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Multimodal nonlinear-optical imaging of nucleoli

M. S. Pochechuev, A. A. Lanin, I. V. Kelmanson, A. S. Chebotarev, E. S. Fetisova, D. S. Bilan, E. K. Shevchenko, A. A. Ivanov, A. B. Fedotov, V. V. Belousov, and A. M. Zheltikov

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Abstract

Multimodal nonlinear microscopy combining third-harmonic generation (THG) with two- and three-photon-excited fluorescence (2PEF and 3PEF) is shown to provide a powerful resource for high-fidelity imaging of nucleoli and nucleolar proteins. We demonstrate that, with a suitably tailored genetically encoded fluorescent stain, the 2PEF/3PEF readout from specific nucleolar proteins can be reliably detected against the extranucleolar 2PEF/3PEF signal, enabling high-contrast imaging of the key nucleolar ribosome biogenesis components, such as fibrillarin. THG is shown to provide a versatile readout for unstained nucleolus imaging in a vast class of biological systems as different as neurons in brain slices and cultured HeLa cells.

© 2021 Optical Society of America

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