Microspectrophotometric measurements of vertebrate photoreceptors using CCD-based detection technology

We have developed a charge-coupled-device (CCD)-based microspectrophotometer (MSP) system and provide the first report on the successful employment of this technology to measure the spectral absorbance properties of vertebrate photoreceptors. The principal difference between the CCD-based MSP system and wavelength-scanning MSP systems, commonly used in vision biology, is that a short duration (800-1200 ms), broad-spectrum flash is employed rather than ascending and descending wavelength scanning. Data acquisition is thus significantly faster, with the added possible advantages of less variance due to movement of target photoreceptors during measurement, reduced spectral distortion due to photoproduct interference and an ability to measure fast, transient changes in absorbance as bleaching proceeds. Rainbow trout photoreceptors, previously measured with a wavelength-scanning MSP system, were again measured using the CCD-based MSP system. Our analysis of optical recordings from 102 photoreceptors corroborated data obtained previously with rainbow trout photoreceptors on lambda(max) (wavelength of maximum absorbance), A(max) (maximum absorbance) and half maximum bandwidth (HBW) of ultraviolet-, blue-, green- and red-sensitive cones and rods. There were slight differences in lambda(max) and half-maximum bandwidth of the ultraviolet-, blue- and green-sensitive cone classes, but this was most probably due to variation in the A(1):A(2) visual pigment ratio of the trout used in the two different studies. However, we were capable of resolving the A(1) and A(2) visual pigment spectra in the red-sensitive cones and the rods.