The distribution of 3-dehydroretinal (Ral2) in dorsal, middle, and ventral slices of eyes of the crayfish Procambarus clarkii was examined by HPLC. No pronounced differences were found. Similar results were obtained when the eyes were cut into anterior, intermediate, and posterior portions.

Dichroic difference spectra were measured in single halves of microvillar layers of isolated rhabdoms and the proportions of rhodopsin (P1) and porphyropsin (P2) estimated by comparison with computer-generated mixtures of these pigments, whose spectra are known from previous work. The fraction of visual pigment that is porphyropsin appears to be uniform throughout individual retinular cells and among the retinular cells of individual rhabdoms, but various substantially among different rhabdoms from the same eye.

The interommatidial variation in the amount of P2 greatly exceeds the gross regional variation in Ral2. This means there is an intermingling of ommatidia with different levels of P2. The variability in P2 among ommatidia is not likely to have important implications for the vision of the crayfish but suggests that in the metabolism of retinoids, individual ommatidia are quasi-independent metabolic units. The results are compatible with a single opsin for both crayfish rhodopsin and porphyropsin.

The habitat occupied by larval winter flounder (Pseudopleuronectes americanus) differs considerably in light regime from that of the adult. To understand how the visual system has adapted to such changes, photoreceptor spectral absorbance was measured microspectrophotometrically in premetamorphic and postmetamorphic specimens of winter flounder. Before metamorphosis, larval flounder retinas contain only one kind of photoreceptor which is morphologically cone-like with peak absorbance at 519 nm. After metamorphosis, the adult retina has three types of photoreceptors: single cones, double cones, and rods. The visual pigment in single cones has a peak absorbance at λmax = 457 nm, the double cones at λmax = 531 and 547 nm, and the rod photoreceptors at λmax = 506 nm. Double cones were morphologically identical, but the two members contained either different (531/547 nm) or identical pigments (531/531 nm). The latter type were found only in the dorsal retina. The measured spectral half-bandwidths (HBW) were typical of visual pigments with chromophores derived from vitamin A1 with the possible exception of the long-wavelength absorbing pigment in double cones which appeared slightly broader. Because the premetamorphic pigment absorbance has a different λmax than those of the postmetamorphic pigments, different opsin genes must be expressed before and after metamorphosis.

Visual pigment, sensitivity, and rhabdomere size were measured throughout a 12-h light/12-h dark cycle in Drosophila. Visual pigment and sensitivity were measured during subsequent constant darkness [dark/dark (D/D)]. MSP (microspectrophotometry) and the ERG (electroretinogram) revealed a cycling of visual pigment and sensitivity, respectively. A visual pigment decrease of 40% was noted at 4 h after light onset that recovered 2–4 h later in white-eyed (otherwise wild-type, w per+) flies. The ERG sensitivity [in w per+ flies in light/dark (L/D)] decreased by 75% at 4 h after light onset, more than expected if mediated by visual pigment (MSP) changes alone. ERG sensitivity begins decreasing 8 h before light onset while decreases in visual pigment begin 2 h after light onset. These cycles continue in constant darkness (D/D), suggesting a circadian rhythm. White-eyed period (per) mutants show similar cycles of visual pigment level and sensitivity in L/D; per's alterations, if any on the D/D cycles were subtle. The cross-sectional areas of rhabdomeres in w per+ were measured using electron micrographic (EM) morphometry. Area changed little through the L/D cycle.

Visual pigments from the red rods of adults of eight species of Australian anuran amphibians, from a variety of habitats, were analyzed by microspectrophotometry. The λmax in all cases fell between 502 nm and 506 nm, and the absorption spectra were well fitted by an A1-based visual pigment template curve. Red rod pigments were also analyzed for a number of tadpoles. In some cases the data were best fitted with an A1based visual pigment template, in other cases with an A2-based template, and finally some tadpoles appeared to have mixtures of the two pigments.

We present microspectrophotometric evidence for the existence of two distinct visual pigments residing in two different morphological types of photoreceptor of the sea lamprey. In the upstream migrant Petromyzon marinus, the pigment found in short receptors has a wavelength of peak absorbance (λmax) of 525 nm, whereas the pigment located in long receptors has a λmax of 600 nm. Although the former appears to be pure porphyropsin, the latter is akin to visual pigments found in the red-absorbing cones of amphibian and teleost retinae. The kinship is more than superficial pertaining to λmax of the a–band absorbance to its native maximum value. The presence of an anion-sensitive and an anion-insensitive pigment in a retina implies the expression of two distinct opsin genes. We infer this from several examples of correlation between anion sensitivity and opsin sequence groupings. Moreover, the presence of two distinct opsin genes expressed throughout six vertebrate classes implies their existence in a common ancestor to all.

Absorbance spectra of rods and some cones were measured by microspectrophotometry in 22 fish species from the brackish-water of the Baltic Sea, and when applicable, in the same species from the Atlantic Ocean (3 spp.), the Mediterranean Sea (1 sp.), or Finnish fresh-water lakes (9 spp.). The main purpose was to study whether there were differences suggesting spectral adaptation of rod vision to different photic environments during the short history (<104 years) of postglacial isolation of the Baltic Sea and the Finnish lakes. Rod absorbance spectra of the Baltic subspecies/populations of herring (Clupea harengus membras), flounder (Platichthys flesus), and sand goby (Pomatoschistus minutus) were all long-wavelength-shifted (9.8, 1.9, and 5.3 nm, respectively, at the wavelength of maximum absorbance, λmax) compared with their truly marine counterparts, consistent with adaptation for improved quantum catch, and improved signal-to-noise ratio of vision in the Baltic light environment. Judged by the shape of the spectra, the chromophore was pure A1 in all these cases; hence the differences indicate evolutionary tuning of the opsin. In no species of fresh-water origin did we find significant opsin-based spectral shifts specific to the Baltic populations, only spectral differences due to varying A1/A2 chromophore ratio in some. For most species, rod λmax fell within a wavelength range consistent with high signal-to-noise ratio of vision in the spectral conditions prevailing at depths where light becomes scarce in the respective waters. Exceptions were sandeels in the Baltic Sea, which are active only in bright light, and all species in a “brown” lake, where rod λmax lay far below the theoretically optimal range.

The Syngnathidae are specialized diurnal feeders that are known to possess a retinal fovea and use independent eye movements to locate, track, and strike individual planktonic prey items. In this study, we have investigated the spectral sensitivities of three syngnathid species: a pipefish and two seahorses. We used spectrophotometry to measure the spectral transmission properties of ocular lenses and microspectrophotometry to measure the spectral absorption characteristics of visual pigments in the retinal photoreceptors. The pipefish, Stigmatopora argus, together with the seahorse Hippocampus subelongatus, is found in “green-water” temperate coastal seagrass habitats, whereas the second seahorse, H. barbouri, originates from a “blue-water” tropical coral reef habitat. All species were found to possess short wavelength absorbing pigment(s) in their lenses, with the 50% cut-off point of S. argus and H. subelongatus at 429 and 425 nm respectively, whereas that of H. barbouri was located at 409 nm. Microspectrophotometry of the photoreceptors revealed that the rods of all three species contained visual pigment with the wavelength of maximum absorption (λmax) at approximately 500 nm. The visual pigment complement of the cones varied between the species: all possessed single cones with a λmax close to 460 nm but H. barbouri also possessed an additional class of single cone with λmax at 430 nm. Three classes of visual pigment were found in the double cones, the λmax being approximately 520, 537, and 560 nm in the two seahorses and 520, 537, and 580 nm in the pipefish. The spectral sensitivities of the syngnathids investigated here do not appear to conform to generally accepted trends for fishes inhabiting different spectral environments. The influence of the specialized feeding regime of the syngnathids is discussed in relation to our findings that ultra-violet sensitivity is apparently not necessary for zooplanktivory in certain habitats.

The four spectral cone types in the zebrafish retina each contribute to photopic visual sensitivity as measured by the b-wave of the electroretinogram (ERG). The goal of the current study was to evaluate a model of photopic b-wave spectral sensitivity in the zebrafish that mapped first-order cellular and biophysical aspects of cone photoreceptors (visual pigment absorbance spectra and cone fractions) onto a second-order physiological aspect of cone-derived neural activity in the retina. Good correspondence between the model and photopic ERG data was attained using new visual pigment absorbance data for zebrafish cones (λmax of the L, M, and S cones were 564, 473, and 407 nm, respectively), visual pigment templates, and linearly gained cone fractions. The model inferred four distinct cone processing channels that contribute to the photopic b-wave, two of which are antagonistic combinations of cone-derived signals (L-M and M-S), and two of which are noncombinatorial signals from S and U cones. The nature of the gains and the processing channels suggested general rules of cone-specific inputs to second-order neurons. The model further suggested that the zebrafish retina utilizes neuronal mechanisms for enhancing sensitivity to luminance contrast at short wavelengths and chromatic contrast at middle and long wavelengths. The results indicated that first-order cellular and biophysical aspects of cone photoreceptors can successfully explain physiological aspects of cone-derived neuronal activity in the zebrafish retina.

Absorbance spectra of cone outer segments and oil droplets were recorded microspectrophotometrically in the retina of the red-eared turtle, Trachemys scripta elegans. There are four cone visual pigments, with λmax = 617 nm (red sensitive), 515 nm (green sensitive), 458 nm (blue sensitive), and 372 nm (UV-sensitive). The red-sensitive pigment resides in single cones with red or orange oil droplets, and in both members of double cones. The principal member of the double cone contains an orange oil droplet, and the accessory member is droplet free. The green-sensitive pigment is situated in single cones with orange/dark yellow droplets. The blue-sensitive pigment is combined with the UV-absorbing oil droplet in single cones. The UV-sensitive pigment resides in single cones with clear oil droplets that exhibited virtually no absorbance down to 325 nm. Thus, seven types of cones can be identified based on their morphology, oil droplet color, and the visual pigment absorbance. At the moment, this is the most complex cone system described for vertebrates.

Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of α and β subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of α and β hemes separately. Previous studies have characterized the oxygen binding by α(Ni2+)2 β(Fe2+)2 crystals. Here, we have determined the three-dimensional (3D) structure and oxygen binding of α(Fe2+)2 β(Ni2+)2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation (p50s) are 95 ± 3 and 87 ± 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 ± 0.06 and 0.90 ± 0.03. Analysis of the binding curves, taking into account the different projections of the α hemes along the optical directions, indicates that the oxygen affinity of α1 hemes is 1.3-fold lower than α2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the β subunits of α(Ni2+)2 β(Fe2+)2 crystals. The average oxygen affinity of the α subunits (p50 = 91 Torr) is about 1.2-fold higher than the β subunits (p50 = 110 Torr). In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T-state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model.