572

J. Physiol. (I955) I27, 572-586

BLEACHING EXPERIMENTS ON EYES OF

LIVING GUINEA-PIGS
BY R. A. WEALE
From the Medical Research Council Group for Research in the Physiology of
Vision, Institute of Ophthalmology, Judd Street, London
(Received 8 September 1954)
When light strikes the fundus of an eye, part of it is reflected and re-emerges
through the pupil. The reflected fraction is smaller or greater accordingly as
the eye is dark- or light-adapted, since the light has to travel through the
retina which will or will not contain photosensitive material. The ratio of the
two fractions determined in these circumstances provides a measure of the
density of such material (Rushton, 1952).
After exposing a cat's eye to a bright source of white light, the intensity of
a monochromatic test-beam was found to diminish either rapidly or slowly
(Weale, 1953a, b). This suggested that a light-absorbing substance was
accumulating in the retina. Observations with lights of different wavelengths
showed that the rapid decrease was maximal for a wavelength of about
550 m,u, the slow change for one of about 500 m,. In fact, the spectral
distribution of the slow effect suggests that, as time goes on, visual purple
accumulates in the retina. This finding confirmed one first made by Rushton,
using bleaching experiments (1952).
Statistically significant differences were, however, found to exist between
the known absorption spectrum of visual purple and that observed by the
above means in the cat.
It was, therefore, decided to examine another species. The guinea-pig was
thought to be suitable for this purpose, although the smaller size of its eye
offers a technical drawback. Histological evidence on the composition of this
animal's retina is controversial. Walls (1942) writes that it 'is more certainly
pure-rod than that of the rabbit'. This view has been confirmed more recently
by Tansley (personal communication). O'Day (1947), on the other hand, states
that it contains cones, and Kolmer (1936) puts the rod-cone ratio as low as
4 or 5 to 1. This is similar to the value he found for the human periphery. If
the guinea-pig retina contains no cones, a pure visual purple bleaching
 PIGMENTS IN THE GUINEA-PIG EYE 573
spectrum might be reasonably expected to result from the present work
(Wald, 1953). On the other hand, if the bleaching spectrum were composite,
support would be provided for the view that the percipient elements of the
guinea-pig do not belong to one type only.

METHOD
Except where otherwise stated the animals were dark-adapted for 16-20 hr. This was necessary
because, in these animals, the regeneration of the bleached pigment after exposure to a bright
light proceeded at too small a rate to make feasible such measurements as those on the cat.
Measurements of regeneration were, therefore, replaced by those of bleaching. In effect, the
measuring procedure was as follows. A monochromatic beam of light was divided in two with a
photometer prism. One half entered the animal's eye, the corneal refraction of which was
neutralized with a contact lens, and focused on the fundus. The portion reflected at the fundus
emerged from the eye, and was juxtaposed to the other half of the original beam of light. The
intensity of the latter could be controlled with a neutral density wedge, and thus set equal to that
of the test-beam. In general, different readings would be obtained before and after exposing the
eye to a bleaching light. The change due to bleaching was computed from these measurements and
represented a measure of the bleachable material present in the eye.
In some experiments, the available pigment supply was exhausted not in one, but in two stages,
first with coloured, then with white lights. Measurements of the reflexion factor were made also
in between the two bleaches so that the change in density due to each could be determined.
As soon as the animal had been taken from its blacked-out cage, where it had spent the previous
night, the eye to be examined was covered with a thick wad of black cotton-wool to prevent any
light from reaching it. While the animal was anaesthetized (25% urethane, 3-6-4-75 ml./06 kg
body wt.), the trachea cannulated, and the animal placed in the requisite position in relation to
the visual reflectometer (Weale, 1953c), the illumination in the laboratory was provided by faint
red or faint blue light.
The trachea was cannulated so that artificial respiration could be provided for the animal, which
was curarized to eliminate eye movements. The fur surrounding the orbit was shaved. Then the
animal's head was immobilized in a large retort clamp so that the test-beam could enter its left
eye. When the head was secured, a hot-water bottle was placed under the animal, the trunk was
covered with cotton-wool, and the black cotton-wool removed from the eye to be examined.
A thin layer of vaseline was spread over the shaved portion of the animal's head and a thick layer
of a mixture of hard and soft paraffin-wax spread on top of the vaseline. The paraffin mixture was
fairly easy to spread, although its softening point was above body temperature. It was shaped
into a half-moon surrounding the eye on the lower side of the orbit. A circular cover-slip was then
fitted to it, and the space between the cornea and the cover-slip carefully filled with normal saline
(0.9 g NaCl/100 ml. of glass-distilled water). The corneal refraction was thus neutralized, and the
test-beam could be focused on the fundus oculi as accurately as the imperfections of the dioptric
media would permit. No drug was required to dilate the pupil.
The spectrophotometer. This part of the apparatus was the same as in the previous investigations.
The neutral density wedge with which the measurements were made was calibrated at every wave-
length both before and after the investigation, and its photometric slope found to be constant to
within ± 1-5%. The calibration of the Hilger Barfit Spectrometer was checked frequently and
found to be constant to within ±0-4 m,u at A=643-8 mA, and ±0-1 m,u at 480-0 m,u. In order to
compensate for the reduction of energy at short wavelengths, the geometrical slit-width was
increased at the blue end of the spectrum. This did not materially affect the optical slit-width
which was 3-7 m, at 546-1 mp, and varied linearly from 2-1 to 5-6 mix between 440 and 490 mp.
The bleaching source. This was the same as had been used in the work on the cat (Weale, 1953a).
The actual light exposures were, however, reduced to the values shown in Table 1.
574 R. A. WEALE
The bleaching process was complete after every total bleach, and after every fractional chromatic
followed by a fractional white bleach. This was confirmed by further exposing the eye for 1 min to
source P.
The characteristics of the chromatic bleaching sources are indicated at the top of Figs. 2-4. The
short vertical line represents the maximum of the product TASA, where TA is the transmissivity
of each filter and SA the energy emitted by a tungsten filament lamp run at a temperature of
28480 K. The horizontal line indicates the range of this product with values higher than 50% of
the maximum. These values cannot be given for the red filter; the short-wave limit, therefore,
represents the wavelength at which this filter transmits less than 1% of the incident light. Since
two such filters were used in series, the bleaching source may be considered to contain effectively
only light of wavelengths longer than 620 m,u.
TABLE 1. Exposures to bleaching light and the luminances of three test-lights, measured as
white-light equivalents* when reflected from a magnesium oxide surface
Luminance Symbol
Type of bleach (ft. lamberts) in text
Fractional and total white 7 for 3 min f1
Fractional yellow-green (Ilford 625) m2
2-1 for 3 min
Fractional blue (Ilford 602) 0-13 for 3 min f3
Fractional red (2 Ilford 608) 25 for 3 min 4
White control m5
631 for 1 min
Test-lights
A in my. Luminance (ft. lamberts)
460 0 01
500 0-023
600 0 037
* The author's colour vision is normal.
The luminance of the monochromatic test-beams was such as to make measurable bleaching
unlikely while readings were taken. The white-light equivalents of three important test wavelengths
are also shown in Table 1. The eyes were exposed to these and other monochromatic weak test-
lights for much briefer periods than to the proper bleaching lights. It is, therefore, not surprising
that the test-lights were without effect on the readings also when repeated measurements were
made at the same wavelength. This bears out a previous observation on the cat; the only occasions
on which the test-light visibly affected readings in those experiments was when the regeneration
of the visual material was rapid (Weale, 1953 b). This has never been observed in any of the
guinea-pigs.
Animals. The results presented below were obtained from forty-two animals out of a total of
105 which were examined. The failure of many experiments was largely due to unstable conditions
(cf. 'Procedure'). The fraction of premature deaths was unexpectedly low. No measurements of
blood pressure were made, but, as far as could be judged by the appearance of the fundus in the
albino animals, it was not abnormally low.
Procedure. The order of test wavelengths for each run was decided before each experiment.
In any one animal, a start was made with a wavelength, chosen at random, and followed by the
next but one on the way to either end of the visible spectrum. When the end was reached, the
omitted wavelengths were used on the return journey, and the alternating order pursued to the
other end of the spectrum. Since some wavelengths would thus be missed on the far side of the
starting point, these would be used after the second 'about turn' till the first wavelength wa s
reached. Measurements were repeated with this and the second, and possibly third wavelength
of the run. Unless the readings agreed within the experimental error with those obtained in the
first instance, the run was rejected. In a dark-adapted animal the run could then be repeated in
the hope that stabler conditions would prevail. This hope was usually in vain. Sometimes, how-
ever, the 'dark-adapted' run would provide reproducible results. After bleaching, on the other
 PIGMENTS IN THE GUINEA-PIG EYE 575
hand, conditions might be unstable, in which case the whole of the experiment would be written
off as a failure. Only those experiments in which reproducible results were obtained for each run
were considered to be successful.
The order of wavelengths used when examining the dark-adapted animal was adhered to with
any one animal after bleaching, but varied from case to case. It depended partly on the original
wavelength and whether one went towards the long or short wavelength end of the spectrum. It
was hoped that any variations in the data, conceivably due to the order in which the readings
were taken, would cancel as a result of both interlacing the readings and altering their pattern.
The reasons for the instability have not been elucidated. Variations in blood pressure may
partly be responsible; in an experiment on a cat, which was very unstable, a close correlation was
observed between readings taken at one wavelength and the blood pressure.
Type8 of experiment8 performed. The pattern underlying this investigation was not planned but
emerged as the data became available. The first experiments were done on pigmented animals
which had been dark-adapted for only an hour after being prepared. No precaution was taken
with the illumination of the room. The bleaching was total and done with white light. Every
bleaching spectrum was so utterly at variance with preconceived ideas that it was decided to
extend the measurements to albino animals under similar conditions, and also to albino and
pigmented guinea-pigs which had been dark-adapted for 16-20 hr. In experiments in which the
animals had been dark-adapted for such a long period, the precautions mentioned previously were
carried out rigorously. Five animals were used in each of the above four sets of experiments.
When the results for the total white bleaches were examined, the question arose whether or not
only a single photochemical substance was being bleached. The method of partial bleaching
(Dartnall, 1952) was simplified in an attempt to provide an answer. The density changes due to
chromatic bleaching and to bleaching to completion with white light were determined in turn.
The chromatic bleaching was done on three sets of five animals, each with yellow-green, blue
and red lights. The light on the operating table, torches, etc., was red in the former two cases, but
blue when the red bleaching light was used, in the hope that this would minimize the effect of
stray light in the spectral regions under special examination. The animals used in the chromatic
bleaching experiments were all albinos and all dark-adapted for 16-20 hr.
A relation was expected to exist between the changes in density due to total white bleaches on
the one hand, and the combined chromatic and fractional white bleaches on the other, since the
initial stage a totally dark-adapted eye and the final stage a totally bleached eye-were
similar in the two groups. Results from fifteen fractional (chromatic and white) and five total
white bleaches, done on albinos after long dark-adaptation, were available for the comparison.
But the former group contained five, prepared in blue light, whereas red light had been used during
the preparation of the latter. To make the two groups more strictly comparable total white bleaches
were done on two more albino animals which had been prepared in blue light.
Once an eye had been exposed to light in order to bleach part or all of its photosensitive contents
the source was switched on in between readings so as to keep the eye in a photochemically steady
state.
The time occupied by the measurements was about 35 min when the bleach was total, and nearly
an hour when two fractional bleaches were done.
Correction factor. The theory underlying the determination of the factor a which takes into
account the fraction of light scattered into the photometer by irregularities in the crystalline lens
is given in the Appendix.

RESULTS
The total white bleaches. The averages of five total white bleaches done on
albino and pigmented animals both after an hour's (LA) and 16-20 hr (DA)
dark-adaptation are shown in Fig. 1. The data for the pigmented animals are
double-humped. In spite of the sizeable standard error (due more to variations
576
576R.AWEL R. A. WEALE
amongst animals than to errors of measurement) the humps are considered to
be significant. Two peaks occurred in every single run in both the LA and DA
set of experiments; the only variation consisted in their precise spectral location.
The individual measurements on the albinos were always maximal at about
500 mu, but all showed slight humps or shoulders at 460-470 m,u.

600 650 450 500

Wavelength (mj.)
Fig. 1. Loss of density ADR at various wavelengths resulting from total bleaching with light
source (fi1). A. Pigmented guinea-pigs, nos. 19, 20, 23, 27 and 28, dark-adapted for
16-20 hr. B. Pigmented guinea-pigs, nos. 1, 4, 5, 18 and 34, dark-adapted for 1 hr. C. Albino
guinea-pigs, nos. 26, 32, 33, 35 and 92, dark-adapted for 16-20 hr. D. Albino guinea-pigs,
nos. 7, 10, 11, 15 and 17, dark-adapted for 1 hr.

The fractional bleaches. Results obtained from chromatic bleaches with

yellow-green light are shown in Fig. 2. The maximum is at about 510 mU,
and there is a subsidiary hump in the orange part of the spectrum. The data
resuilting from subsequent white bleaches are shown below the former; they
are maximal at about 460 m,u (this was observed in every run).
Fig. 3 shows results obtained when the bleaching light was blue. There is
a sharp peak at 460 m,u (obtained in every run) with a broad hump at longer
wavelengths. This secondary maximum was ill-defined in the individual
experiments. The subsequent white bleaches produced points maximal at
about 500 mu and a faint shoulder at about 540 m,u.
 PIGMENTS IN THE GUINEA-PIG EYE 577

Yellow-green
006 followed by white
004 _
MMI LF-
A
._

006

, 004 F
0o
0021 0
B
0 I

-0-02
I I
450 500 550 600 650
Wavelength (m,u)
Fig. 2. Loss of density ADA at various wavelengths resulting from A, partial bleaching with light
source f2; B, bleaching to completion with light source p. Albino guinea-pigs, nos. 51, 53,
55, 60 and 62.

008 Blue followed

006 bywhite
0*04
0-02
40
c

-55 0-12-
0510 4 0
004
0-08- \

0-06

0*04-
0-02 -

450 500 550 600 650

Wavelength (m,u)
Fig. 3. Loss of density AD3 at various wavelengths resulting from A, partial bleaching;with light
source P.; B, bleaching to completion with light source I,L. Albino guinea-pigs, nos. 65, 66,
69, 79 and 80.
37 PHYSIO. CXXVII
578 R. A. WBAlB
The data yielded from red-light bleaches are shown in Fig. 4. At short
wavelengths the trend is negative (possibly a substance was being regenerated).
But bleaching has taken place in the orange part of the spectrum maximally
at about 610 m,u. Subsequent bleaching with white light produced a maximum
at 500 mu together with a well-marked minimum at 580 m,t, which occurred
in every individual run.
The curves in Figs. 1-4 were drawn freehand. Representative samples of
standard errors are indicated in a few instances.

002 Red followed A

by white
0
-002
012
<1
0) 10
-C
50-08-
c 0-06 -

0*04
002 -

0 0
450 S00 550 600 650
Wavelength (m,n)
Fig. 4. Loss of density ADA at various wavelengths resulting from A, partial bleaching with light
source i4,; B, bleaching to completion with light source #1. Albino guinea-pigs, nos. 81, 82,
83, 85, and 90.
DISCUSSION
Seasonal variation. The experiments were begun on 7 December 1953 and
concluded on 16 July 1954. Tansley (1931) found that the rate of regeneration
of visual purple in the rat showed a seasonal variation, and some seasonal
effect might have been expected in the present measurements. The rate of
regeneration, however, is not necessarily related to the total available amount
of visual purple, which may explain why no measurable seasonal variation in
the amounts of pigments bleached could be detected here.
Application of the correction factor a. Since equation (3) (see Appendix)
contains the antilogarithm of the measured density change ADM, the correc-
tion factor oc should be applied to every reading obtained at each wavelength.
It was found, however, that when the individual values so corrected were
 PIGMENTS IN THE GUINEA-PIG EYE 579
averaged, the difference between this mean and that resulting from an applica-
tion of cx to the uncorrected average of five values affected only the fourth
decimal place. Such a difference is insignificant. The procedure of correcting
mean values was therefore adhered to throughout the calculation of the final
results.
Although a (Fig. 7) shows a pronounced spectral variation, it has no marked effect on the
character of the bleaching curves. In some cases the difference between corrected and uncorrected
data is as much as 75%. But in no case does the application of a to the raw results lead to the
appearance or disappearance of any characteristic feature.
The total white bleaches. Experiments done on the cat (Rushton, 1952;
Weale, unpublished observations) and the rabbit (Hagins & Rushton, 1953)
with white light led to bleaching curves which could be explained by assuming
that the substance bleached was visual purple. It is clear, however, that the
double-humped data on the pigmented animals are not comparable with the
absorption spectrum of visual purple (cf. the curves shown in Fig. 5); as
mammalian visual purple differs from amphibian (Crescitelli & Dartnall, 1953),
it is visual pigment 497 which forms the basis of the present comparisons.
Although the experiments done on the albino animals provide data similar
to the absorption spectrum of visual purple, both (LA) and (DA) show a
shoulder in the blue part of the spectrum. There is little doubt that this is
connected with the hump found in this spectral region when the pigmented
animals were examined, but that the peak is, as it were, nearly submerged in
a sea of visual purple.
This raises the interesting question of the relative amount of bleaching
which occurs in these experiments. While there is only a small difference
between the ordinate values when the (DA) are compared with the (LA)
animals, the differences are sizeable when the albinos are compared with the
pigmented guinea-pigs. This suggests, at first sight, that albinos have more of
the light-sensitive substance. But it is hard to find a reason for such a state of
affairs. One would expect the reverse to be the case. Assuming the visual tasks
performed by these animals in everyday life to be similar, the unpigmented
iris of the albino will transmit more light than the pigmented one of the other
animal. The light absorption due to haemoglobin in the iris will not seriously
affect the bleaching value of the light, and thus the albino retina will receive
more light than that of the pigmented animal. But if the stimulus is stronger
the concentration of visual pigments could be smaller. In fact, the opposite
appears to be the case.
An alternative explanation suggests itself. The amounts of bleachable
material may well be the same in both types of eye. But the presence of
pigment in the pigment epithelium in the 'pigmented' eye ensures that a much
smaller fraction of the light transmitted by the retina is reflected. Thus the
bleaching in the albino is much more efficient than in the pigmented eye. This
37-2
580 R. A. WEALE
interpretation has to face the difficulty of explaining why efficiency should
matter when bleaching is complete and regeneration very slow. Further
evidence on this point is required.
Perhaps it may be emphasized that the difference between the two types is not likely to be due
to some obvious technical cause, such as bleaching by the test-lights. It has previously been shown
that this was unlikely to occur in any of the experiments. In any case, owing to the greater
reflectivity of the albino fundus it was possible to use lights about half as intense as those employed
in the pigmented eyes. In such circumstances, a greater density change would be expected to
occur in these eyes than in the albino. Since the reverse is true, additional evidence is provided
for the belief that the test-lights have no measurable effect on the observed density changes.
The partial bleaches. The total white bleaches seemed to indicate that visual
pigment 497 could not be the only photosensitive component present in the
guinea-pig retina: what other photosensitive pigments were present? Their
existence was shown more clearly in the data on the pigmented than in those
on the albino animals. But the measurements on the former were so arduous
because of the small amount of light reflected from the pigmented fundus that
it was decided to continue the work on albinos only. This decision involved a
gamble because it was taken for granted that there was no essential photo-
chemical difference between the two types of eye.
Dartnal (1952) has shown that when a thermally stable photosensitive solution is exposed in
turn to two or more bleaching lights of different spectral composition, conclusive evidence can be
obtained as to the minimum number of independent photosensitive components present in the
solution. If the photosensitive contents of the retina can be bleached in vivo, there is good reason
to believe that this can be done also in stages with different illuminants, provided the preparation
remains stable. It was assumed in these as in the total white bleaches that if the preparation
was stable during the measurements, the only change due to bleaching was photochemical in
nature.
The first chromatic (partial) bleaching was done with yellow-green light; the
bleaching was completed with white light. The results shown in Fig. 2A,
which represent the change due to illumination with yellow-green light, are
maximal at about 510 mpt and differ from the absorption spectrum of visual
pigment 497. The additional hump in the orange part of the spectrum is pre-
sumably due to another light-sensitive component. The reason for the con-
siderable negative values at 640 and 650 m,u is unknown. Generally, such
negative data were obtained in this region only when the animal was prepared
in red light. They occurred also in the cat (Weale, 1953 a), but it is not known
at present whether or not they are due to the accumulation of some substance
owing to bleaching.
The subsequent white bleaches are maximal in the region of 460 mp. At
first sight, it might be thought that this is adequate proof of the existence of
a 'blue' pigment in its own right, and that this accounts for the humps and
shoulders obtained in this spectral region when the bleaching was white and
total.
 PIGMENTS IN THE GUINEA-PIG EYE 581
However, the displacement of the maximum from 500 to 510 m,u (in the
yellow-green bleach) might be accidental, and the peak at 460 m,t might
result merely from a secondary change occurring in a product due to the
first bleaching (cf. Wald, 1953).
To put these views to a test, a second series of experiments was carried out
in which the eyes were first exposed to blue and then to white light. Fig. 3
shows the results obtained under these conditions; the peak in the former data
is in the same spectral region as that due to the second bleach in Fig. 2. The
data obtained from the subsequent white bleach are maximal at 500 mit. The
shoulder shown at about 540 mu is consistent with the displacement of the
maximum from 500 to 510 m,u as found in the yellow-green bleaches (i.e.
Fig. 2A), but it is uncertain whether this is significant.
The narrowness of the peak at 460 mut in both Figs. 2B and 3A calls for
some comment. It is impossible to say whether or not it represents a 'narrow-
band' pigment (cf. Arden, 1954 a). In the present experiments, measurements
were made of difference spectra. Although such spectra may be similar to
absorption spectra under certain conditions (cf. Arden, 1954 b), the eyes under
examination do not contain only one photosensitive component. Thus the
accumulation of a decomposition product of one component-which would
normally be manifested as an increase in density, i.e. by negative AXDM
values-may partly mask the disappearance of another light-sensitive sub-
stance, represented by positive ADM values. For example, the temporary
accumulation of a decomposition product due to a pigment absorbing
maximally, say at 540 m,, could, as it were, sharpen the spectral variation
of the disappearance of one absorbing maximally at 460 m,.
This seems to be the case when the initial bleaching light is red (Fig. 4).
Selective bleaching occurs in the orange part of the spectrum. However, the
intensity employed in these experiments does not appear to have denuded the
eye of all the red-sensitive substance present. On subsequently bleaching the
eye with white light, when the main density change occurred at about 500 mp,
there was a further small change between 600 and 650 mp,, but at 580 m,u there
was an increase in density (the standard error at this wavelength is smaller
than the symbol used for the point); values at 540 and 560 m,u are also some-
what lower than would be expected on the basis of the other measurements.
This may be due to the accumulation of a product of the red-sensitive com-
ponent cancelling the density decrease due to the disappearance of a green-
sensitive component. There is no suggestion here that this cancellation is
photochemical in nature; it is caused, in all probability, by a densitometric
effect.
Comparison of total and combined partial bleaches. As decomposition products
due to the chromatic bleach may be present during the subsequent white
bleach, a rigorous correlation between the sum of the two partial bleaches and
582 R. A. WEALE
the total white bleach (Fig. 1C) would not necessarily be expected. On the
other hand, if the three types of chromatic and partial white bleaches are
averaged, the above secondary effects will lose much of their significance. Such
an averaging has been done for fifteen animals in Fig. 5a (white circles), where

014 -* White bleaches -

012 ( 0 Fractional
*1 9r \\bleaches
X010 _ // \\ --- Absorption
<I// \\ * spectrum of visual
0, 008 / lVo pigment 497
CZ
-C
u 0-06

0-04
002 - o
0
0
0
I,, ,,,, I,,
,, I
450 S00 550 600 650
Wavelength (mis)

Regions
003 - (b) of modulator
activity in
, 002 the guinea-pig

0-01

450 500 550 600 650

Wavelength (mu)
Fig. 5. (a) Comparison of the sums of the partial with total bleaches. 0, bleaches with light source
&, albino guinea-pigs, nos. 26, 32, 33, 35, 93, 97 and 105. 0, mean of sums of partial bleaches
obtained from Figs. 2-4. - - -, absorption spectrum of visual pigment 497 corresponding
to a density of 0-13. , ditto for 0-12 (for details see text). (b). Difference between the
averages of 0 and 0, and the template curve for a density of 0-12 in Fig. 5a. The horizontal
lines represent regions of modulator activity found in the guinea-pig eye by Granit (1942).

the data are compared with seven total white bleaches (black circles). The
latter contain five data for animals prepared in red, and two prepared in blue
light, so as to make the results more nearly comparable. Perhaps it should be
emphasized that both sets of points represent the actual density changes to
which o has been applied; the ordinates of neither set have been scaled to
 PIGMENTS IN THE GUINEA-PIG EYE 583
facilitate the comparison. The agreement between the two sets is as satisfactory
as can be expected from the conditions of the experiments.
Comparison with the absorption spectrum of visual pigment 497. All mam-
malian retinae from which extracts have been made and examined have been
found to contain a visual pigment whose maximum of absorption occurs at
a wavelength of about 497 m,. The absorption spectrum of this substance,
which presumably exists also in the guinea-pig retina, provides a yard-stick
with which to assess the present data. In all tests of this nature an arbitrary
assumption is made as regards the relative scaling of the data to be examined.
In the present case, the amount of presumed pigment chosen was such as to
avoid negative values when the density of the pigment was subtracted from
the experimental data. That is to say, the curve was equated to the data at
A= 480 m,u. Its maximum thus corresponds to a density of 0412.
The agreement is poor, and cannot be improved by increasing the presumed
amount of visual pigment 497 to correspond to a value of, say, 0 13 (cf. Fig. 5a).
While the density change due to bleaching is about 0413-0-14, it is impossible
to decide on the contribution of visual pigment 497 to this value. The necessity
to effect partial bleaching in a relatively short time enforces the use of rather
impure chromatic light. This renders a precise characterization of the indivi-
dual photosensitive components of a retina impossible. While the results of
Figs. 2-4 have failed in accurately describing the pigments present in the
guinea-pig retina, it may be said to contain at least three components other
than visual pigment 497.
It also follows that the sum of the absorptions of several pigments can
approximately mimic the smooth absorption spectrum of a single substance,
and-unless partial bleaching is carried out in vivo as well as in vitro-mis-
leading deductions can be made from the appearance of both absorption and
bleaching spectra.
Comparison with electrophysiological work. Granit (1942) has published data
on the spectral variation of single-fibre potentials in light- and dark-adapted
guinea-pig retinae. The sensitivity curve for dark-adapted receptor-fibres
agrees well with the absorption spectrum of visual pigment 497 except in the
region of 450 m,u. It also agrees with the data shown in Fig. 1 C. According
to Granit, 'the large value in 0'450,u comes from two series with a markedly
blue-sensitive element despite dark-adaptation'. He also found evidence for
modulator-type elements active mainly in the spectral regions indicated in
Fig. 5b. If D., the density of the visual pigment 497, is subtracted from the
mean of the experimental data in Fig. 5 a, residuals are obtained which are
plotted in Fig. 5 b. Their spectral variation, which varies only slowly with the
maximum assumed for Dp, suggests that the photochemical components
revealed by partially bleaching the guinea-pig eye (Figs. 2-4) may perhaps
provide a basis for the modulators which Granit found in this eye.
584 R. A. WEALE

SUMMARY
1. Bleaching spectra were measured in twenty-seven albino and ten
pigmented anaesthetized, curarized guinea-pigs.
2. The bleaching sources were: white, red, yellow-green, or blue.
3. Prolonged dark-adaptation of the animals before the experiment led to
greater density changes than in the absence of dark-adaptation.
4. The white bleaches suggested the presence of more than one photo-
sensitive component in the guinea-pig's retina.
5. This was confirmed by the chromatic bleaches. It is thought that four
such components may be present in this eye.
6. The data are compared with others obtained in photochemical and
electrophysiological investigations.
I should like to thank Miss Daphne Taylor for her able assistance in these experiments.

APPENDIX
Theory. Brindley & Willmer (1952) have suggested that, when the intensity of light reflected
from the human fundus is measured, allowance may have to be made for the light scattered into
the detector by the ocular media. Hagins (personal communication) determined the fraction of
light scattered in the lens of the albino rabbit by injecting the vitreous with indian ink.

isIi
I
lgl\TI\ s ~I, /,'X1
II~~RT(
-) //I

(a) (b)
Fig. 6. See text.

A simple theory shows that the operative factor is not so much the amount of light scattered by
the lens, as the ratio of this quantity to that reflected from the fundus. In the present apparatus,
in which a narrow pencil of light enters the eye, and the minute amount of light reflected at the
air-eye surface can be deflected away from the apparatus simply by tilting the cover-slip over the
eye, this ratio can be determined directly. Let I be the intensity of the monochromatic test-beam
incident on the eye (Fig. 6a). The amount scattered backward within a small solid angle dO is Is.
The intensity on the posterior side of the lens is therefore I (1 - S), where S represents the total
amount of light scattered. In the dark-adapted eye, the retina transmits a fraction TD of the
 PIGMENTS IN THE GUINEA-PIG EYE 585
incident light to the fundus, where a fraction R is reflected towards the detector. Hence the
measured intensity of the returning beam is I[RT2 (1 - S)2 + 8]. A similar expression is obtained
for the bleached eye, the retina transmitting a fraction TL of the incident light (Fig. 6b). Thus the
measured, as distinct from the actual, change in the transmissivity of the retina, which occurs
during bleaching, is given by the ratio of these quantities, namely,
T2m RTD(1-8)2+s 1)
RT2(1-S)2+s (

If 8 is very small as compared with the first terms in the numerator and denominator respectively,
T2 T12DI/ITL, or the measured is equal to the actual change in transmissivity. This obtains when
R is large, e.g. when the fundus is covered with a tapetum.
When the eye was viewed, not through the photometer, but from a point above, and to one side
of, the path of the test-beam, one could see two linear patches of light. The anterior one was due
to light scattered in the lens, the posterior to the reflexion at the fundus. The separation between
the two enabled one to compare their brightness.

* Pigmented guinea-pigs
06 - o Albino guinea-pigs

0.5
0-4

0-3

0-2

01 /\
0
450 500 550 600
Wavelength (mg)
Fig. 7. Values of oc. Pigmented guinea-pigs nos. 98, 99, 100,
albino guinea-pigs nos. 102, 103 and 105.

Visual examination showed that within an angle of ±20' from the direction of the incident
beam there was no appreciable variation with viewing angle of the amounts of light scattered by
the lens and reflected by the fundus respectively. It could thus be assumed that the ratio (light
scattered by the lens) + (light reflected from fundus) when measured at an angle of about 150 would
be the same as along the direction of the incident beam.
The brightnesses of the two beams were equated by placing in the path of the brighter beam
a neutral filter of suitable density. A series of such filters whose nominal values increased by
0*1 was calibrated at each wavelength (Weale, 1951). Let the transmissivity of the filter equating
the two brightnesses be oc. It follows from Fig. 6b that
8 (2)
RT2 (1 - S)2'
if the eye remains completely bleached. This was ensured by switching on source Pr, between the
matches, a precaution which may well be thought unnecessary in view of the slowness of regenera-
tion of photochemical substances in the guinea-pig retina.
586 R. A. WEALE
Substituting (2) in (1),
Xm l+x
D _T2. (1 +ot) -a,

ADR=ADM- ilog1O (1 +am-2) (3)

where ADR and ADM are the real and measured density changes respectively. In effect, the real
is always greater than the measured change, no matter whether ADM is positive or negative.
Re&ults. The correction factor a was determined on three pigmented (40) and three albino (0)
guinea-pigs respectively (Fig. 7). The two bands superimposed on the general trend of the curves
at 540 and 580 mu are due to haemoglobin. In the region of the haemoglobin bands light is
absorbed strongly and a correspondingly smaller proportion reflected. But, according to equation
(2), a reduction in B leads to an increase in a, which is in fact observed. The poor reflexion in
pigmented eyes also explains why a for these animals is so much higher than for the albinos.

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