Investigation of the effect of simulated lens yellowing, transparency loss and refractive error on in vivo resonance Raman spectroscopy.

PURPOSE: To separately investigate the impact of simulated age-related lens yellowing, transparency loss and refractive error on measurements of macular pigment (MP) using resonance Raman spectroscopy. METHODS: Two healthy young subjects with clear media underwent Raman spectroscopy under the following conditions: age-related lens yellowing was simulated using seven broad-band yellow filters with transmittance at 488 nm ranging from 0.54 to 0.90; cataract was simulated using five white filters of increasing opacity (scatter filters), the transmittance of which ranged from 0.42 to 0.86, each of which reduced peak contrast sensitivity by approximately 0.1 log units over the previous filter. Refractive error up to +6.25 D was achieved using soft contact lenses. RESULTS: The Raman signal declined steadily to an average value of 43% of the starting value with the densest yellow filter in place. The white scatter filters produced a progressive linear reduction in signal resulting in almost complete signal loss with the densest filter. Refractive error resulted in an initial slight improvement in Raman count up to a value of +2.00 D followed by a decline thereafter. CONCLUSIONS: These results imply that lens yellowing and increasing scatter has an influence on the Raman signal and suggest that studies using this technology to estimate MP levels in older populations should carefully account for the status of the lens.

In vivo macular pigment measurements: a comparison of resonance Raman spectroscopy and heterochromatic flicker photometry.

AIM: To investigate whether two methods of measuring macular pigment-namely, heterochromatic flicker photometry (HFP) and resonance Raman spectroscopy (RRS)--yield comparable data. METHODS: Macular pigment was measured using HFP and RRS in the right eye of 107 participants aged 20-79 years. Correlations between methods were sought and regression models generated. RRS was recorded as Raman counts and HFP as macular pigment optical density (MPOD). The average of the top three of five Raman counts was compared with MPOD obtained at 0.5 degrees eccentricity, and an integrated measure (spatial profile; MPODsp) computed from four stimulus sizes on HFP. RESULTS: The coefficient of variation was 12.0% for MPODsp and 13.5% for Raman counts. MPODsp exhibited significant correlations with Raman counts (r = 0.260, p = 0.012), whereas MPOD at 0.5 degrees did not correlate significantly (r = 0.163, p = 0.118). MPODsp was not significantly correlated with age (p = 0.062), whereas MPOD at 0.5 degrees was positively correlated (p = 0.011). Raman counts showed a significant decrease with age (p = 0.002) and were significantly lower when pupil size was smaller (p = 0.015). CONCLUSIONS: Despite a statistically significant correlation, the correlations were weak, with those in excess of 90% of the variance between MPODsp and Raman counts remaining unexplained, meriting further research.

[Invariants of spatial summation for S (short wavelength) cone vision]. / Invarianty prostranstvennoi summatsii dlia S (korotkovolnovogo) kolbochkovogo zreniia.

Spatial summation in the human visual system was studied as a function of retinal eccentricity upon selective stimulation of the short-wavelength sensitive cones. The area of complete spatial summation (Ricco's area) was found to increase with retinal eccentricity while the threshold of stimuli equal in size with Ricco's area remained constant. Comparisons with known morphology of the small bistratified retinal ganglion cells, the only cells known to be excited by S-one ON stimulation, showed that Ricco's area included 2-4 such cells and is up to 1.5 times larger than the dendritic field of a single cell. These relationships were relatively constant within the eccentricity range tested (5-20 deg along the temporal horizontal meridian) and might be the source of threshold invariance of stimuli matching Ricco's area.

The contrast sensitivity function for detection and resolution of blue-on-yellow gratings in foveal and peripheral vision.

Previous studies using polychromatic gratings have shown that the peripheral grating contrast sensitivity function is significantly different when the task is resolution rather than detection. Specifically, in the middle frequency range, while resolution acuity drops suddenly to zero, detection performance continues up to much higher frequencies, accompanied by observations of aliasing. We wanted to determine if the same holds true for blue-cone isolating gratings in either foveal or peripheral vision. Contrast sensitivity function (CSFs) were measured at the fovea and 20 degrees eccentricity in the temporal retina under conditions of short-wavelength-sensitive (SWS)-cone pathway isolation using a two-alternative forced choice paradigm. The detection and resolution CSF were identical at the low frequency end but at higher frequencies resolution sensitivity falls abruptly while contrast detection remained possible till higher frequencies [cut-off frequencies: fovea detection 6.0 cycles (degree)(-1), resolution 4.6 cycles (degree)(-1); periphery detection 1.6 cycles (degree)(-1), resolution 1.05 cycles (degree)(-1)]. Aliasing was observable when spatial frequency exceeded the resolution limit. Medium/high contrast blue-cone-mediated resolution acuity is sampling limited in both the fovea and periphery. Previous studies of blue-cone contrast sensitivity which employed a detection task do not reflect the true resolution limit.

Binocular summation for grating detection and resolution in foveal and peripheral vision.

Peripheral monocular grating resolution has been shown to be limited by the sampling density of the underlying retinal ganglion cells. We wanted to determine if peripheral resolution is also sampling limited binocularly; and, if so, how great is any improvement in either detection or resolution when viewing binocularly? We measured detection and resolution acuity for sinusoidal gratings in foveal and peripheral vision both monocularly and binocularly. Detection and resolution acuity were very similar in foveal vision and displayed a binocular improvement of 5% over best monocular acuity. However, in peripheral vision, while detection acuity improved by 6% binocularly, resolution acuity improved by 16%, with a subsequently smaller aliasing zone. This improvement was greater than predicted by probability summation and implies that the two monocular ganglion cell sampling arrays combine at a higher level resulting in a higher binocular sampling density.

Reversed-phase high-performance liquid chromatography for evaluating the distribution of pharmaceutical substances in suppository base-phosphate buffer system.

The aim of this study was to assess the potency of the reversed-phase high-performance liquid chromatography (RP-HPLC) for in vitro evaluation of the distribution behavior of common drugs between one of the generally used suppository bases Witepsol H15 and the rectal liquid which is imitated by a phosphate buffer, pH 7.2. The distribution coefficients (log K) of nine compounds--paracetamol, caffeine, diclofenac, propyphenazone, indomethacin, codeine base, codeine phosphate, phenobarbital acid and phenobarbital sodium salt were determined by the classical shake-flask' method followed by RP-HPLC quantitative assay. The capacity factors log k' of the compounds were determined on reversed-phase C18 column at a number of methanol-5 mM phosphate buffer, pH 7.2 mobile phases containing different percentages of methanol (phiMeOH). The apparent capacity factors log k(w)app were derived by extrapolation of the methanol concentration to zero and using the correction for ionization, the real capacity factors log k'(w) were calculated. The lipophilicity of the compounds was assessed by the partition coefficients CLOGP and the distribution coefficients CLOGD7.2, calculated for the n-octanol-water system. Correlations between log k'(w) and CLOGP, log k(w)app and CLOGD7.2, log k(w)app and log K were found. The last correlation indicated that the parameter log k(w)app was suitable for evaluating the distribution behavior of the studied drugs in the examined Witepsol H15-rectal liquid system. The predictive power of this correlation was tested by a set of nine non-congeners. It was shown that the classical 'shake-flask' method for determination of the distribution behavior of the studied drugs between the suppository base Witepsol H15 and the phosphate buffer, pH 7.2 might be replaced by the RP-HPLC technique due to its priorities of rapid, stable and reproducible experiments.

Temporal characteristics of line orientation identification.

The hypothesis that identification of line orientation is based on different mechanisms--a detector mechanism at large orientation differences and a computational one at small orientation differences--was tested in three experiments. The first two experiments compared reaction time and time of complete temporal summation (tc) in two tasks, line detection and line orientation identification. Identification at orientation differences 15 degrees or more was similar to detection in several respects, suggesting that it was accomplished according to the principle of "labeled lines." In agreement with the initial hypothesis, identification at differences smaller than 15 degrees had a slower time course and could not be explained by the "labeled lines" principle. Experiment 3 explored the orientation acuity as a function of exposure duration and stimulus energy. Energy could not completely substitute for time in providing high orientation acuity, a result suggesting the involvement of neurophysiological mechanisms of large time constants.

Spatial summation of blue-on-yellow light increments and decrements in human vision.

In the primate retina, blue-OFF cells are less numerous than blue-ON cells but no psychophysical equivalent of this asymmetry has been found so far. The hypothesis put forward in the present study is that the ON-OFF asymmetry should manifest itself in the size and effectiveness of spatial summation of S-cone signals of opposite polarity. To test this hypothesis upon selective stimulation of the S-cones in man, a 3 cd/m(2) blue light was superimposed on a 300 cd/m(2) yellow background and the test stimulus consisted in a luminance increment or decrement of the blue light from its steady level over a circular area of variable size. The test stimuli were presented at 12.5 degrees retinal eccentricity. Within the test-stimulus spectral band, sensitivity was that of Stiles' pi(1) mechanism. Increasing stimulus area reduced more the decrement threshold than the increment threshold, and Ricco's area was larger for luminance decrements (0.8-2 degrees ) than for increments (0.6-0.9 degrees ). Experiments with red-on-red stimuli confirmed that the large summation area and stimulus-polarity-dependent spatial summation are specific for the isolated S-cone signals. The sign-dependency of spatial summation is probably a psychophysical correlate of the asymmetry of the ON- and OFF- visual pathways receiving S-cone input.

Orientation identification at different background levels: its precision and distortions.

Orientation identification was studied at high (1000 td) and low (1 td) background illuminations. The subject had to estimate in degrees the orientation of a line presented in one of 18 orientations, 10 deg apart. Unlike other spatial discriminations, the precision of orientation judgments was not significantly impaired at low background if a suprathreshold stimulation was used. However, our earlier data showed that the identification at threshold was deteriorated at low background level. Different mechanisms of threshold and suprathreshold identification were suggested. The mechanism of suprathreshold identification was thought to contribute to the stability of the orientation perception during adaptation. The deviations of the subjective estimates from real orientations were also studied. The bias of perceived orientation toward the vertical increased at low background. This bias remained the same in egocentric coordinates, when the subjects were in supine position.

Effect of light adaptation on the orientation acuity of human cone vision.

At the detection threshold, a line orientation is identified by the accuracy of 15-20 deg. Deviations from this rule are observed for short lines presented on a dim background. We checked up for one possible source of deviation: the substitution for foveal cone vision by peripheral rod vision. Long wavelength (red) light was used for the test line. In a control experiment such light did not stimulate rods. The test line was 4 min of arc long. It was presented for 20 ms on a white background of 1 or 1000 trolands and was viewed foveally. In a 2-alternative forced-choice experiment the psychometric functions for detection and orientation identification (vertical v. horizontal) were compared. In another experiment, the line was presented randomly in one of 18 orientations in the range 0-180 deg and the subject was asked to determine the orientation. In both experiments orientation perception was impaired at the dim background in comparison with the results at the bright background. This suggests that light-adaptational changes of the mechanisms responsible for orientation perception exist within the photopic vision.

Length and width summation in human vision at different background levels.

The effect of background intensity on the spatial summation of rectangular stimuli of varying length and width was studied in human psychophysical experiments and compared with the known effects of light adaptation on the spatial summation of circular stimuli. Both the detection threshold and the threshold of orientation identification were measured. In agreement with previous data, summation was more efficient along the line stimulus than across it. This asymmetry was found to exist at all adaptation levels studied (1-1000 trolands). The adaptation level affected both length and width summation; the change in the length of summation was twice as great as the change in the diameter of summation with circular stimuli. Orientation selectivity was reduced for short lines presented on a dim background. The results suggest the existence of mechanisms of light adaptation at the level of cortical orientation-selective units.

Reaction time and critical duration for lines' detection and identification of their orientation.

Reaction time (RT) and duration of complete temporal summation (tc) were studied for both detection of lines and identification of their orientation. The RT was studied as a function of stimulus intensity, number of orientations and angular difference between them. The temporal summation was studied for detection and identification at several angular differences. It was found that the intensity-dependent part of RT for identification at angular difference of 22 degrees equaled that for detection but considerably lengthened at smaller angular differences (10 degrees). The intensity-independent part of RT was minimal for detection and maximal for identification with eight alternatives. The tc increased from 30 msec for detection and identification with the accuracy of 22 degrees to 100 msec for identification with the accuracy of 5 degrees. The results support the conclusion that detection and coarse identification differ only in their later stages and that accurate identification involves additional processes with longer time-constants compared to coarse identification.

Temporal summation and orientation acuity.

The effect of stimulus exposure time (ET) on the threshold intensity, necessary for fine orientation discrimination was compared with the effect of ET on the detection threshold. Both discrimination and detection performances were studied concurrently in a two-by-two forced choice paradigm. The results suggest the existence of a mechanism with much longer temporal summation for the discrimination of small orientation differences than for the detection of the stimulus. Orientation acuity improves with ET.

Perception of orientation of short lines: its dependence on line's length and intensity.

The threshold intensity necessary for correct identification of orientation of a briefly presented line was measured as a function of line's lenght and number of possible orientations (2 orientations differing by 90 degrees, 4 differing by 45 degrees, and 8 differing by 22.5 degrees). The thresholds were the same for 2, 4 and 8 orientations when the lines were longer than 20 min of arc. On the contrary, in the case of short lines, the correct report of 8 orientations required much more intensive stimulation than the report of 2 orientations. The discrimination of small differences in orientation of short lines involves additional processes to those for their detection and coarse perception of their orientation.