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.
Highlights
Biological samples often contain highly ordered molecular structures and microcrystalline aggregates that play important structural and physiological roles
The second and third harmonic generation signals generated at the focus of the objective in the sample are collected in the forward direction by a custom-built UV-transmitting objective
This study shows that both the molecular organization in ordered structures and the orientation of the marker-molecules within the labeled structure can be conveniently examined with differential polarization microscopy
Summary
Biological samples often contain highly ordered molecular structures and microcrystalline aggregates that play important structural and physiological roles Examples of these structures are ordered protein assemblies, such as collagen fibers and myosin filaments in striated muscle, as well as polysaccharide structures, such as starch granules and cellulose fibers. The adaptive optics mitigate the problem of beam overlap, which is usually required for a multibeam system This is especially important and difficult if multiplexed beam differential polarization microscopy is to be used for super-resolution investigations. We present a multibeam differential polarization microscopy setup and provide details on the implementation of the deformable mirrors as well as their performance in overlapping the two excitation beams, while simultaneously correcting for optical aberrations in the setup. We show that differential polarization microscopy with multiplexed beams is a powerful technique for structural investigations of molecular orientations in ordered structures and sidesteps common issues such as photobleaching and sample movement artifacts in polarization measurements
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