Multimodal nonlinear-optical imaging of nucleoli.

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.

Cell-specific three-photon-fluorescence brain imaging: neurons, astrocytes, and gliovascular interfaces.

We present brain imaging experiments on rat cortical areas, demonstrating that, when combined with a suitable high-brightness, cell-specific genetically encoded fluorescent marker, three-photon-excited fluorescence (3PEF), enables subcellular-resolution, cell-specific 3D brain imaging that is fully compatible and readily integrable with other nonlinear-optical imaging modalities, including two-photon-fluorescence and harmonic-generation microscopy. With laser excitation provided by sub-100-fs, 1.25-µm laser pulses, cell-specific 3PEF from astrocytes and their processes detected in parallel with a three-photon-resonance-enhanced third harmonic from blood vessels is shown to enable a high-contrast 3D imaging of gliovascular interfaces.

Stain-free subcellular-resolution astrocyte imaging using third-harmonic generation.

We demonstrate stain-free, high-contrast, subcellular-resolution imaging of astroglial cells using epi-detected third-harmonic generation (THG). The astrocyte-imaging capability of THG is verified by colocalizing THG images with fluorescence images of astrocytes expressing a genetically encodable fluorescent reporter. We show that THG imaging with an optimized point-spread function can reliably detect significant subcellular features of astrocytes, including cell nuclei, as well as the soma shape and boundaries.