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Microspectrophotometric measurements of vertebrate photoreceptors using CCD-based detection technology

Craig W. Hawryshyn^*, Theodore J. Haimberger and Mark E. Deutschlander

Department of Biology, University of Victoria, PO Box 3020 STN CSC, Victoria, British Columbia, Canada V8W 3N5
Present address: Rochester Institute of Technology, Rochester, NY 14623-5603, USA

View larger version (16K): [in a new window]   Fig. 1. Schematic diagram of the CCD microspectrophotometer. BI, back illumination; BIR, infrared filter; BS, beam splitter; CCD, charge-coupled device camera; CCS, custom computer software; CRO, reflective objective as condenser; FO, fibre optic cable; M, microscope; O, objective lens; PREP, preparation; S, xenon lamp (light source); SH, shutter; SIR, infrared filter mounted on a swing arm; SP, spectrograph; VA, X–Y variable aperture; XYS, X–Y stage.

View larger version (174K): [in a new window]   Fig. 2. Representative images of rainbow trout photoreceptors taken under infrared illumination. (A) The arrow points to a blue-sensitive cone (also shown in B and C). (D) Ultraviolet-sensitive cone. (E) Rod photoreceptors. (F) Double cone. Additional images were used in the calculation of mean outer segment diameter, which was used to determine specific absorbance (see Table1). Scale bars, 10 µm.

View larger version (30K): [in a new window]   Fig. 3. Representative absorbance spectra of rainbow trout cone photoreceptors. (A–D) Mean relative absorbance for the ultraviolet- (A), blue- (B), green- (C) and red-sensitive cones (D, two spectra represent curves dominated by the A1 and A2 visual pigments). The red line shows the mean absorbance spectrum and the blue line shows the eighth-order polynomial template. Note that the two spectra (red lines) in D represent the A1-based visual pigment red-sensitive cones (curve peaking at 574nm) fitted by an eighth-order template and the A2-based visual pigment red-sensitive cones (curve peaking at 623nm) fitted by a Fourier transform function. (E–H) The unbleached (red line) and bleached (blue line) absorbance of a single cone recording for the ultraviolet- (E), blue- (F), green- (G) and red-sensitive (H) cones. See Table1 for additional spectral data of cones.

View larger version (14K): [in a new window]   Fig. 4. Representative absorbance spectra of rainbow trout rod photoreceptors. (A) Mean absorbance spectrum of rhodopsin-based rods (black line). This spectrum (red diamonds) and those in B (red triangles) and C (red circles) are fitted with long-wavelength limb templates taken from Munz and Beatty (Munz and Beatty, 1965). (B) Mean absorbance spectrum for mixed rhodopsin porphyropsin rods. (C) Mean absorbance spectrum for porphyropsin-based rods.