Low-cost monochromatic microsecond flash microbeam apparatus for single-cell photolysis of rhodopsin or other photolabile pigments

Delivery of intense, brief flashes of monochromatic light are required in single-cell physiological experiments to photolyze cellular chromophores or pigments. In the xenon flash instrument constructed, flashes are collimated, made monochromatic with selectable bandpass filters and imaged into a small-core fiber. The flash is transmitted over meters to the epiflourescent port of a microscope where additional optics again collimate the beam. The objective lens of the microscope functions to condense flash energy into a microbeam in the specimen (field) plane and to image the cell under parafocal conditions. Spot diameters are 228 and 166 μm (full width half maximum) for 40X and 60X objectives. Flash intensities can be measured with this instrument during experiments using the microscope phase/differential interference contrast condenser to couple the microbeam to a calibrated photodiode. Flash intensities between 10⁸ and 10⁹ photons/μm² were achieved across the near-ultraviolet/visible spectrum. Flash durations were under 20 μs with a short-arc 7 J flash tube. Shielding and fiber transfer permit delivery of intense flashes without electromagnetic noise to the electrophysiological recording apparatus. Flashes generated with this instrument activated intramolecular charge motions (early receptor currents) in the visual pigment, rhodopsin, expressed from transgenes in single cultured cells.