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Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy
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Multimodal nonlinear-optical imaging of nucleoli.

M S Pochechuev, A A Lanin, I V Kelmanson

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    |July 30, 2021
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    Summary

    Multimodal nonlinear microscopy offers high-fidelity imaging of nucleoli and nucleolar proteins. This technique combines third-harmonic generation (THG) with two- and three-photon-excited fluorescence (2PEF/3PEF) for clear visualization.

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    Area of Science:

    • Biophysics
    • Cell Biology
    • Microscopy

    Background:

    • Nucleoli are crucial for ribosome biogenesis.
    • High-resolution imaging of nucleoli and their proteins is essential for understanding cellular function.
    • Existing imaging techniques face challenges in achieving high contrast and specificity for nucleolar components.

    Purpose of the Study:

    • To develop and demonstrate a multimodal nonlinear microscopy technique for high-fidelity imaging of nucleoli and nucleolar proteins.
    • To enable high-contrast visualization of specific nucleolar proteins, such as fibrillarin, involved in ribosome biogenesis.
    • To showcase the versatility of third-harmonic generation (THG) for unstained nucleolus imaging.

    Main Methods:

    • Utilizing a multimodal nonlinear microscopy approach combining third-harmonic generation (THG) with two- and three-photon-excited fluorescence (2PEF and 3PEF).
    • Employing a genetically encoded fluorescent stain tailored for specific nucleolar protein detection.
    • Applying the technique to image nucleoli and nucleolar proteins in biological samples, including neurons and HeLa cells.

    Main Results:

    • Demonstrated reliable detection of 2PEF/3PEF signals from specific nucleolar proteins against background signals.
    • Achieved high-contrast imaging of key ribosome biogenesis components like fibrillarin.
    • Showcased THG as a versatile method for unstained nucleolus imaging in diverse biological systems.

    Conclusions:

    • Multimodal nonlinear microscopy (THG, 2PEF, 3PEF) is a powerful tool for high-fidelity nucleolus and nucleolar protein imaging.
    • Genetically encoded fluorescent stains combined with 2PEF/3PEF enable specific, high-contrast visualization of nucleolar components.
    • THG microscopy provides a valuable approach for unstained nucleolus imaging across various cell types and tissues.