A novel rapid scanning microspectrophotometer and its use in measuring rhodopsin photoproduct pathways and kinetics in frog retinas.

A novel rapid scanning microspectrophotometer is described which utilizes a cathode ray tube as a measuring light source. Spectral scanning is accomplished electronically with a sampling time of 600 mus for each waveband. The cathode ray tube emission is chopped electronically into two separate beams, 180 degrees out of phase, resulting in a dual-beam configuration. A lock-in amplifier functions as a coherent detector to recover separately the signals from the two beams. The instrument generates separate voltage outputs, one proportional to the transmittance of a single sample and the other to the difference between two samples. A computer calculates both absorption and difference spectra directly from voltage measurements. A demonstration of the instrument's use to study kinetics of visual pigment photoproducts is presented. Two models of photoproduct sequence and kinetics were examined to determine which better represents the experimental data. The experiments show that environmental factors, such as pH, metabolic and respiratory state, interact in complex ways to determine the pathways and kinetics of photoproducts of rhodopsin in intact vertebrate eyes.