Abstract: Methods and compositions to activate a genetically designated target cell (or population of target cells) artificially, in vivo or in vitro, employ triggering of heterologous stimulus-gated ion channels to activate the cells. The stimulus-gated ion channels are suitably TRPV1, TRPM8 or P2X2. A stimulus which leads to opening or “gating” of the ion channel can be a physical stimulus or a chemical stimulus. Physical stimuli can be provided by heat, or mechanical force, while chemical stimuli can suitably be a ligand, such as capsaicin for TRPV1 or ATP for P2X2, or a “caged ligand,” for example a photolabile ligand derivative, in which case a physical signal in the form of light is used to provide the chemical signal. Selective activation of the cell may be used for various applications including neuronal and neuroendocrine mapping and drug screening.

Abstract: Cells are rendered sensitive to stimulation by introducing into a non-photoreceptor cell nucleic acid sequences encoding at least an opsin gene product, an arrestin gene product, and the alpha subunit of the heterotrimeric G protein of the Gq family. The introduced sequences are expressed by the cell to yield at least the opsin gene product, the arrestin gene product, and the alpha subunit of the heterotrimeric G protein of the Gq family. Retinal or a derivative thereof capable of bonding with the opsin gene product to form a rhodopsin is provided to the cell. The cell is then irradiated with light having a wavelength capable of converting the rhodopsin to metarhodopsin. The conversion of rhodopsin to metarhodopsin triggers a cascade of intracellular responses within the cell resulting in an increased intracellular concentration of IP3 and calcium ions.

Abstract: The invention relates to methods and compositions which utilize the emission of light to monitor changes in microenvironments involving cells. The invention is especially useful for monitoring exocytotic activity such as detecting quantal release of synaptic vesicles. Fusion proteins of Cypridina luciferase and synaptotagmin-I or VAMP/synaptobrevin-2 were targeted to synaptic vesicles and, upon exocytosis, formed light-emitting complexes with luciferin present in the extracellular medium. Photon emissions in the presence of a depolarizing stimulus can be observed with these systems. pH-sensitive mutants of green fluorescent protein are also provided, which are useful for visualizing exocytosis and for imaging and measuring the pH of intracellular compartments.

Abstract: Method and apparatus for steering a beam of light. The method and apparatus are based on the discovery that the spectral dispersion of multi-chromatic light pulses by an acousto-optical deflector can be significantly ameliorated by positioning a dispersive element, such as a prism, along the path of the multi-chromatic light pulses in such a way that the dispersive element disperses the multi-chromatic light pulses in a direction opposite to the spectral dispersion caused by the acousto-optical deflector. The dispersive element may be positioned either before or after the acousto-optical deflector. The method and apparatus are particularly well-suited for use with ultrashort laser pulses in the visible and infrared ranges having a bandwidth of up to about 40 nm. The method and apparatus have applicability in, among other things, multi-photon laser scanning microscopy.

Abstract: The invention relates to methods and compositions which utilize the emission of light to monitor changes in microenvironments involving cells. The invention is especially useful for monitoring exocytotic activity such as detecting quantal release of synaptic vesicles. Fusion proteins of Cypridina luciferase and synaptotagmin-I or VAMP/synaptobrevin-2 were targeted to synaptic vesicles and, upon exocytosis, formed light-emitting complexes with luciferin present in the extracellular medium. Photon emissions in the presence of a depolarizing stimulus can be observed with these systems. pH-sensitive mutants of green fluorescent protein are also provided, which are useful for visualizing exocytosis and for imaging and measuring the pH of intracellular compartments.

Abstract: Methods and compositions to activate a genetically designated target cell (or population of target cells) artificially, in vivo or in vitro, employ triggering of heterologous stimulus-gated ion channels to activate the cells. The stimulus-gated ion channels are suitably TRPV1, TRPM8 or P2X2. A stimulus which leads to opening or “gating” of the ion channel can be a physical stimulus or a chemical stimulus. Physical stimuli can be provided by heat, or mechanical force, while chemical stimuli can suitably be a ligand, such as capsaicin for TRPV1 or ATP for P2X2, or a “caged ligand,” for example a photolabile ligand derivative, in which case a physical signal in the form of light is used to provide the chemical signal. Selective activation of the cell may be used for various applications including neuronal and neuroendocrine mapping and drug screening.

Abstract: The invention relates to methods and compositions which utilize the emission of light to monitor changes in microenvironments involving cells. The invention is especially useful for monitoring exocytotic activity such as detecting quantal release of synaptic vesicles. Fusion proteins of Cypridina luciferase and synaptotagmin-I or VAMP/synaptobrevin-2 were targeted to synaptic vesicles and, upon exocytosis, formed light-emitting complexes with luciferin present in the extracellular medium. Photon emissions in the presence of a depolarizing stimulus can be observed with these systems. pH-sensitive mutants of green fluorescent protein are also provided, which are useful for visualizing exocytosis and for imaging and measuring the pH of intracellular compartments.

Abstract: Method for rendering a cell sensitive to stimulation. In a preferred embodiment, the method comprises introducing, into a cell, nucleic acid sequences encoding at least an opsin gene product, an arrestin gene product, and the alpha subunit of the heterotrimeric G protein of the Gq family. The introduced nucleic acid sequences are then expressed by the non-photoreceptor cell to yield at least the opsin gene product, the arrestin gene product, and the alpha subunit of the heterotrimeric G protein of the Gq family. Retinal or a derivative thereof capable of bonding with the opsin gene product to form a rhodopsin is then provided to the non-photoreceptor cell. The non-photoreceptor cell is then irradiated with light having a wavelength capable of converting the rhodopsin to metarhodopsin. The conversion of rhodopsin to metarhodopsin triggers a cascade of intracellular responses within the cell resulting in an increased intracellular concentration of IP3 and calcium ions leading to an action potential in the cell.

Abstract: Method and apparatus for steering a beam of light. The method and apparatus are based on the discovery that the spectral dispersion of multi-chromatic light pulses by an acousto-optical deflector can be significantly ameliorated by positioning a dispersive element, such as a prism, along the path of the multi-chromatic light pulses in such a way that the dispersive element disperses the multi-chromatic light pulses in a direction opposite to the spectral dispersion caused by the acousto-optical deflector. The dispersive element may be positioned either before or after the acousto-optical deflector. The method and apparatus are particularly well-suited for use with ultrashort laser pulses in the visible and infrared ranges having a bandwidth of up to about 40 nm. The method and apparatus have applicability in, among other things, multi-photon laser scanning microscopy.