Multimodal nonlinear-optical imaging of nucleoli.

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.