Design and development of a multimodal nonlinear optical microscope for applications in biosciences

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Date
2024-12-16
Authors
Montero Collado, Vladimir
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Abstract

Nonlinear optical microscopy is an expanding field in the area of optical microscopy applied to biosciences. Methods such as Two-Photon Excited Fluorescence (2PEF or TPEF), Second Harmonic Generation (SHG), Third Harmonic Generation (THG), and Coherent Anti-Stokes Raman Scattering (CARS) constitute an improvement over classical (single-photon) fluorescence microscopy. With them, the need for the use of exogenous labelling agents disappears together with issues such as phototoxicity and photobleaching. These techniques can provide detailed structural images and chemical information from unstained samples. The laser wavelengths used in these methods penetrate deeper into the biological tissues and allow us to obtain images with sub-micron lateral and axial resolution. In the present work, a multimodal nonlinear optical microscope was designed and developed. In the instrument, the following nonlinear methods were implemented: 2PEF, SHG, THG with CARS being added in the near future. A compact, reliable, and cost-efficient Yb:KGW laser was used as a light source. The low energy (12 nJ) per pulse decreases the amount of damage to the surrounding sample areas. The ultrashort laser pulses provide us with a broad spectrum that makes it easier to find lines that coincide with the absorption spectra of different biological materials. The software for controlling the instrument was also developed in two versions (single-tile and multi-tile). The multi-tile version allows the scanning of larger sample areas by joining several tiles. Finally, conditions were created for future implementation of CARS using the Yb:KGW radiation to provide the Stokes beam and a Photonic Crystal Fiber to generate the pump beam.

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Keywords
Nonlinear Optical Microscopy, Nonlinear Optical Imaging, Second Harmonic Generation, Third Harmonic Generation, Two-Photon Excited Fluorescence, Coherent Anti-Stokes Raman Scattering, Multimodal, Laser Scanning Microscopy
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