Abstract

A bipartite field (retinal irradiance ratio 2:1 on the two sides) was imaged on the retina of an albino rat. Following exposure to the visual stimulus, the retina was dissected from the eye and placed in an incubation medium which selectively stains the ellipsoid portion of photoreceptors which have been exposed to light. In this study, the optical density of stain was measured as a function of stimulus magnitude. It was shown that the density of stain increased with increasing retinal irradiance over the range of values tested. The indicator system tended to underestimate differences in stimulus magnitude, and showed evidence of approaching an asymptote at higher stimulus levels. Many technical problems encountered in this experiment made this a difficult exercise.

© 1966 Optical Society of America

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Corrections

Jay M. Enoch, "Errata: Validation of an Indicator of Mammalian Retinal Receptor Response: Density of Stain as a Function of Stimulus Magnitude.," J. Opt. Soc. Am. 56, 529-529 (1966)
https://www.osapublishing.org/josa/abstract.cfm?uri=josa-56-4-529

References

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  1. J. M. Enoch, Invest. Ophthalmol. 2, 16 (1963).
  2. Somewhat similar incubation media are used to localize the sites of activity of the enzyme, succinic dehydrogenase. When succinate, the substrate, is present (in the medium in which the tissue is incubated), the nitro-blue tetrazolium is reduced at some point in the succinoxidose system, and a blue-violet insoluble pigment (dinitro-formazan) is deposited, most probably at the point where the reaction occurs.
  3. J. M. Enoch, J. Opt. Soc. Am. 54, 368 (1964).
    [CrossRef] [PubMed]
  4. J. M. Enoch, J. Opt. Soc. Am. 54, 1027 (1964).
    [CrossRef] [PubMed]
  5. W. Sickel (Johns Hopkins University) writes that he has detected the reaction in frogs.
  6. J. M. Enoch, Am. J. Optom. 42, 63 (1965).
    [CrossRef]
  7. W. L. Fowlks, Proc. Soc. Exp. Biol. Med. 118, 491 (1965).
    [CrossRef] [PubMed]
  8. W. L. Fowlks, Invest. Ophthalmol. 3, 550 (1964).
  9. I. Osipova and S. Shukoljukov, Cytologia 6, 626 (1964), in Russian.
  10. B. Commoner and D. Lipkin, Science 110, 41 (1949).
    [CrossRef] [PubMed]
  11. This medium was employed because it has been shown that it provides lower stain thresholds, and results obtained seem more sharply delineated than those evoked when we used the second medium we developed.4We have obtained quantitative stability using the Sigma product. In the past, we have found it necessary to keep our NBT cold, dark, and dry. We divide a lot into several dark bottles because we find that after a sample has been used some unspecified number of times the NBT rather rapidly loses potency. A stored, second unopened bottle of the same lot has approximately original potency. (The first few mixtures made from a new bottle have an apparent increased potency over that usually measured.) We usually are able to make at least sixty valid determinations per bottle. Potency is defined in terms of threshold stimulus magnitude required to produce a stained image in our preparations.
  12. A. Pearse, Histochemistry, Theoretical and Applied (Little, Brown and Company, Boston, 1961), p. 543.
  13. R. Cone, J. Gen. Physiol. 46, 1267 (1963).See comments on this paper in Ref. 3. The reader is referred to Cone’s paper for an error analysis concerning each of the factors employed. In recent conversations with Dr. Cone, he has pointed out that he has reproduced the experiment by Lewis mentioned in Ref. 13 [D. Lewis, J. Physiol. (London) 136, 615 (1957)], and found Lewis’s values to be valid. Lewis suggested that 0.50 of the energy incident at the retina was absorbed by the rhodopsin at λ=500 nm. The value employed in Table II (0.23) was taken from measurements made by Paul Brown (Harvard University) for Dr. Cone. Brown used excised retinas, Lewis used whole excised eyes. In dead and dying retina there is increased scatter, swelling, and reduced transmittance of light.6 The orientation of the photosensitive pigment molecules may be altered. The rod receptors are physically separated, and part of the energy transmitted in a waveguide is carried outside of the cell. Lewis’s data are more subject to stray-light effects because of the integrating-sphere-like action of the rat eye. In that experiment, bleaching in an image per se cannot be distinguished from that produced by stray light on the remaining retina. I believe that the value provided by Brown may be somewhat more applicable to the measurements made here. Hence, Brown’s estimate is retained in this analysis. Obviously, this is a weak point in the treatment and it needs to be better defined.
  14. J. Dowling, J. Gen. Physiol. 46, 1287 (1963).
  15. The stray light caused by the more intensely stimulated side of the image would have a slightly greater effect upon the less stimulated side than vice versa.
  16. K. Patau, Chromosoma 5, 341 (1952).
    [CrossRef]
  17. L. Ornstein, Lab. Invest. 1, 250 (1952).
  18. W. Marks, W. Dobelle, and E. MacNichol, Science 143, 1181 (1964).
    [CrossRef] [PubMed]
  19. P. Brown and G. Wald, Science 144, 45 (1964).
    [CrossRef] [PubMed]

1965 (2)

J. M. Enoch, Am. J. Optom. 42, 63 (1965).
[CrossRef]

W. L. Fowlks, Proc. Soc. Exp. Biol. Med. 118, 491 (1965).
[CrossRef] [PubMed]

1964 (6)

W. L. Fowlks, Invest. Ophthalmol. 3, 550 (1964).

I. Osipova and S. Shukoljukov, Cytologia 6, 626 (1964), in Russian.

W. Marks, W. Dobelle, and E. MacNichol, Science 143, 1181 (1964).
[CrossRef] [PubMed]

P. Brown and G. Wald, Science 144, 45 (1964).
[CrossRef] [PubMed]

J. M. Enoch, J. Opt. Soc. Am. 54, 368 (1964).
[CrossRef] [PubMed]

J. M. Enoch, J. Opt. Soc. Am. 54, 1027 (1964).
[CrossRef] [PubMed]

1963 (3)

J. M. Enoch, Invest. Ophthalmol. 2, 16 (1963).

R. Cone, J. Gen. Physiol. 46, 1267 (1963).See comments on this paper in Ref. 3. The reader is referred to Cone’s paper for an error analysis concerning each of the factors employed. In recent conversations with Dr. Cone, he has pointed out that he has reproduced the experiment by Lewis mentioned in Ref. 13 [D. Lewis, J. Physiol. (London) 136, 615 (1957)], and found Lewis’s values to be valid. Lewis suggested that 0.50 of the energy incident at the retina was absorbed by the rhodopsin at λ=500 nm. The value employed in Table II (0.23) was taken from measurements made by Paul Brown (Harvard University) for Dr. Cone. Brown used excised retinas, Lewis used whole excised eyes. In dead and dying retina there is increased scatter, swelling, and reduced transmittance of light.6 The orientation of the photosensitive pigment molecules may be altered. The rod receptors are physically separated, and part of the energy transmitted in a waveguide is carried outside of the cell. Lewis’s data are more subject to stray-light effects because of the integrating-sphere-like action of the rat eye. In that experiment, bleaching in an image per se cannot be distinguished from that produced by stray light on the remaining retina. I believe that the value provided by Brown may be somewhat more applicable to the measurements made here. Hence, Brown’s estimate is retained in this analysis. Obviously, this is a weak point in the treatment and it needs to be better defined.

J. Dowling, J. Gen. Physiol. 46, 1287 (1963).

1952 (2)

K. Patau, Chromosoma 5, 341 (1952).
[CrossRef]

L. Ornstein, Lab. Invest. 1, 250 (1952).

1949 (1)

B. Commoner and D. Lipkin, Science 110, 41 (1949).
[CrossRef] [PubMed]

Brown, P.

P. Brown and G. Wald, Science 144, 45 (1964).
[CrossRef] [PubMed]

Commoner, B.

B. Commoner and D. Lipkin, Science 110, 41 (1949).
[CrossRef] [PubMed]

Cone, R.

R. Cone, J. Gen. Physiol. 46, 1267 (1963).See comments on this paper in Ref. 3. The reader is referred to Cone’s paper for an error analysis concerning each of the factors employed. In recent conversations with Dr. Cone, he has pointed out that he has reproduced the experiment by Lewis mentioned in Ref. 13 [D. Lewis, J. Physiol. (London) 136, 615 (1957)], and found Lewis’s values to be valid. Lewis suggested that 0.50 of the energy incident at the retina was absorbed by the rhodopsin at λ=500 nm. The value employed in Table II (0.23) was taken from measurements made by Paul Brown (Harvard University) for Dr. Cone. Brown used excised retinas, Lewis used whole excised eyes. In dead and dying retina there is increased scatter, swelling, and reduced transmittance of light.6 The orientation of the photosensitive pigment molecules may be altered. The rod receptors are physically separated, and part of the energy transmitted in a waveguide is carried outside of the cell. Lewis’s data are more subject to stray-light effects because of the integrating-sphere-like action of the rat eye. In that experiment, bleaching in an image per se cannot be distinguished from that produced by stray light on the remaining retina. I believe that the value provided by Brown may be somewhat more applicable to the measurements made here. Hence, Brown’s estimate is retained in this analysis. Obviously, this is a weak point in the treatment and it needs to be better defined.

Dobelle, W.

W. Marks, W. Dobelle, and E. MacNichol, Science 143, 1181 (1964).
[CrossRef] [PubMed]

Dowling, J.

J. Dowling, J. Gen. Physiol. 46, 1287 (1963).

Enoch, J. M.

Fowlks, W. L.

W. L. Fowlks, Proc. Soc. Exp. Biol. Med. 118, 491 (1965).
[CrossRef] [PubMed]

W. L. Fowlks, Invest. Ophthalmol. 3, 550 (1964).

Lipkin, D.

B. Commoner and D. Lipkin, Science 110, 41 (1949).
[CrossRef] [PubMed]

MacNichol, E.

W. Marks, W. Dobelle, and E. MacNichol, Science 143, 1181 (1964).
[CrossRef] [PubMed]

Marks, W.

W. Marks, W. Dobelle, and E. MacNichol, Science 143, 1181 (1964).
[CrossRef] [PubMed]

Ornstein, L.

L. Ornstein, Lab. Invest. 1, 250 (1952).

Osipova, I.

I. Osipova and S. Shukoljukov, Cytologia 6, 626 (1964), in Russian.

Patau, K.

K. Patau, Chromosoma 5, 341 (1952).
[CrossRef]

Pearse, A.

A. Pearse, Histochemistry, Theoretical and Applied (Little, Brown and Company, Boston, 1961), p. 543.

Shukoljukov, S.

I. Osipova and S. Shukoljukov, Cytologia 6, 626 (1964), in Russian.

Sickel, W.

W. Sickel (Johns Hopkins University) writes that he has detected the reaction in frogs.

Wald, G.

P. Brown and G. Wald, Science 144, 45 (1964).
[CrossRef] [PubMed]

Am. J. Optom. (1)

J. M. Enoch, Am. J. Optom. 42, 63 (1965).
[CrossRef]

Chromosoma (1)

K. Patau, Chromosoma 5, 341 (1952).
[CrossRef]

Cytologia (1)

I. Osipova and S. Shukoljukov, Cytologia 6, 626 (1964), in Russian.

Invest. Ophthalmol. (2)

W. L. Fowlks, Invest. Ophthalmol. 3, 550 (1964).

J. M. Enoch, Invest. Ophthalmol. 2, 16 (1963).

J. Gen. Physiol. (2)

R. Cone, J. Gen. Physiol. 46, 1267 (1963).See comments on this paper in Ref. 3. The reader is referred to Cone’s paper for an error analysis concerning each of the factors employed. In recent conversations with Dr. Cone, he has pointed out that he has reproduced the experiment by Lewis mentioned in Ref. 13 [D. Lewis, J. Physiol. (London) 136, 615 (1957)], and found Lewis’s values to be valid. Lewis suggested that 0.50 of the energy incident at the retina was absorbed by the rhodopsin at λ=500 nm. The value employed in Table II (0.23) was taken from measurements made by Paul Brown (Harvard University) for Dr. Cone. Brown used excised retinas, Lewis used whole excised eyes. In dead and dying retina there is increased scatter, swelling, and reduced transmittance of light.6 The orientation of the photosensitive pigment molecules may be altered. The rod receptors are physically separated, and part of the energy transmitted in a waveguide is carried outside of the cell. Lewis’s data are more subject to stray-light effects because of the integrating-sphere-like action of the rat eye. In that experiment, bleaching in an image per se cannot be distinguished from that produced by stray light on the remaining retina. I believe that the value provided by Brown may be somewhat more applicable to the measurements made here. Hence, Brown’s estimate is retained in this analysis. Obviously, this is a weak point in the treatment and it needs to be better defined.

J. Dowling, J. Gen. Physiol. 46, 1287 (1963).

J. Opt. Soc. Am. (2)

Lab. Invest. (1)

L. Ornstein, Lab. Invest. 1, 250 (1952).

Proc. Soc. Exp. Biol. Med. (1)

W. L. Fowlks, Proc. Soc. Exp. Biol. Med. 118, 491 (1965).
[CrossRef] [PubMed]

Science (3)

B. Commoner and D. Lipkin, Science 110, 41 (1949).
[CrossRef] [PubMed]

W. Marks, W. Dobelle, and E. MacNichol, Science 143, 1181 (1964).
[CrossRef] [PubMed]

P. Brown and G. Wald, Science 144, 45 (1964).
[CrossRef] [PubMed]

Other (5)

The stray light caused by the more intensely stimulated side of the image would have a slightly greater effect upon the less stimulated side than vice versa.

Somewhat similar incubation media are used to localize the sites of activity of the enzyme, succinic dehydrogenase. When succinate, the substrate, is present (in the medium in which the tissue is incubated), the nitro-blue tetrazolium is reduced at some point in the succinoxidose system, and a blue-violet insoluble pigment (dinitro-formazan) is deposited, most probably at the point where the reaction occurs.

This medium was employed because it has been shown that it provides lower stain thresholds, and results obtained seem more sharply delineated than those evoked when we used the second medium we developed.4We have obtained quantitative stability using the Sigma product. In the past, we have found it necessary to keep our NBT cold, dark, and dry. We divide a lot into several dark bottles because we find that after a sample has been used some unspecified number of times the NBT rather rapidly loses potency. A stored, second unopened bottle of the same lot has approximately original potency. (The first few mixtures made from a new bottle have an apparent increased potency over that usually measured.) We usually are able to make at least sixty valid determinations per bottle. Potency is defined in terms of threshold stimulus magnitude required to produce a stained image in our preparations.

A. Pearse, Histochemistry, Theoretical and Applied (Little, Brown and Company, Boston, 1961), p. 543.

W. Sickel (Johns Hopkins University) writes that he has detected the reaction in frogs.

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Figures (4)

Fig. 1
Fig. 1

(A) A bipartite field was imaged on the rat retina. The numbers indicate the schematic distribution of transmittance measurements made in the two parts of the image and image surround. These determinations were used in computing optical density of the stain. (B) The shape of the image and the relative stimulus magnitudes employed are shown. (C) and (D). These schematic drawings relate to an experimental design (see Discussion) which may be used to test the response characteristics of more than one set of receptors. Figure 1(B) duplicates the test requirements of configuration 1(C).

Fig. 2
Fig. 2

Plot of the density of formazan stain measured versus computed quanta absorbed (or molecules of rhodopsin bleached) per retinal rod per second, for a 300-sec exposure. Computed percent rhodopsin bleached is also plotted. The points indicate the measured values, and the lines connect the individual pairs of values determined for each preparation. The larger open circles represent data taken from class A retinal specimens, and the smaller dots indicate the determinations made on class B specimens.

Fig. 3
Fig. 3

Same as Fig. 2 except that the abscissa is plotted on a logarithmic scale. The abscissa is elongated in order that the slopes of the lines connecting the data points correspond to the tangent values plotted in Fig. 4. For comparison purposes, the line to the left has a slope (tangent) equal to 1.0 (45°). If the abscissa were shortened so that these data extend over the same proportional distance across the graph as in Fig. 2, no conclusions would be altered. Large open circles represent data taken from class A specimens and small dots indicate results determined on class B specimens.

Fig. 4
Fig. 4

Slopes of lines in Figure 3. Slopes are shown in terms of ΔD (to the left), and the tangent of the slope angle (to the right). There was a difference in stimulus magnitude of 0.3 log units on the two sides of the stimulus [Fig. 1(b)]. Class A retinas are indicated by large open circles and class B specimens by small dots. The individual points are plotted on a logarithmic scale at the geometric mean of the two values of quanta absorbed per rod per second (Qa) computed for each retina.

Tables (4)

Tables Icon

Table II Values employed in computations.

Tables Icon

Table III Stain threshold.

Tables Icon

Table IV Data and computed values.

Equations (3)

Equations on this page are rendered with MathJax. Learn more.

D = f 1 ( c ) ;             c = f 2 ( r ) ;             f 3 ( Q a ) ;             Q a = f 4 ( H τ ) .
R = exp [ - Q a t / u ] .
tan θ = [ ( D 2 - D 1 ) / ( log Q a 2 - log Q a 1 ) ] = ( Δ D / 0.3 ) .