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 th at 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 spectr al distortion due to photoproduct interference and an ability to measure fa st, transient changes in absorbance as bleaching proceeds. Rainbow trout ph otoreceptors, previously measured with a wavelength-scanning MSP system, we re again measured using the CCD-based MSP system. Our analysis of optical r ecordings from 102 photoreceptors corroborated data obtained previously wit h rainbow trout photoreceptors on lambda (max) (wavelength of maximum absor bance), A(max) (maximum absorbance) and half maximum bandwidth (HBW) of ult raviolet-, blue-, green- and red-sensitive cones and rods. There were sligh t differences in Amax and half-maximum bandwidth of the ultraviolet-, blue- and green-sensitive cone classes, but this was most probably due to variat ion in the A(1):A(2) visual pigment ratio of the trout used in the two diff erent studies. However, we were capable of resolving the A(1) and A(2) visu al pigment spectra in the red-sensitive cones and the rods.