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

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  1. T. O. Caspersson, "Methods for the determination of the absorption spectra of cell structures," J. R. Micr. Soc. 60, 8–25(1940).
  2. B. Chance, R. Perry, L. Akerman, and B. Thorell, "Highly sensitive recording microspectrophotometer," Rev. Sci. Instrum. 30, 735–741(1959).
  3. P. K. Brown, "A system for microspectrophotometry employing a commercial recording microspectrophotometer," J. Opt. Soc. Am. 51, 1000–1008(1961).
  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).
  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, Jr., "Dichroic microspectrophotometer: A computer-assisted, rapid, wavelength-scanning photometer for measuring linear dichroism in single cells," J. Opt. Soc. Am. 64, 903–918(1974).
  7. T. Hanaoka and K. Fujimoto, "Absorption spectrum of a single cone in carp retina," Jpn. J. Physiol. 7, 276–285(1957).
  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).
  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).
  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).
  12. R. N. Frank, "Photoproducts of rhodopsin bleaching in the isolated, perfused frog retina," Vision Res. 9, 1415–1433(1969).
  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).
  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).
  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).
  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).

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).

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).

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

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).

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).

1972 (4)

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).

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

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. N. Frank, "Photoproducts of rhodopsin bleaching in the isolated, perfused frog retina," Vision Res. 9, 1415–1433(1969).

R. A. Cone and W. H. Cobbs, "Rhodopsin cycle in the living eye of the rat," Nature 221, 820–822 (1969).

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).

1957 (1)

T. Hanaoka and K. Fujimoto, "Absorption spectrum of a single cone in carp retina," Jpn. J. Physiol. 7, 276–285(1957).

1940 (1)

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

Akerman, L.

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

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).

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).

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).

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).

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).

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).

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).

Chance, B.

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

Cobbs, W. H.

R. A. Cone and W. H. Cobbs, "Rhodopsin cycle in the living eye of the rat," Nature 221, 820–822 (1969).

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).

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).

Fujimoto, K.

T. Hanaoka and K. Fujimoto, "Absorption spectrum of a single cone in carp retina," Jpn. J. Physiol. 7, 276–285(1957).

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).

Hanaoka, T.

T. Hanaoka and K. Fujimoto, "Absorption spectrum of a single cone in carp retina," Jpn. J. Physiol. 7, 276–285(1957).

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).

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).

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).

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).

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).

MacNichol, Jr., 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).

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).

Perry, R.

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

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).

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).

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).

Thorell, B.

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

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)

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).

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).

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).

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).

R. A. Cone and W. H. Cobbs, "Rhodopsin cycle in the living eye of the rat," Nature 221, 820–822 (1969).

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).

Rev. Sci. Instrum. (1)

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

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).

R. N. Frank, "Photoproducts of rhodopsin bleaching in the isolated, perfused frog retina," Vision Res. 9, 1415–1433(1969).

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).

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).

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.

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.

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

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).

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