A LED light calibration source for dual-wavelength microscopy

An LED light source for dual-wavelength calibration of light microscope recording systems is described. A 1 mm2 circular field of bi-chromatic illumination is formed by passing the crossed beams of two light emitting diodes (LEDS) through an optical diffuser and circular mask. The source is constructed using LEDs which emit light at wavelengths overlapping those of many of the dual-emission molecular probes used for measurement of calcium, pH and membrane potential. The light output can be independently varied to set the ratio of light intensities recorded at two wavelengths. Internally sampled voltages which are proportional to LED light output intensity provide an internal reference for comparison with optical recordings. The LED source is useful for obtaining the optical response of dual emission recording equipment for specific recording conditions defined by the filtering and light detection components of the system.