure changes in the nonlinear signal anisotropy in dynamically changing in vivo structures.
Keywords: differential microscopy; nonlinear microscopy; multiharmonic generation; genetic algorithms; adaptive optics
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Samim, M.; Sandkuijl, D.; Tretyakov, I.; Cisek, R.; Barzda, V. Differential Polarization Nonlinear Optical Microscopy with Adaptive Optics Controlled Multiplexed Beams. Int. J. Mol. Sci. 2013, 14, 18520-18534.
Samim M, Sandkuijl D, Tretyakov I, Cisek R, Barzda V. Differential Polarization Nonlinear Optical Microscopy with Adaptive Optics Controlled Multiplexed Beams. International Journal of Molecular Sciences. 2013; 14(9):18520-18534.
Samim, Masood; Sandkuijl, Daaf; Tretyakov, Ian; Cisek, Richard; Barzda, Virginijus. 2013. "Differential Polarization Nonlinear Optical Microscopy with Adaptive Optics Controlled Multiplexed Beams." Int. J. Mol. Sci. 14, no. 9: 18520-18534.