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 40× and 60× 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 dura...

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