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.

Short-circuited neuron: a note.

Here, we demonstrate in a direct electrophysiological experiment that a neuron can form electrical connections to itself. An isolated identified neuron with a long axon was plated in culture and the axon was looped so that its distal end contacted the cell body. After two days in culture, the cell body and the axon were both impaled with microelectrodes and the axon segment between the recording electrodes was cut. Electrotonic coupling was revealed between the separated cell compartments immediately after axon transection. In contrast to an earlier publication [Guthrie P. B. et al. (1994) J. Neurosci. 14, 1477-1485], no constraints on the formation of the electrical connections between different parts of the same neuron were revealed in our experiments.Thus, these experiments demonstrate that in vitro culture of a single neuron can form reflexive electrical connections which may strongly affect the basic properties of the neuron and should be taken into account in both experimental and model electrophysiological studies.