Abstract: Provided herein are devices, methods, and systems for in situ gene sequencing of a target nucleic acid in a cell in an intact tissue. Methods of screening a candidate agent to determine whether the candidate agent modulates gene expression of a nucleic acid in a cell in an intact tissue are also provided herein.

Abstract: Various systems and methods are implemented for controlling stimulus of a cell. One such method is implemented for optical stimulation of a cell expressing an NpHR ion pump. The method includes the step of providing a sequence of stimuli to the cell. Each stimulus increases the probability of depolarization events occurring in the cell. Light is provided to the cell to activate the expressed NpHR ion pump, thereby decreasing the probability of depolarization events occurring in the cell.

Abstract: The present disclosure provides methods of modulating a feature of a behavioral state. The methods involve inhibiting or activating the activity of a bed nucleus of stria terminalis (BNST) neuron, a BNST subnucleus, or a neuronal output to or from a BNST neuron. Animals encounter environmental conditions that require rapid switching among different behavioral states to increase the likelihood of survival and reproduction.

Abstract: Provided herein are methods of inducing psychosis in animals using light-responsive opsins and methods of identifying or screening compounds that may be useful in treating psychosis.

Abstract: Compositions and methods are provided for TEMPEST (Target-Element Modification by Physical and Enduring Structural Transmutation), a method for creating durable structures in vivo in a cell-type and/or circuit specific manner via the use of insoluble polymers. TEMPEST provides a way to functionally remove cells while preserving their “shadow” for easy post-experiment detection and classification. The method of the invention are of particular interest for modifying neurons, which may be central nervous system or peripheral nervous system cells, however the approach may be applied to other cellular systems as well, either in culture system models or in animals.

Abstract: Methods for selectively stimulating a plurality of light-responsive neurons in a sample are provided. Methods according to certain embodiments include irradiating a sample comprising a plurality of light-responsive neurons with a plurality of holographic images that are each configured to stimulate one or more light-responsive neurons in the sample, wherein the holographic images are created by light projection system that includes a plurality of light sources; a plurality of optical adjustment components; a plurality of spatial light modulators; a controller; a processor; and a computer-readable medium comprising instructions that, when executed by the processor, cause the controller to operate the light sources, optical adjustment components and spatial light modulators to generate and display a plurality of holographic images; direct each of the holographic images to a projection location; and project the holographic images onto the sample at a rate greater than 1 kHz.

Abstract: Disclosed herein are systems and methods involving the use of magnetic resonance imaging and optogenetic neural stimulation. Aspects of the disclosure include modifying a target neural cell population in a first region of a brain to express light-responsive molecules. Using a light pulse, the light-responsive molecules in the target neural cell population are stimulated. Multiple regions of the brain are scanned via magnetic resonance imaging. The scans allow for observation of a neural reaction in response to the stimulation in at least one of the multiple regions of the brain.

Abstract: Provided herein is a method and system for imaging a biological sample. The method may include scanning a biological sample using one or more light sheets, where the biological sample is in a field of view of a microscope that includes an objective and a direction of observation of the objective defines a z-axis, where a point spread function of the microscope is elongated in the z-axial direction, and the biological sample is at a z-axial distance from the objective, thereby illuminating a plurality of z-axial slices of the biological sample, and recording a plurality of images corresponding to a plurality of z-axial slices of the sample, where the images are generated by light patterns emitted from the scanned biological sample, thereby generating an image stack that includes a plurality of images of the biological sample.

Abstract: The present disclosure provides nucleic acid activity-dependent expression vectors and activity-dependent expression cassettes for the activity-dependent expression of an encoded polypeptide. Also provided are recombinant adenoassociated viruses (AAV) containing an expression vector comprising an activity-dependent expression cassette for the activity-dependent expression of an encoded polypeptide by cells infected with the AAV vector. The present disclosure also provides methods for the activity-dependent labeling of cells in vitro or in vivo by introducing into the cells an expression vector containing an activity-dependent regulatory sequence driving expression of a labeling polypeptide. Also provided are methods for the activity-dependent control of cells in vitro or in vivo by introducing into the cells an expression vector containing an activity-dependent regulatory sequence driving expression of a light-responsive polypeptide.

Abstract: Methods and devices for conducting high-speed, high-resolution imaging of large intact tissue samples are provided. Aspects of the methods include placing a sample in an optically homogenous sample manipulation component, performing a calibration procedure to align a light sheet and a detection focal plane at a plurality of locations within the sample, and performing an imaging procedure on the sample to collect an image from each location. The collected images are reconstructed to form a three-dimensional image of the sample. Devices for carrying out the steps of the methods are also provided.

Abstract: According to one aspect and example, a method for facilitating cellular interactions in biological tissue provides controllable activation of a selected type of stem cell among a plurality of cell types present in the tissue. The method includes various steps including the introduction of a microbial opsin into a region of the tissue that includes a selected type of stem cell, by expressing the microbial opsin in the stem cell. A light source is then introduced near the stem cell, and the light source is used to controllably activate the light source to direct pulses of illumination from the light source to the selected type of stem cell, for selectively controlling the growth and development of the stem cell in a manner that is independent of the growth and development of the other types of cells.

Abstract: Provided herein is a method of optically recording neural activity in one or more regions of a target tissue. Also provided is a method of optically modulating the activity of a neural tissue. Further provided is a system that finds use in performing the present methods.

Abstract: Various systems and methods are implemented for in vivo use in a living animal. One such method involves stimulating target cells having light-responsive proteins and includes providing an elongated light-delivery structure in a narrow passageway in the animal, the elongated light-delivery structure having separately-activatable light sources located along the length of the elongated light-delivery structure. The method also includes activating less than all the light sources to deliver light to light-responsive proteins adjacent to the activated light sources along the length of the elongated light-delivery structure, thereby stimulating target cells in vivo.

Abstract: Aspects of the present disclosure include a method for visualizing a fiber-like structure in a biological specimen, the method comprising: clearing the biological specimen comprising a fiber-like structure, wherein the fiber-like structure is detectably labeled; illuminating the cleared biological specimen with two light sheets from a first side and a second side to produce an image volume, wherein the second side is opposite to the first side and wherein the image volume comprises a representation of the fiber-like structure; defining a plurality of voxels within the representation of the fiber-like structure; processing each of the plurality of voxels to estimate a plurality of principal fiber-like structure orientations; and defining a starting point on the representation of the fiber-like structure and propagating a plurality of streamlines from the starting point, according to the plurality of principal fiber-like structure.

Abstract: The present disclosure provides compositions and methods for controlling pain. The present disclosure provides methods for identifying agents that control pain.

Abstract: The present invention provides compositions and methods for light-activated cation channel proteins and their uses within cell membranes and subcellular regions. The invention provides for proteins, nucleic acids, vectors and methods for genetically targeted expression of light-activated cation channels to specific cells or defined cell populations. In particular the invention provides millisecond-timescale temporal control of cation channels using moderate light intensities in cells, cell lines, transgenic animals, and humans. The invention provides for optically generating electrical spikes in nerve cells and other excitable cells useful for driving neuronal networks, drug screening, and therapy.

Abstract: The present disclosure provides methods of preparing a biological specimen for imaging analysis, comprising fixing and clearing the biological specimen and subsequently analyzing the cleared biological specimen using microscopy. Also included are methods of quantifying cells, for example, active populations of cells in response to a stimulant. The present disclosure also provides devices for practicing the described methods. A flow-assisted clearing device provides rapid clearing of hydrogel-embedded biological specimens without the need of specialized equipment such as electrophoresis or perfusion devices.

Abstract: Stimulation of target cells using light, e.g., in vitro, is implemented using a variety of methods and devices. One example involves a vector for delivering a light-activated NpHR-based molecule comprising a nucleic acid sequence that codes for light-activated NpHR-based molecule and a promoter. Either a high expression of the molecule manifests a toxicity level that is less than about 75%, or the light-activated NpHR-based proteins are expressed using at least two NpHR-based molecular variants. Each of the variants characterized in being useful for expressing a light-activated NpHR-based molecule that responds to light by producing an inhibitory current to dissuade depolarization of the neuron. Other aspects and embodiments are directed to systems, methods, kits, compositions of matter and molecules for ion pumps or for controlling inhibitory currents in a cell (e.g., in in vivo and in vitro environments).

Abstract: A variety of methods, devices, systems and arrangements are implemented for stimulation of the peripheral nervous system. Consistent with one embodiment of the present invention, method is implemented in which light-responsive channels or pumps are engineered in a set of motor units that includes motor units of differing physical volumes. Optical stimuli are also provided to the light-responsive channels or pumps at an optical intensity that is a function of the size of motor units to be recruited. In certain implementations, the intensity of the optical stimuli is increased so as to recruit increasingly larger motor units.

Abstract: A variety of applications, systems, methods and constructs are implemented for use in connection with screening of ion-channel modulators. Consistent with one such system, drug candidates are screened to identify their effects on cell membrane ion channels and pumps. The system includes screening cells having light responsive membrane ion switches, voltage-gated ion switches and fluorescence producing voltage sensors. A chemical delivery device introduces the drug candidates to be screened. An optical delivery device activates the light responsive ion switches. An optical sensor monitors fluorescence produced by the voltage sensors. A processor processes data received from the optical sensor. A memory stores the data received from the optical sensor.