30 September 2005 Applications of nonlinear microscopy for studying the structure and dynamics in biological systems
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Laser scanning nonlinear optical microscopy is used to study structure and dynamics of cellular and sub-cellular structures in vivo. Under tight focusing conditions with a high numerical aperture objective, nonlinear optical signals such as third harmonic generation (THG), second harmonic generation (SHG), and multiphoton excitation fluorescence (MPF) are simultaneously produced. MPF is extensively used in biological imaging. Unfortunately, fluorescence is accompanied by heat dissipation in the sample and photobleaching effects. On the other hand, parametric processes such as SHG and THG are free of photobleaching since they involve only virtual electronic states where there is no transfer of energy into the medium. There are many naturally occurring structures that exhibit harmonic generation effects, and hence, do not require dyes that can potentially disrupt the normal functionality of the system. SHG is efficiently generated in non-centrosymmetric media, such as chiral structures and interfaces. The THG signal is generated due to a break in symmetry at interfaces and can be enhanced by the presence of multilamellar structures, as in the mitochondria or chloroplasts. Many interesting biological processes, such as signal transduction in neurons or ATP synthesis in mitochondria, involve the movement of ions across membranes. THG and SHG are sensitive to changing electric potential gradients, and hence are ideally suited for dynamical investigations of these biological processes. The present work will expose the structural factors and conditions that influence THG and SHG generation efficiencies in biological samples. Examples of visualizing chloroplasts and mitochondria will illustrate the advantages of harmonic generation microscopy for studying structural and functional properties of the in vivo systems.
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Nicole Prent, Nicole Prent, Richard Cisek, Richard Cisek, Catherine Greenhalgh, Catherine Greenhalgh, Raymond Sparrow, Raymond Sparrow, Neeresh Rohitlall, Neeresh Rohitlall, Maike-Svenja Milkereit, Maike-Svenja Milkereit, Chantal Green, Chantal Green, Virginijus Barzda, Virginijus Barzda, "Applications of nonlinear microscopy for studying the structure and dynamics in biological systems", Proc. SPIE 5971, Photonic Applications in Nonlinear Optics, Nanophotonics, and Microwave Photonics, 597106 (30 September 2005); doi: 10.1117/12.628801; https://doi.org/10.1117/12.628801

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