Holographic photolysis of caged neurotransmitters

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Abstract
Holographic illumination allows the production of complex, user-defined, two-dimensional illumination patterns. Used to manipulate light-sensitive molecules in cells, this system permits their simultaneous excitation at multiple locations of arbitrary shape and size—facilitating spatial and temporal regulation of cell function. Stimulation of light-sensitive chemical probes has become a powerful tool for the study of dynamic signaling processes in living tissue. Classically, this approach has been constrained by limitations of lens-based and point-scanning illumination systems. Here we describe a microscope configuration that incorporates a nematic liquid-crystal spatial light modulator to generate holographic patterns of illumination. This microscope can produce illumination spots of variable size and number, and in patterns shaped to precisely match user-defined elements in a specimen. Using holographic illumination to photolyze caged glutamate in brain slices, we show that shaped excitation on segments of neuronal dendrites and simultaneous, multispot excitation of different dendrites enables precise spatial and rapid temporal control of glutamate receptor activation. By allowing the excitation volume shape to be tailored precisely, the holographic microscope provides an extremely flexible method for activation of various photosensitive proteins and small molecules.