Abstract

A novel rapid scanning microspectrophotometer is described which utilizes a cathode ray tube as a measuring light source. Spectral scanning is accomplished electronically with a sampling time of 600μs for each waveband. The cathode ray tube emission is chopped electronically into two separate beams, 180° out of phase, resulting in a dual-beam configuration. A lock-in amplifier functions as a coherent detector to recover separately the signals from the two beams. The instrument generates separate voltage outputs, one proportional to the transmittance of a single sample and the other to the difference between two samples. A computer calculates both absorption and difference spectra directly from voltage measurements. A demonstration of the instrument’s use to study kinetics of visual pigment photoproducts is presented. Two models of photoproduct sequence and kinetics were examined to determine which better represents the experimental data. The experiments show that environmental factors, such as pH, metabolic and respiratory state, interact in complex ways to determine the pathways and kinetics of photoproducts of rhodopsin in intact vertebrate eyes.

© 1978 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. T. O. Caspersson, “Methods for the determination of the absorption spectra of cell structures,” J. R. Micr. Soc. 60, 8–25(1940).
    [CrossRef]
  2. B. Chance, R. Perry, L. Akerman, and B. Thorell, “Highly sensitive recording microspectrophotometer,” Rev. Sci. Instrum. 30, 735–741(1959).
    [CrossRef]
  3. P. K. Brown, “A system for microspectrophotometry employing a commercial recording microspectrophotometer,” J. Opt. Soc. Am. 51, 1000–1008(1961).
    [CrossRef]
  4. P. A. Liebman and G. Entine, “Sensitive low-light-level microspectrophotometer: Detection of photosensitive pigments of retinal cones,” J. Opt. Soc. Am. 54, 1451–1459(1964).
    [CrossRef] [PubMed]
  5. W. B. Marks, “Difference spectra of the visual pigments in single goldfish cones,” Ph.D. thesis (Johns Hopkins University, 1963); W. B. Marks, “Visual pigments of single goldfish cones,” J. Physiol. 178, 14–32(1965).
  6. F. I. Harosi and E. F. MacNichol, “Dichroic microspectrophotometer: A computer-assisted, rapid, wavelength-scanning photometer for measuring linear dichroism in single cells,” J. Opt. Soc. Am. 64, 903–918(1974).
    [CrossRef] [PubMed]
  7. T. Hanaoka and K. Fujimoto, “Absorption spectrum of a single cone in carp retina,” Jpn. J. Physiol. 7, 276–285(1957).
    [CrossRef] [PubMed]
  8. G. E. Busch, M. L. Applebury, A. A. Lamola, and P. M. Rentzepis, “Formation and decay of prelumirhodopsin at room temperature,” Proc. Natl. Acad. Sci. (U.S.) 69, 2802–2806(1972).
    [CrossRef]
  9. P. A. Liebman, W. S. Jagger, M. W. Kaplan, and F. G. Bargoot, “Membrane structure changes in rod outer segments associated with rhodopsin bleaching,” Nature 251, 31–36(1974).
    [CrossRef] [PubMed]
  10. C. Baumann, “The formation of metarhodopsin 380 in the retinal rods of the frog,” J. Physiol. 259, 357–366(1976).
  11. J. Koszewski, J. Jasny, and Z. R. Grabowski, “Rapid scan flash spectrophotometer with a flying spot light source and magnetic storage,” Appl. Opt. 7, 2178–2183(1968).
    [CrossRef] [PubMed]
  12. R. N. Frank, “Photoproducts of rhodopsin bleaching in the isolated, perfused frog retina,” Vision Res. 9, 1415–1433(1969).
    [CrossRef] [PubMed]
  13. C. Baumann, “Kinetics of slow thermal reactions during the bleaching of rhodopsin in the perfused frog retina,” J. Physiol. 222, 643–663(1972).
  14. G. Gyllenberg, T. Reuter, and H. Sippel, “Long-lived photoproducts of rhodopsin in the retina of the frog,” Vision Res. 14, 1349–1357(1974).
    [CrossRef] [PubMed]
  15. H. H. A. Dartnall, “The photobiology of visual processes,” in The Eye, edited by H. Davson, Part II, Vol. 2, (Academic, New York, 1962) pp. 321–533.
  16. R. A. Cone and W. H. Cobbs, “Rhodopsin cycle in the living eye of the rat,” Nature 221, 820–822 (1969).
    [CrossRef] [PubMed]
  17. K. P. Brin and H. Ripps, “Rhodopsin photoproducts and rod sensitivity in the skate retina,” J. Gen. Physiol. 69, 97–120 (1977).
  18. G. D. Knott and D. K. Reece, “MLAB: A civilized curve-fitting system,” Proc. ONLINE ’72 Intl. Conf., Brunel Univ. Eng. 1, 497–526(1972).
  19. All coefficients are normalized with respect to the amount of rhodopsin bleached to allow for comparisons among different experiments.
  20. J. I. Korenbrot and R. A. Cone, “Dark ionic flux and the effects of light in isolated rod outer segments,” J. Gen. Physiol. 60, 20–45(1972).
  21. H. S. Maker and G. M. Lehrer, “Carbohydrate chemistry of brain,” in Basic Neurochemistry, edited by R. W. Albers, G. J. Siegel, R. Katzmann, and B. W. Agranoff (Little, Brown, Boston, 1972), pp. 169–190.
  22. A. L. Lehninger, in Biochemistry, 2nd ed. (Worth, New York, 1975), pp. 417–542.
  23. A. N. Wick, D. R. Dury, M. I. Nakada, and J. B. Wolfe, “Localization of the primary metabolic block produced by 2-deoxyglucose,” J. Biol. Chem. 224, 963–969(1967).
  24. J. K. Bowmaker, “The photoproducts of retinal-based visual pigments in situ: A contrast between Rana pipiens and Gekko gekko,” Vision Res. 13, 1227–1240(1973).
    [CrossRef] [PubMed]
  25. Because of measurement limitations of the RMSP in the ultraviolet spectrum, it was not possible to detect the formation of retinol, the final stable photoproduct with absorption maximum at 32 nm.
  26. R. Paulsen, J. A. Miller, A. E. Brodie, and M. D. Bownds, “The decay of long-lived photoproducts in the isolated bullfrog rod outer segment: Relationship to other dark reactions,” Vision Res. 15, 1325–1332(1975).
    [CrossRef] [PubMed]

1977 (1)

K. P. Brin and H. Ripps, “Rhodopsin photoproducts and rod sensitivity in the skate retina,” J. Gen. Physiol. 69, 97–120 (1977).

1976 (1)

C. Baumann, “The formation of metarhodopsin 380 in the retinal rods of the frog,” J. Physiol. 259, 357–366(1976).

1975 (1)

R. Paulsen, J. A. Miller, A. E. Brodie, and M. D. Bownds, “The decay of long-lived photoproducts in the isolated bullfrog rod outer segment: Relationship to other dark reactions,” Vision Res. 15, 1325–1332(1975).
[CrossRef] [PubMed]

1974 (3)

G. Gyllenberg, T. Reuter, and H. Sippel, “Long-lived photoproducts of rhodopsin in the retina of the frog,” Vision Res. 14, 1349–1357(1974).
[CrossRef] [PubMed]

P. A. Liebman, W. S. Jagger, M. W. Kaplan, and F. G. Bargoot, “Membrane structure changes in rod outer segments associated with rhodopsin bleaching,” Nature 251, 31–36(1974).
[CrossRef] [PubMed]

F. I. Harosi and E. F. MacNichol, “Dichroic microspectrophotometer: A computer-assisted, rapid, wavelength-scanning photometer for measuring linear dichroism in single cells,” J. Opt. Soc. Am. 64, 903–918(1974).
[CrossRef] [PubMed]

1973 (1)

J. K. Bowmaker, “The photoproducts of retinal-based visual pigments in situ: A contrast between Rana pipiens and Gekko gekko,” Vision Res. 13, 1227–1240(1973).
[CrossRef] [PubMed]

1972 (4)

C. Baumann, “Kinetics of slow thermal reactions during the bleaching of rhodopsin in the perfused frog retina,” J. Physiol. 222, 643–663(1972).

G. E. Busch, M. L. Applebury, A. A. Lamola, and P. M. Rentzepis, “Formation and decay of prelumirhodopsin at room temperature,” Proc. Natl. Acad. Sci. (U.S.) 69, 2802–2806(1972).
[CrossRef]

G. D. Knott and D. K. Reece, “MLAB: A civilized curve-fitting system,” Proc. ONLINE ’72 Intl. Conf., Brunel Univ. Eng. 1, 497–526(1972).

J. I. Korenbrot and R. A. Cone, “Dark ionic flux and the effects of light in isolated rod outer segments,” J. Gen. Physiol. 60, 20–45(1972).

1969 (2)

R. A. Cone and W. H. Cobbs, “Rhodopsin cycle in the living eye of the rat,” Nature 221, 820–822 (1969).
[CrossRef] [PubMed]

R. N. Frank, “Photoproducts of rhodopsin bleaching in the isolated, perfused frog retina,” Vision Res. 9, 1415–1433(1969).
[CrossRef] [PubMed]

1968 (1)

1967 (1)

A. N. Wick, D. R. Dury, M. I. Nakada, and J. B. Wolfe, “Localization of the primary metabolic block produced by 2-deoxyglucose,” J. Biol. Chem. 224, 963–969(1967).

1964 (1)

1961 (1)

1959 (1)

B. Chance, R. Perry, L. Akerman, and B. Thorell, “Highly sensitive recording microspectrophotometer,” Rev. Sci. Instrum. 30, 735–741(1959).
[CrossRef]

1957 (1)

T. Hanaoka and K. Fujimoto, “Absorption spectrum of a single cone in carp retina,” Jpn. J. Physiol. 7, 276–285(1957).
[CrossRef] [PubMed]

1940 (1)

T. O. Caspersson, “Methods for the determination of the absorption spectra of cell structures,” J. R. Micr. Soc. 60, 8–25(1940).
[CrossRef]

Akerman, L.

B. Chance, R. Perry, L. Akerman, and B. Thorell, “Highly sensitive recording microspectrophotometer,” Rev. Sci. Instrum. 30, 735–741(1959).
[CrossRef]

Applebury, M. L.

G. E. Busch, M. L. Applebury, A. A. Lamola, and P. M. Rentzepis, “Formation and decay of prelumirhodopsin at room temperature,” Proc. Natl. Acad. Sci. (U.S.) 69, 2802–2806(1972).
[CrossRef]

Bargoot, F. G.

P. A. Liebman, W. S. Jagger, M. W. Kaplan, and F. G. Bargoot, “Membrane structure changes in rod outer segments associated with rhodopsin bleaching,” Nature 251, 31–36(1974).
[CrossRef] [PubMed]

Baumann, C.

C. Baumann, “The formation of metarhodopsin 380 in the retinal rods of the frog,” J. Physiol. 259, 357–366(1976).

C. Baumann, “Kinetics of slow thermal reactions during the bleaching of rhodopsin in the perfused frog retina,” J. Physiol. 222, 643–663(1972).

Bowmaker, J. K.

J. K. Bowmaker, “The photoproducts of retinal-based visual pigments in situ: A contrast between Rana pipiens and Gekko gekko,” Vision Res. 13, 1227–1240(1973).
[CrossRef] [PubMed]

Bownds, M. D.

R. Paulsen, J. A. Miller, A. E. Brodie, and M. D. Bownds, “The decay of long-lived photoproducts in the isolated bullfrog rod outer segment: Relationship to other dark reactions,” Vision Res. 15, 1325–1332(1975).
[CrossRef] [PubMed]

Brin, K. P.

K. P. Brin and H. Ripps, “Rhodopsin photoproducts and rod sensitivity in the skate retina,” J. Gen. Physiol. 69, 97–120 (1977).

Brodie, A. E.

R. Paulsen, J. A. Miller, A. E. Brodie, and M. D. Bownds, “The decay of long-lived photoproducts in the isolated bullfrog rod outer segment: Relationship to other dark reactions,” Vision Res. 15, 1325–1332(1975).
[CrossRef] [PubMed]

Brown, P. K.

Busch, G. E.

G. E. Busch, M. L. Applebury, A. A. Lamola, and P. M. Rentzepis, “Formation and decay of prelumirhodopsin at room temperature,” Proc. Natl. Acad. Sci. (U.S.) 69, 2802–2806(1972).
[CrossRef]

Caspersson, T. O.

T. O. Caspersson, “Methods for the determination of the absorption spectra of cell structures,” J. R. Micr. Soc. 60, 8–25(1940).
[CrossRef]

Chance, B.

B. Chance, R. Perry, L. Akerman, and B. Thorell, “Highly sensitive recording microspectrophotometer,” Rev. Sci. Instrum. 30, 735–741(1959).
[CrossRef]

Cobbs, W. H.

R. A. Cone and W. H. Cobbs, “Rhodopsin cycle in the living eye of the rat,” Nature 221, 820–822 (1969).
[CrossRef] [PubMed]

Cone, R. A.

J. I. Korenbrot and R. A. Cone, “Dark ionic flux and the effects of light in isolated rod outer segments,” J. Gen. Physiol. 60, 20–45(1972).

R. A. Cone and W. H. Cobbs, “Rhodopsin cycle in the living eye of the rat,” Nature 221, 820–822 (1969).
[CrossRef] [PubMed]

Dartnall, H. H. A.

H. H. A. Dartnall, “The photobiology of visual processes,” in The Eye, edited by H. Davson, Part II, Vol. 2, (Academic, New York, 1962) pp. 321–533.

Dury, D. R.

A. N. Wick, D. R. Dury, M. I. Nakada, and J. B. Wolfe, “Localization of the primary metabolic block produced by 2-deoxyglucose,” J. Biol. Chem. 224, 963–969(1967).

Entine, G.

Frank, R. N.

R. N. Frank, “Photoproducts of rhodopsin bleaching in the isolated, perfused frog retina,” Vision Res. 9, 1415–1433(1969).
[CrossRef] [PubMed]

Fujimoto, K.

T. Hanaoka and K. Fujimoto, “Absorption spectrum of a single cone in carp retina,” Jpn. J. Physiol. 7, 276–285(1957).
[CrossRef] [PubMed]

Grabowski, Z. R.

Gyllenberg, G.

G. Gyllenberg, T. Reuter, and H. Sippel, “Long-lived photoproducts of rhodopsin in the retina of the frog,” Vision Res. 14, 1349–1357(1974).
[CrossRef] [PubMed]

Hanaoka, T.

T. Hanaoka and K. Fujimoto, “Absorption spectrum of a single cone in carp retina,” Jpn. J. Physiol. 7, 276–285(1957).
[CrossRef] [PubMed]

Harosi, F. I.

Jagger, W. S.

P. A. Liebman, W. S. Jagger, M. W. Kaplan, and F. G. Bargoot, “Membrane structure changes in rod outer segments associated with rhodopsin bleaching,” Nature 251, 31–36(1974).
[CrossRef] [PubMed]

Jasny, J.

Kaplan, M. W.

P. A. Liebman, W. S. Jagger, M. W. Kaplan, and F. G. Bargoot, “Membrane structure changes in rod outer segments associated with rhodopsin bleaching,” Nature 251, 31–36(1974).
[CrossRef] [PubMed]

Knott, G. D.

G. D. Knott and D. K. Reece, “MLAB: A civilized curve-fitting system,” Proc. ONLINE ’72 Intl. Conf., Brunel Univ. Eng. 1, 497–526(1972).

Korenbrot, J. I.

J. I. Korenbrot and R. A. Cone, “Dark ionic flux and the effects of light in isolated rod outer segments,” J. Gen. Physiol. 60, 20–45(1972).

Koszewski, J.

Lamola, A. A.

G. E. Busch, M. L. Applebury, A. A. Lamola, and P. M. Rentzepis, “Formation and decay of prelumirhodopsin at room temperature,” Proc. Natl. Acad. Sci. (U.S.) 69, 2802–2806(1972).
[CrossRef]

Lehninger, A. L.

A. L. Lehninger, in Biochemistry, 2nd ed. (Worth, New York, 1975), pp. 417–542.

Lehrer, G. M.

H. S. Maker and G. M. Lehrer, “Carbohydrate chemistry of brain,” in Basic Neurochemistry, edited by R. W. Albers, G. J. Siegel, R. Katzmann, and B. W. Agranoff (Little, Brown, Boston, 1972), pp. 169–190.

Liebman, P. A.

P. A. Liebman, W. S. Jagger, M. W. Kaplan, and F. G. Bargoot, “Membrane structure changes in rod outer segments associated with rhodopsin bleaching,” Nature 251, 31–36(1974).
[CrossRef] [PubMed]

P. A. Liebman and G. Entine, “Sensitive low-light-level microspectrophotometer: Detection of photosensitive pigments of retinal cones,” J. Opt. Soc. Am. 54, 1451–1459(1964).
[CrossRef] [PubMed]

MacNichol, E. F.

Maker, H. S.

H. S. Maker and G. M. Lehrer, “Carbohydrate chemistry of brain,” in Basic Neurochemistry, edited by R. W. Albers, G. J. Siegel, R. Katzmann, and B. W. Agranoff (Little, Brown, Boston, 1972), pp. 169–190.

Marks, W. B.

W. B. Marks, “Difference spectra of the visual pigments in single goldfish cones,” Ph.D. thesis (Johns Hopkins University, 1963); W. B. Marks, “Visual pigments of single goldfish cones,” J. Physiol. 178, 14–32(1965).

Miller, J. A.

R. Paulsen, J. A. Miller, A. E. Brodie, and M. D. Bownds, “The decay of long-lived photoproducts in the isolated bullfrog rod outer segment: Relationship to other dark reactions,” Vision Res. 15, 1325–1332(1975).
[CrossRef] [PubMed]

Nakada, M. I.

A. N. Wick, D. R. Dury, M. I. Nakada, and J. B. Wolfe, “Localization of the primary metabolic block produced by 2-deoxyglucose,” J. Biol. Chem. 224, 963–969(1967).

Paulsen, R.

R. Paulsen, J. A. Miller, A. E. Brodie, and M. D. Bownds, “The decay of long-lived photoproducts in the isolated bullfrog rod outer segment: Relationship to other dark reactions,” Vision Res. 15, 1325–1332(1975).
[CrossRef] [PubMed]

Perry, R.

B. Chance, R. Perry, L. Akerman, and B. Thorell, “Highly sensitive recording microspectrophotometer,” Rev. Sci. Instrum. 30, 735–741(1959).
[CrossRef]

Reece, D. K.

G. D. Knott and D. K. Reece, “MLAB: A civilized curve-fitting system,” Proc. ONLINE ’72 Intl. Conf., Brunel Univ. Eng. 1, 497–526(1972).

Rentzepis, P. M.

G. E. Busch, M. L. Applebury, A. A. Lamola, and P. M. Rentzepis, “Formation and decay of prelumirhodopsin at room temperature,” Proc. Natl. Acad. Sci. (U.S.) 69, 2802–2806(1972).
[CrossRef]

Reuter, T.

G. Gyllenberg, T. Reuter, and H. Sippel, “Long-lived photoproducts of rhodopsin in the retina of the frog,” Vision Res. 14, 1349–1357(1974).
[CrossRef] [PubMed]

Ripps, H.

K. P. Brin and H. Ripps, “Rhodopsin photoproducts and rod sensitivity in the skate retina,” J. Gen. Physiol. 69, 97–120 (1977).

Sippel, H.

G. Gyllenberg, T. Reuter, and H. Sippel, “Long-lived photoproducts of rhodopsin in the retina of the frog,” Vision Res. 14, 1349–1357(1974).
[CrossRef] [PubMed]

Thorell, B.

B. Chance, R. Perry, L. Akerman, and B. Thorell, “Highly sensitive recording microspectrophotometer,” Rev. Sci. Instrum. 30, 735–741(1959).
[CrossRef]

Wick, A. N.

A. N. Wick, D. R. Dury, M. I. Nakada, and J. B. Wolfe, “Localization of the primary metabolic block produced by 2-deoxyglucose,” J. Biol. Chem. 224, 963–969(1967).

Wolfe, J. B.

A. N. Wick, D. R. Dury, M. I. Nakada, and J. B. Wolfe, “Localization of the primary metabolic block produced by 2-deoxyglucose,” J. Biol. Chem. 224, 963–969(1967).

Appl. Opt. (1)

J. Biol. Chem. (1)

A. N. Wick, D. R. Dury, M. I. Nakada, and J. B. Wolfe, “Localization of the primary metabolic block produced by 2-deoxyglucose,” J. Biol. Chem. 224, 963–969(1967).

J. Gen. Physiol. (2)

J. I. Korenbrot and R. A. Cone, “Dark ionic flux and the effects of light in isolated rod outer segments,” J. Gen. Physiol. 60, 20–45(1972).

K. P. Brin and H. Ripps, “Rhodopsin photoproducts and rod sensitivity in the skate retina,” J. Gen. Physiol. 69, 97–120 (1977).

J. Opt. Soc. Am. (3)

J. Physiol. (2)

C. Baumann, “The formation of metarhodopsin 380 in the retinal rods of the frog,” J. Physiol. 259, 357–366(1976).

C. Baumann, “Kinetics of slow thermal reactions during the bleaching of rhodopsin in the perfused frog retina,” J. Physiol. 222, 643–663(1972).

J. R. Micr. Soc. (1)

T. O. Caspersson, “Methods for the determination of the absorption spectra of cell structures,” J. R. Micr. Soc. 60, 8–25(1940).
[CrossRef]

Jpn. J. Physiol. (1)

T. Hanaoka and K. Fujimoto, “Absorption spectrum of a single cone in carp retina,” Jpn. J. Physiol. 7, 276–285(1957).
[CrossRef] [PubMed]

Nature (2)

P. A. Liebman, W. S. Jagger, M. W. Kaplan, and F. G. Bargoot, “Membrane structure changes in rod outer segments associated with rhodopsin bleaching,” Nature 251, 31–36(1974).
[CrossRef] [PubMed]

R. A. Cone and W. H. Cobbs, “Rhodopsin cycle in the living eye of the rat,” Nature 221, 820–822 (1969).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. (U.S.) (1)

G. E. Busch, M. L. Applebury, A. A. Lamola, and P. M. Rentzepis, “Formation and decay of prelumirhodopsin at room temperature,” Proc. Natl. Acad. Sci. (U.S.) 69, 2802–2806(1972).
[CrossRef]

Proc. ONLINE ’72 Intl. Conf., Brunel Univ. Eng. (1)

G. D. Knott and D. K. Reece, “MLAB: A civilized curve-fitting system,” Proc. ONLINE ’72 Intl. Conf., Brunel Univ. Eng. 1, 497–526(1972).

Rev. Sci. Instrum. (1)

B. Chance, R. Perry, L. Akerman, and B. Thorell, “Highly sensitive recording microspectrophotometer,” Rev. Sci. Instrum. 30, 735–741(1959).
[CrossRef]

Vision Res. (4)

G. Gyllenberg, T. Reuter, and H. Sippel, “Long-lived photoproducts of rhodopsin in the retina of the frog,” Vision Res. 14, 1349–1357(1974).
[CrossRef] [PubMed]

R. N. Frank, “Photoproducts of rhodopsin bleaching in the isolated, perfused frog retina,” Vision Res. 9, 1415–1433(1969).
[CrossRef] [PubMed]

J. K. Bowmaker, “The photoproducts of retinal-based visual pigments in situ: A contrast between Rana pipiens and Gekko gekko,” Vision Res. 13, 1227–1240(1973).
[CrossRef] [PubMed]

R. Paulsen, J. A. Miller, A. E. Brodie, and M. D. Bownds, “The decay of long-lived photoproducts in the isolated bullfrog rod outer segment: Relationship to other dark reactions,” Vision Res. 15, 1325–1332(1975).
[CrossRef] [PubMed]

Other (6)

Because of measurement limitations of the RMSP in the ultraviolet spectrum, it was not possible to detect the formation of retinol, the final stable photoproduct with absorption maximum at 32 nm.

H. S. Maker and G. M. Lehrer, “Carbohydrate chemistry of brain,” in Basic Neurochemistry, edited by R. W. Albers, G. J. Siegel, R. Katzmann, and B. W. Agranoff (Little, Brown, Boston, 1972), pp. 169–190.

A. L. Lehninger, in Biochemistry, 2nd ed. (Worth, New York, 1975), pp. 417–542.

All coefficients are normalized with respect to the amount of rhodopsin bleached to allow for comparisons among different experiments.

H. H. A. Dartnall, “The photobiology of visual processes,” in The Eye, edited by H. Davson, Part II, Vol. 2, (Academic, New York, 1962) pp. 321–533.

W. B. Marks, “Difference spectra of the visual pigments in single goldfish cones,” Ph.D. thesis (Johns Hopkins University, 1963); W. B. Marks, “Visual pigments of single goldfish cones,” J. Physiol. 178, 14–32(1965).

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (14)

FIG. 1
FIG. 1

Diagram of rapid scanning microspectrophotometer. See text for explanation.

FIG. 2
FIG. 2

Emission spectrum of cathode ray tube. The ordinate represents photons (× 107) delivered to the specimen during a measurement interval (600 μs) at each wavelength. Triangles, CRT unclamped, circles, CRT clamped. See text and Fig. 5 for description of the clamping operation. Calibration employed United Detector #5183 Photodiode.

FIG. 3
FIG. 3

Timing of sampling process. The RMSP square-wave clock operates at 33 kHz. Integration and first-stage sampling occur during the first 19 clock cycles. During the 20th cycle, the first-stage output is sampled by the second-stage amplifier, and then the integrator is reset. The second-stage amplifier holds this voltage level for a total of 20 clock cycles (600 μs) for each wavelength, gated by the “hold” signal. The voltage change to the CRT horizontal plates is synchronized with the sampling procedure.

FIG. 4
FIG. 4

Block diagram of lock-in amplifier operation. (A) The PM anode current, consisting of the summation of out-of-phase transmittances from both spots of the specimen, is preamplified in composite form. The output of the preamplifier is switched by S1 between two integrators, synchronously with application of a square-wave deflection voltage to the CRT vertical plates. This separately recovers the signal from each spot. Switch S1 is driven electronically by the RMSP clock signal at 33 kHz. VS1(λ) and VS2(λ) are recorded for data reduction. VΔ(λ), the arithmetic difference between integrator outputs A1 and A2, is amplified and utilized during an experiment as an approximate means of assessing the quality of the experiment. (B) Separate recovery of signals proportional to the incident light fluxes, diverted by the beam splitter, is obtained as in (A). Switch S2 is set manually to select which spot is the “sample.” VL(λ), the output of system B1, is recorded. The sum of integrator outputs B1 and B2 is amplified and applied as a feedback clamping signal to the grid of the CRT to improve emission characteristics.

FIG. 5
FIG. 5

Clamping of cathode ray tube in dual-beam configuration. (A) The open-loop voltage resulting from the preamplified PM anode current is illustrated at a single wavelength for a sampling interval of 20 clock cycles, or 600 μs. The imbalance between respective half-cycles is due to inhomogeneity of the CRT phosphors of the two spots. SB and SE denote the first and last clock cycles in the interval for the “sample” spot. RB and RE indicate the same for the reference spot. (B) Same as (A), except with the feedback loop to the CRT grid closed. The clamping operation is an attempt to equalize the total flux delivered during a sampling interval by each of the two chopped CRT spots. This correction process is indicated by the increase in output level from SB to SE, with corresponding decrease in output level from RB to RE. Also shown is the RMSP reset voltage, in which the beginning and end of the sampling interval are indicated by pulses R1 and R2, respectively.

FIG. 6
FIG. 6

Block diagram of wavelength scanning system. The scanning operation begins manually or automatically through a one-shot by enabling a Schmitt trigger, which produces a square-wave clock signal at 33 kHz. The vertical amplifier drive signal is described in the text. The duration of a single sampling interval is 20 clock cycles (600 μs). Sampling and integrator reset are performed after the 19th clock cycle in each interval. The horizontal counter is incremented from the preset start-point magnitude by the step-size amount at the conclusion of each sampling interval. The output of the horizontal counter is converted to an analog voltage, which is applied to the horizontal deflection plates of the CRT to change the wavelength. A scan is completed when the horizontal count reaches the preset stop-point magnitude. One scan consists of (stop point − start point)/(step size) sampling intervals. The entire process is repeated until the scans count equals the preset number of scans, whereupon the clock enable is reset.

FIG. 7
FIG. 7

Timing diagram of RMSP operation. The start pulse enables opening of the bleaching light shutter. The PM shutters, disabled by the interlock during bleaching, do not open until the bleaching light shutter has closed completely. After a suitable delay to insure that the PM shutters are completely open, CRT brightening and horizontal scanning are begun. The PM shutters close upon completion of the scanning operation.

FIG. 8
FIG. 8

Block diagram of RMSP signal processing. See text and Fig. 4 for explanation. (A) Single-beam operation. VS(λ) is designated to represent VS1(λ) of Fig. 4(A). (B) Dual-beam operation.

FIG. 9
FIG. 9

Typical example of digitized RMSP raw data output voltages for single-beam configuration. (A) Record of raw data voltages from typical 400 μm diameter retinal patch, measured prior to bleaching. VS1(λ) (triangles) represents digitized waveform from the transmitted-light sample-and-hold amplifier [see Fig. 4(A)]. Also shown in VS0 (circles), which represents the baseline level of the electronics. The wavelength range is 370–640 nm. (B) Typical record of raw data voltage from the same retinal patch, measured subsequent to bleaching. (C) Record of sample-and-hold amplifier output VSB(λ) measured and recorded through a blank area of the chamber. Light attenuated by 0.6 neutral density filter. (D) Record of sample-and-hold output VL(λ) representing a fraction of the incident light which is diverted by the beam splitter.

FIG. 10
FIG. 10

Record of typical absorption spectra after data reduction. Triangles represent absorptance, in logarithmic optical density units, of a patch of in a dark-adapted retina. Circles illustrate optical density measured 100 ms after the completion of a 300 ms bleach. Squares portray a measurement 650 s post-bleach. Lines connecting data points in this figure were generated by linear interpolation.

FIG. 11
FIG. 11

Records of typical difference spectra after data reduction. (A) Difference in optical density resulting from subtraction of pre-bleach measurement (triangles of Fig. 10) from an early post-bleach measurement (100 ms; circles of Fig. 10). The loss in optical density at 500 nm is due to the amount of rhodopsin bleached (see Fig. 13). (B) Superimposed difference spectra. Triangles: Difference in optical density resulting from the subtraction of a late post-bleach reference measurement (650 s; squares of Fig. 10) from an early post-bleach measurement (100 ms; circles of Fig. 10). Circles: Same late post-bleach reference measurement, subtracted from a measurement 60 s post-bleach. Squares: Same reference, subtracted from a measurement 180 s post-bleach. Lines connecting data points were generates by linear interpolation.

FIG. 12
FIG. 12

Record of typical isolated-wavelength kinetics. (A) Wavelengths displayed are selected by locating maximum and minimum of difference spectra such as those illustrated in Fig. 11, in which final, late post-bleach measurement is utilized as a reference curve for the subtraction. Optical density differences at these specific wavelengths are retrieved from each difference spectrum (calculated sequentially for each scan) and are displayed here as the ordinate function. A negative intercept on the ordinate axis is with respect to the reference measurement (see text). The abscissa represents the time of measurement of a particular scan following completion of the bleach. Triangles, 380 nm. Circles, 470 nm. Lines connecting data points were generated by linear interpolation. (B) Solid lines represent a simulation of the best fit of the data of (A) to the kinetic model described in the text.

FIG. 13
FIG. 13

Possible sequences of rhodopsin photoproducts in isolated, intact retinas. The wavelength of maximum absorption of each species is indicated in parentheses. Photoproducts appearing earlier than MII are excluded. R = Rhodopsin; MII = Metarhodopsin II; MIII = Metarhodopsin III; RAL = Retinal; ROL = Retinol. Rate constants ki are indicated for each model. (A) Route according to Baumann (Ref. 13) in frog (Rana pipiens). (B) Route according to Brin and Ripps (Ref. 16) in skate (Raja erinacea).

FIG. 14
FIG. 14

Representative relationships between MII, MIII, and retinal kinetics. Optical density changes measured at 380 nm (triangles) and 470 nm (circles) are illustrated as a function of post-bleach time. The rapid phase of the 380 nm decay in (A) is attributed to MII; the slow phase in (C) to retinal; (B) illustrates the combined decay of both pigments. MIII is present in decreasing concentrations in (A) through (C). Continuous lines illustrate the best fit of the data to the parameters of single, linear, first-order processes described in Eqs. (18) (380 nm) and (19) (470 nm), as calculated by MLAB software curve-fitting routines on a PDP-10 computer. All optical density values are normalized with respect to the amount of rhodopsin bleached. See Table III for the distribution of perfusate compositions with respect to each of the three categories.

Tables (3)

Tables Icon

TABLE I Concentrations of ions and buffering agents of perfusate solutions at each pH of study. See text.

Tables Icon

TABLE II Summary of parameter values of curve-fitting to model. Values indicated in columns A through C correspond to data of Figs. 14(A) through 14(C), respectively. T1 through T4 represent half-times, in seconds, of processes k1 through k4, respectively, illustrated in the model of Fig. 13(A). Also indicated are the proportions of MIII and retinal (RAL), normalized with respect to the amount of rhodopsin bleached, which are formed from MII.

Tables Icon

TABLE III Distribution of environmental conditions for categories in Fig. 14.

Equations (29)

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

absorptance = 1 - transmitted flux incident flux .
V S ( λ ) = G S [ P S ( λ ) ϕ S ( λ ) - I S ] .
V L ( λ ) = G L [ P L ( λ ) ϕ L ( λ ) - I L ] .
f ( λ ) = ϕ L ( λ ) / ϕ S ( λ ) .
V L ( λ ) = G L [ P L ( λ ) f ( λ ) ϕ S ( λ ) - I L ] .
V S 0 ( λ ) = - G S I S = V S 0 ;
V L 0 ( λ ) = - G L I L = V L 0 ;
V S ( λ ) - V S 0 = G S P S ( λ ) ϕ S ( λ ) ;
V L ( λ ) - V L 0 = G L P L ( λ ) ϕ S ( λ ) f ( λ ) .
A S ( λ ) = 1 - ϕ S ( λ ) / ϕ S ( λ ) = 1 - G L P L ( λ ) f ( λ ) G S P S ( λ ) V S ( λ ) - V S 0 V L ( λ ) - V L 0 .
V S B ( λ ) - V S 0 = G S P S ( λ ) ϕ S B ( λ ) ;
V L B ( λ ) - V L 0 = G L P L ( λ ) f ( λ ) ϕ S B ( λ ) .
A S ( λ ) = 1 - V L B ( λ ) - V L 0 V S B ( λ ) - V S 0 V S ( λ ) - V S 0 V L ( λ ) - V L 0 ;
D S ( λ ) = - log [ 1 - A S ( λ ) ] ;
= log V S B ( λ ) - V S 0 V L B ( λ ) - V L 0 V L ( λ ) - V L 0 V S ( λ ) - V S 0 .
V S 1 ( λ ) = G 1 S [ P S ( λ ) ϕ 1 S ( λ ) - I S ] ;
V S 2 ( λ ) = G 2 S [ P S ( λ ) ϕ 2 S ( λ ) - I S ] ;
V S 1 ( λ ) - V S 10 = G 1 S P S ( λ ) ϕ 1 S ( λ ) ;
V S 2 ( λ ) - V S 20 = G 2 S P S ( λ ) ϕ 2 S ( λ ) .
Δ D S ( λ ) = - log [ ϕ 2 S ( λ ) / ϕ 2 S ( λ ) ] - { - log [ ϕ 1 S ( λ ) / ϕ 1 S ( λ ) ] } = log { [ ϕ 1 S ( λ ) / ϕ 1 S ( λ ) ] [ ϕ 2 S ( λ ) / ϕ 2 S ( λ ) ] } .
V S B 1 ( λ ) - V S 10 = G 1 S P S ( λ ) ϕ S B 1 ( λ ) ;
V S B 2 ( λ ) - V S 20 = G 2 S P S ( λ ) ϕ S B 2 ( λ ) .
Δ D S ( λ ) = log V S 1 ( λ ) - V S 10 V S B 1 ( λ ) - V S 10 V S B 2 ( λ ) - V S 20 V S 2 ( λ ) - V S 20 .
V Δ ( λ ) = G D P S ( λ ) [ ϕ 2 S ( λ ) - ϕ 1 S ( λ ) ] .
Δ D a ( 380 ) = e - ( k 1 a + k 4 a ) t × ( 1 + k 1 a k 2 a + k 4 a ( k 2 a - k 1 a - k 4 a ) ( k 2 a - k 1 a - k 4 a ) ( k 3 a - k 1 a - k 4 a ) ) + k 1 a k 2 a ( k 1 a + k 4 a - k 2 a ) ( k 3 a - k 2 a ) e - k 2 a t + k 1 a k 2 a + k 4 a ( k 2 a - k 3 a ) ( k 1 a + k 4 a - k 3 a ) ( k 2 a - k 3 a ) e - k 3 a t + 0.25 Δ D a ( 470 ) + δ 380 a ;
Δ D a ( 470 ) = k 1 a k 2 a - ( k 1 a + k 4 a ) × ( e - ( k 1 a + k 4 a ) t - e - k 2 a t ) + δ 470 a ;
Δ D b ( 380 ) = e - ( k 1 b + k 4 b ) t + k 4 b k 3 b - ( k 1 b + k 4 b ) × ( e - k 1 b + k 4 b ) t - e - k 3 b t ) + δ 380 b ;
Δ D b ( 470 ) = k 2 b k 2 b - ( k 1 b + k 4 b ) × ( e - ( k 1 b + k 4 b ) t - e - k 2 b t ) + δ 470 b .
T i = 1 / k ln 2.