Abstract
Laser scanning microscopy using high-peak-power ultrashort near infrared light pulses can visualize biological microstructures by utilizing non-linear optical processes, such as multi-photon excitation and sum frequency generation. Here we introduced a polarization-resolving detection methodology for a laser scanning microscopy system equipped with a spinning disk confocal scanner. The developed system achieved high-speed intravital imaging of living tissues with resolving their signals to orthogonally polarized components. First, we applied the system to a liposomal vesicle labeled with the fluorescent lipophilic dye and confirmed the orientation map of the lipid bilayer. Next, by detecting polarization-resolved second harmonic generation signals, the structural orientations of the collagen fibers in fixed mouse tissues were visualized without exogenous or genetic fluorophore labeling. Finally, we demonstrated in vivo polarization-resolved second harmonic generation imaging of the collagen fibers in the mouse skeletal muscles at a 56-Hz temporal resolution. We expect that our developed methodology can achieve real-time visualization, thus, revealing the conformational changes of supramolecular structures in living animals.
Highlights
Second harmonic generation (SHG) is a non-linear optical process that can be applied for bioimaging [1, 2]
We developed a novel polarization-resolved imaging methodology based on the TPM-SD system
AG and KO conducted the experiments and image acquisitions
Summary
Second harmonic generation (SHG) is a non-linear optical process that can be applied for bioimaging [1, 2]. The probability of SHG occurring largely depends on the relative orientation between the polarization of the incident light beam and the orientation axis of the targeted molecules. Polarization-based SHG imaging, the concept of which is based on analyzing the polarization anisotropy of SHG light or SHG signal intensity as a function of the polarization state of the incident light beam, can be. Most of polarizationbased SHG microscopy requires a specialized optical setup to irradiate the sample with the excitation laser light beam, capture, and/or analyze the SHG light signals
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