Manipulation Of Neurons With Precisely Controlled Illumination In Space And Time

Manipulation of cells with light has become a powerful and widely used tool in neuroscience. For this approach to reach its full potential, two conditions need to be met: the precise spatial and temporal control of light delivery inside the sample, and the availability of light-controllable molecules that can modify crucial cellular and neuronal functions. We present a novel holographic illumination system that can be coupled to existing microscopes (e.g. two-photon microscopes) which provides high resolution control for photomanipulation experiments. The system is optimized for two-photon excitation to improve axial confinement of the excitation patterns as well as tissue penetration. 4d light patterns (x,y,z,t) can be defined by the user based on fluorescence images of the sample. In addition to a technical characterization of the system we present biological applications of the illumination module using various types of light-sensitive molecules, including caged compounds, photo-switchable ligands of neuronal receptors and optogenetics molecules.