Implantable graded-index fibers for neural-dynamics-resolving brain imaging in awake mice on an air-lifted platform.

We demonstrate a versatile framework for cellular brain imaging in awake mice based on suitably tailored segments of graded-index (GRIN) fiber. Closed-form solutions to ray-path equations for graded-index waveguides are shown to offer important insights into image-transmission properties of GRIN fibers, suggesting useful recipes for optimized GRIN-fiber-based deep-brain imaging. We show that the lengths of GRIN imaging components intended for deep-brain studies in freely moving rodents need to be chosen as a tradeoff among the spatial resolution, the targeted imaging depth and the degree of fiber-probe invasiveness.

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.

Enhanced-contrast two-photon optogenetic pH sensing and pH-resolved brain imaging.

We present experiments on cell cultures and brain slices that demonstrate two-photon optogenetic pH sensing and pH-resolved brain imaging using a laser driver whose spectrum is carefully tailored to provide the maximum contrast of a ratiometric two-photon fluorescence readout from a high-brightness genetically encoded yellow-fluorescent-protein-based sensor, SypHer3s. Two spectrally isolated components of this laser field are set to induce two-photon-excited fluorescence (2PEF) by driving SypHer3s through one of two excitation pathways-via either the protonated or deprotonated states of its chromophore. With the spectrum of the laser field accurately adjusted for a maximum contrast of these two 2PEF signals, the ratio of their intensities is shown to provide a remarkably broad dynamic range for pH measurements, enabling high-contrast optogenetic deep-brain pH sensing and pH-resolved 2PEF imaging within a vast class of biological systems, ranging from cell cultures to the living brain.

Multisite cell- and neural-dynamics-resolving deep brain imaging in freely moving mice with implanted reconnectable fiber bundles.

We demonstrate a reconnectable implantable ultraslim fiber-optic microendoscope that integrates a branching fiber bundle (BFB) with gradient-index fiber lenses, enabling a simultaneous fluorescence imaging of individual cells in distinctly separate brain regions, including brain structures as distant as the neocortex and hippocampus. We show that fluorescence images of individual calcium-indicator-expressing neurons in the brain of freely moving transgenic mice can be recorded, via the implanted BFB probe, in parallel with time- and cell-resolved traces of calcium signaling, thus enabling correlated circuit-dynamics studies at -multiple sites within the brain of freely moving animals.

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.

Two- and three-photon absorption cross-section characterization for high-brightness, cell-specific multiphoton fluorescence brain imaging.

We demonstrate an accurate quantitative characterization of absolute two- and three-photon absorption (2PA and 3PA) action cross sections of a genetically encodable fluorescent marker Sypher3s. Both 2PA and 3PA action cross sections of this marker are found to be remarkably high, enabling high-brightness, cell-specific two- and three-photon fluorescence brain imaging. Brain imaging experiments on sliced samples of rat's cortical areas are presented to demonstrate these imaging modalities.

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.

Nonlinear-optical stain-free stereoimaging of astrocytes and gliovascular interfaces.

Methods of nonlinear optics provide a vast arsenal of tools for label-free brain imaging, offering a unique combination of chemical specificity, the ability to detect fine morphological features, and an unprecedentedly high, subdiffraction spatial resolution. While these techniques provide a rapidly growing platform for the microscopy of neurons and fine intraneural structures, optical imaging of astroglia still largely relies on filament-protein-antibody staining, subject to limitations and difficulties especially severe in live-brain studies. Once viewed as an ancillary, inert brain scaffold, astroglia are being promoted, as a part of an ongoing paradigm shift in neurosciences, into the role of a key active agent of intercellular communication and information processing, playing a significant role in brain functioning under normal and pathological conditions.

Reconnectable fiberscopes for chronic in vivo deep-brain imaging.

Reconnectable bundles consisting of thousands of optical fibers are shown to enable high-quality image transmission, offering a platform for the creation of implantable fiberscopes for minimally invasive in vivo brain imaging. Experiments on various lines of transgenic mice verify the performance of this fiberscope as a powerful tool for chronic in vivo neuroimaging using genetically encoded calcium indicators, neuronal activity markers as well as axon growth regulators and brain-specific protein drivers in deep regions of live brain.

Two-photon imaging of fiber-coupled neurons.

Optical coupling between a single, individually addressable neuron and a properly designed optical fiber is demonstrated. Two-photon imaging is shown to enable a quantitative in situ analysis of such fiber-single-neuron coupling in the live brain of transgenic mice. Fiber-optic interrogation of single pyramidal neurons in mouse brain cortex is performed with the positioning of the fiber probe relative to the neuron accurately mapped by means of two-photon imaging.

Quantitative cognitive-test characterization of reconnectable implantable fiber-optic neurointerfaces for optogenetic neurostimulation.

Cognitive tests on representative groups of freely behaving transgenic mice are shown to enable a quantitative characterization of reconnectable implantable fiber-optic neurointerfaces for optogenetic neurostimulation. A systematic analysis of such tests provides a robust quantitative measure for the cognitive effects induced by fiber-optic neurostimulation, validating the performance of fiber-optic neurointerfaces for long-term optogenetic brain stimulations and showing no statistically significant artifacts in the behavior of transgenic mice due to interface implantation.

Three-dimensional fiber-optic readout of single-neuron-resolved fluorescence in living brain of transgenic mice.

A bundle of individually addressable optical fibers is shown to enable three-dimensional optical readout from single neurons in live brain, as well as in intact brain extracted from transgenic mice. With individual fibers in the bundle being only a few microns in diameter, single neurons are readily resolved in brain images transmitted by the fiber bundle. The third dimension is added by scanning the fiber in the longitudinal direction, with the fluorescence return read out from only one of the fibers in the bundle.