Abstract

A novel microspectrophotometer is described, which simultaneously resolves cell absorption into two mutually orthogonal components allowing the determination of linear dichroism as a function of wavelength in the range of 325–695 nm. This instrument uses a single plane-polarized light beam and a small general-purpose digital computer, and is equipped with a photo-flash apparatus for rapid photolysis. Following visual pigment bleaching, it can detect changes occurring on a time scale of seconds in the orientation and spectral character of chromophores in isolated cells. The spectral scanning is performed in either single or multiple sweeps which may be unidrectional or bidirectional. The scanning rate is set to 500 nm/s. Spectral resolution is 5 nm. Its signal and data processing are discussed. Its performance is illustrated on subcellular organelles of retinal photoreceptors from turtle and frog. Rhodopsin and its photoproducts are shown to lend dichroism to frog rod outer segments. Metarhodopsin II, when formed, is transversely dichroic as rhodopsin. The late products (retinol, retainal oxime, etc.) show axial dichroism. The corrected specific optical density (transverse component) of frog rod outer segments (in hydroxylamine) is found to be 0.0182±0.002/μm. The average absorption spectrum is presented for in situ rhodopsin.

© 1974 Optical Society of America

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  1. A. C. Hardy, J. Opt. Soc. Am. 28, 360 (1938).
    [Crossref]
  2. T. Caspersson, J. Roy. Microscop. Soc. 60, 8 (1940).
    [Crossref]
  3. C. C. Yang and V. Legallais, Rev. Sci. Instr. 25, 801 (1954); B. Chance, R. Perry, L. Åkerman, and B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
    [Crossref]
  4. P. K. Brown, J. Opt. Soc. Am. 51, 1000 (1961).
    [Crossref]
  5. P. A. Liebman and G. Entine, J. Opt. Soc. Am. 54, 1451 (1964).
    [Crossref] [PubMed]
  6. W. B. Marks, Ph.D. dissertation (The Johns Hopkins University, Baltimore, Md., 1963); W. B. Marks, J. Physiol. (Lond.) 178, 14 (1965).
  7. J. J. Wolken, R. Forsberg, G. Gallik, and R. Florida, Rev. Sci. Instr. 39, 1734 (1968).
    [Crossref]
  8. T. Hanaoka and K. Fujimoto, Jap. J. Physiol. 7, 276 (1957).
    [Crossref]
  9. W. A. Hagins and W. H. Jennings, Discuss. Faraday Soc. 27, 180 (1959).
    [Crossref]
  10. P. A. Liebman, Biophys. J. 2, 161 (1962).
  11. G. Wald, P. K. Brown, and I. R. Gibbons, J. Opt. Soc. Am. 53, 20 (1963).
    [Crossref] [PubMed]
  12. W. B. Marks, W. H. Dobelle, and E. F. MacNichol, Science 143, 1181 (1964).
    [Crossref] [PubMed]
  13. P. K. Brown and G. Wald, Science 144, 45 (1964); G. Wald and P. K. Brown, Cold Spring Harbor Symp. Quant. Biol. 30, 345 (1965).
    [Crossref] [PubMed]
  14. P. A. Liebman and G. Entine, Vision Res. 8, 761 (1968).
    [Crossref] [PubMed]
  15. W. H. Dobelle, W. B. Marks, and E. F. MacNichol, Science 166, 1508 (1969).
    [Crossref] [PubMed]
  16. P. A. Liebman, in Handbook of Sensory Physiology, Vol. VII/1. Photochemistry of Vision, edited by H. J. A. Dartnall (Springer, Berlin, Heidelberg, New York, 1972), Ch. 12, p. 481.
    [Crossref]
  17. W. J. Schmidt, Kolloid-Z. 85, 137 (1938).
    [Crossref]
  18. E. J. Denton, J. Physiol. (Lond.) 124, 16P and 17P (1954); E. J. Denton, Proc. R. Soc. (Lond.) B150, 78 (1959).
  19. G. Svaetichin, K. Negishi, and R. Fatehchand, in Ciba Foundation Symposium on Colour Vision: Physiology and Experimental Psychology, edited by A. V. S. DeReuck and J. Knight (Little, Brown, Boston, 1965), p. 178.
  20. F. S. Sjöstrand, J. Cellular Comp. Physiol. 42, 15 (1953); S. E. G. Nilsson, J. Ultrastr. Res. 12, 207 (1965).
    [Crossref]
  21. M. F. Moody, Biol. Rev. 39, 43 (1964).
    [Crossref]
  22. See, for example, the following reviews: E. W. Abrahamson and S. E. Ostroy, Progr. Biophys. Molec. Biol. 17, 181 (1967); G. Wald, Nature 219, 800 (1968); R. A. Morton and G. A. J. Pitt, Adv. Enzymol. 32, 97 (1969).
    [Crossref] [PubMed]
  23. See, for example, the recent review by R. A. Morton, in Handbook of Sensory Physiology, Vol VII/1. Photochemistry of Vision, edited by H. J. A. Dartnall (Springer, Berlin, Heidelberg, New York, 1972), Ch. 2, p. 33.
    [Crossref]
  24. It has been shown that the quantum efficiency of bleaching is about 23 for all visual pigments; e.g., H. J. A. Dartnall, in Handbook of Sensory Physiology, Vol. VII/1. Photochemistry of Vision, edited by H. J. A. Dartnall (Springer, Berlin, Heidelberg, New York, 1972), Ch. 4, p. 122.
    [Crossref]
  25. With this statement, we imply that long-term fluctuations are either non-existent in the DMSP, or that their effects are detectable so that all records subject to drift may be discarded. Because of its permanent memory, the DMSP is well suited to test for slow drifts arising from any source in the instrument by computer comparison of reference transmittances recorded at different times during an experiment. The acceptance or rejection of records we decide by our selection rule (Ref. 33), which states that a spectral recording may be regarded free of distortions if the absorptance trace falls within about ±1% of the zero line, before as well as after photolysis, in spectral regions where no absorption is expected. This procedure sets also the limit on the error that may result from the reference being a clear area in the preparation instead of the cell when devoid of pigment. These issues are discussed in more detail in Ref. 33.
  26. F. I. Hárosi, Ph.D. dissertation (The Johns Hopkins University, Baltimore, Md., 1971).
  27. E. F. MacNichol, R. Feinberg, and F. I. Hárosi, in Colour 73 (A. Hilger, Rank Precision Industries, London, 1973), p. 191.
  28. J. L. Michaelson, J. Opt. Soc. Am. 28, 365 (1938).
    [Crossref]
  29. W. A. H. Rushton, F. W. Campbell, W. A. Hagins, and G. S. Brindley, Opt. Acta 1, 183 (1955).
    [Crossref]
  30. The effective bandwidth of 18 Hz is obtained with reference to a single-time-constant (τ) integrator (low-pass filter) for which the upper corner frequency (fc) is given by the relationship 2πfo= 1/τ. The DMSP is operated such that a 5-nm-wide spectral segment is swept in 10 ms. Since about 90% of this time is spent for summing repeated samples of the same signal (120 times on each channel), the integration time is about 9 ms. If τ= 9 × 10−3 s, fc≅ 18 Hz. Note that a simple low-pass filter with τ= RC has a slow roll-off characteristic (20 dB/decade) and hence a broader bandwidth than the fc= 1/2 πτ formula indicates. Digital summation, on the other hand, approaches ideal integration by virtue of its high speed and the lack of phase distortion. Because of these, it is possible to process in the DMSP the separated fast modulation and the slowly varying average signal with essentially no phase shift between them (except for a fixed lag of about 35 μ s due to multiplexing the two analog channels).
  31. The time required for punching the data of one measurement is 12–13 s (on the high-speed paper-tape punch).
  32. See, for example, Fig. 1 of G. Wald, Doc. Ophthalmol. 3, 94 (1949).
    [Crossref]
  33. F. I. Hárosi and E. F. MacNichol, J. Gen. Physiol. 63, 279 (1974).
    [Crossref]
  34. G. Wald and R. Hubbard, Proc. Natl. Acad. Sci. USA 36, 92 (1950); G. Wald and P. K. Brown, Proc. Natl. Acad. Sci. USA 36, 84 (1950); H. J. A. Dartnall, Vision Res. 8, 339 (1968).
    [Crossref] [PubMed]
  35. According to H. Shichi, Biochem. 9, 1973 (1970), the density ratio of the β band peak to the α band peak is 0.29 for extracted, purified cattle rhodopsin. In the publication of H. Shichi, M. S. Lewis, F. Irreverre, and A. L. Stone, J. Biol. Chem. 244, 529 (1969), the ratio of molar extinction coefficients of the β band and the α band is 0.266 for pure cattle rhodopsin. On the other hand, the same ratio obtained from the same preparation by H. Shichi appears to be close to 0.21, in one of his more-recent records that he provided for us. Our choice of 0.25 for this figure represents not only an average to the above numbers, but also our best estimate of the density ratio of the, β and α band peaks for in situ frog rhodopsin.
    [Crossref]
  36. The assumption, that the excess density measured at 370 nm over the expected density of rhodopsin is due to retinal oxime, should be valid only if other photoproducts do not persist long enough to interfere with the measurement. Although we can detect the formation of some Meta III, and the brief presence of some Meta II, at 5- and 10-mM hydroxylamine concentrations, at 75-mM concentration of this reagent the oxime formation appears to be faster than the time resolution of the DMSP.
  37. On the basis of a theoretical analysis by Hárosi and Malerba we derived an equation that relates the different absorbing states of the same cell in terms of molar extinction coefficients (∊′, ∊″), molecular extinctions (M′, M″), cellular optical density components (D||′, D||″), and dichroic ratios (R′ = D⊥′/D||′, R″ = D⊥″/D||″), as ∊′∊″=M′M″=D‖′[1+R′(2-3b)]D‖″[1+R″(2-3b)].Coefficient b is related to the numerical aperture (NA) of the microscope condenser (with aperture cone 2α) as b= tan2(α/2). For an oil-immersion-type condenser (n= 1.455) of (NA) = 0.4, b= 0.0197, and thus ∊′∊″=D‖′(1+1.941R′)D‖″(1+1.941R″).We use this equation to compute photoproduct densities from presumed molar extinction coefficients, or vice versa, and assume unchanging pigment concentrations and path lengths of measuring light within the cell.
  38. G. Wald and P. K. Brown, J. Gen. Physiol. 37, 189 (1953).
  39. H. J. A. Dartnall, Br. Med. Bull. 9, 24 (1953).
  40. See, for example, E. L. Crow, F. A. Davis, and M. W. Maxfield, Statistics Manual (Dover, New York, 1960).

1974 (1)

F. I. Hárosi and E. F. MacNichol, J. Gen. Physiol. 63, 279 (1974).
[Crossref]

1970 (1)

According to H. Shichi, Biochem. 9, 1973 (1970), the density ratio of the β band peak to the α band peak is 0.29 for extracted, purified cattle rhodopsin. In the publication of H. Shichi, M. S. Lewis, F. Irreverre, and A. L. Stone, J. Biol. Chem. 244, 529 (1969), the ratio of molar extinction coefficients of the β band and the α band is 0.266 for pure cattle rhodopsin. On the other hand, the same ratio obtained from the same preparation by H. Shichi appears to be close to 0.21, in one of his more-recent records that he provided for us. Our choice of 0.25 for this figure represents not only an average to the above numbers, but also our best estimate of the density ratio of the, β and α band peaks for in situ frog rhodopsin.
[Crossref]

1969 (1)

W. H. Dobelle, W. B. Marks, and E. F. MacNichol, Science 166, 1508 (1969).
[Crossref] [PubMed]

1968 (2)

J. J. Wolken, R. Forsberg, G. Gallik, and R. Florida, Rev. Sci. Instr. 39, 1734 (1968).
[Crossref]

P. A. Liebman and G. Entine, Vision Res. 8, 761 (1968).
[Crossref] [PubMed]

1967 (1)

See, for example, the following reviews: E. W. Abrahamson and S. E. Ostroy, Progr. Biophys. Molec. Biol. 17, 181 (1967); G. Wald, Nature 219, 800 (1968); R. A. Morton and G. A. J. Pitt, Adv. Enzymol. 32, 97 (1969).
[Crossref] [PubMed]

1964 (4)

M. F. Moody, Biol. Rev. 39, 43 (1964).
[Crossref]

P. A. Liebman and G. Entine, J. Opt. Soc. Am. 54, 1451 (1964).
[Crossref] [PubMed]

W. B. Marks, W. H. Dobelle, and E. F. MacNichol, Science 143, 1181 (1964).
[Crossref] [PubMed]

P. K. Brown and G. Wald, Science 144, 45 (1964); G. Wald and P. K. Brown, Cold Spring Harbor Symp. Quant. Biol. 30, 345 (1965).
[Crossref] [PubMed]

1963 (1)

1962 (1)

P. A. Liebman, Biophys. J. 2, 161 (1962).

1961 (1)

1959 (1)

W. A. Hagins and W. H. Jennings, Discuss. Faraday Soc. 27, 180 (1959).
[Crossref]

1957 (1)

T. Hanaoka and K. Fujimoto, Jap. J. Physiol. 7, 276 (1957).
[Crossref]

1955 (1)

W. A. H. Rushton, F. W. Campbell, W. A. Hagins, and G. S. Brindley, Opt. Acta 1, 183 (1955).
[Crossref]

1954 (2)

C. C. Yang and V. Legallais, Rev. Sci. Instr. 25, 801 (1954); B. Chance, R. Perry, L. Åkerman, and B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[Crossref]

E. J. Denton, J. Physiol. (Lond.) 124, 16P and 17P (1954); E. J. Denton, Proc. R. Soc. (Lond.) B150, 78 (1959).

1953 (3)

F. S. Sjöstrand, J. Cellular Comp. Physiol. 42, 15 (1953); S. E. G. Nilsson, J. Ultrastr. Res. 12, 207 (1965).
[Crossref]

G. Wald and P. K. Brown, J. Gen. Physiol. 37, 189 (1953).

H. J. A. Dartnall, Br. Med. Bull. 9, 24 (1953).

1950 (1)

G. Wald and R. Hubbard, Proc. Natl. Acad. Sci. USA 36, 92 (1950); G. Wald and P. K. Brown, Proc. Natl. Acad. Sci. USA 36, 84 (1950); H. J. A. Dartnall, Vision Res. 8, 339 (1968).
[Crossref] [PubMed]

1949 (1)

See, for example, Fig. 1 of G. Wald, Doc. Ophthalmol. 3, 94 (1949).
[Crossref]

1940 (1)

T. Caspersson, J. Roy. Microscop. Soc. 60, 8 (1940).
[Crossref]

1938 (3)

Abrahamson, E. W.

See, for example, the following reviews: E. W. Abrahamson and S. E. Ostroy, Progr. Biophys. Molec. Biol. 17, 181 (1967); G. Wald, Nature 219, 800 (1968); R. A. Morton and G. A. J. Pitt, Adv. Enzymol. 32, 97 (1969).
[Crossref] [PubMed]

Brindley, G. S.

W. A. H. Rushton, F. W. Campbell, W. A. Hagins, and G. S. Brindley, Opt. Acta 1, 183 (1955).
[Crossref]

Brown, P. K.

P. K. Brown and G. Wald, Science 144, 45 (1964); G. Wald and P. K. Brown, Cold Spring Harbor Symp. Quant. Biol. 30, 345 (1965).
[Crossref] [PubMed]

G. Wald, P. K. Brown, and I. R. Gibbons, J. Opt. Soc. Am. 53, 20 (1963).
[Crossref] [PubMed]

P. K. Brown, J. Opt. Soc. Am. 51, 1000 (1961).
[Crossref]

G. Wald and P. K. Brown, J. Gen. Physiol. 37, 189 (1953).

Campbell, F. W.

W. A. H. Rushton, F. W. Campbell, W. A. Hagins, and G. S. Brindley, Opt. Acta 1, 183 (1955).
[Crossref]

Caspersson, T.

T. Caspersson, J. Roy. Microscop. Soc. 60, 8 (1940).
[Crossref]

Crow, E. L.

See, for example, E. L. Crow, F. A. Davis, and M. W. Maxfield, Statistics Manual (Dover, New York, 1960).

Dartnall, H. J. A.

H. J. A. Dartnall, Br. Med. Bull. 9, 24 (1953).

It has been shown that the quantum efficiency of bleaching is about 23 for all visual pigments; e.g., H. J. A. Dartnall, in Handbook of Sensory Physiology, Vol. VII/1. Photochemistry of Vision, edited by H. J. A. Dartnall (Springer, Berlin, Heidelberg, New York, 1972), Ch. 4, p. 122.
[Crossref]

Davis, F. A.

See, for example, E. L. Crow, F. A. Davis, and M. W. Maxfield, Statistics Manual (Dover, New York, 1960).

Denton, E. J.

E. J. Denton, J. Physiol. (Lond.) 124, 16P and 17P (1954); E. J. Denton, Proc. R. Soc. (Lond.) B150, 78 (1959).

Dobelle, W. H.

W. H. Dobelle, W. B. Marks, and E. F. MacNichol, Science 166, 1508 (1969).
[Crossref] [PubMed]

W. B. Marks, W. H. Dobelle, and E. F. MacNichol, Science 143, 1181 (1964).
[Crossref] [PubMed]

Entine, G.

Fatehchand, R.

G. Svaetichin, K. Negishi, and R. Fatehchand, in Ciba Foundation Symposium on Colour Vision: Physiology and Experimental Psychology, edited by A. V. S. DeReuck and J. Knight (Little, Brown, Boston, 1965), p. 178.

Feinberg, R.

E. F. MacNichol, R. Feinberg, and F. I. Hárosi, in Colour 73 (A. Hilger, Rank Precision Industries, London, 1973), p. 191.

Florida, R.

J. J. Wolken, R. Forsberg, G. Gallik, and R. Florida, Rev. Sci. Instr. 39, 1734 (1968).
[Crossref]

Forsberg, R.

J. J. Wolken, R. Forsberg, G. Gallik, and R. Florida, Rev. Sci. Instr. 39, 1734 (1968).
[Crossref]

Fujimoto, K.

T. Hanaoka and K. Fujimoto, Jap. J. Physiol. 7, 276 (1957).
[Crossref]

Gallik, G.

J. J. Wolken, R. Forsberg, G. Gallik, and R. Florida, Rev. Sci. Instr. 39, 1734 (1968).
[Crossref]

Gibbons, I. R.

Hagins, W. A.

W. A. Hagins and W. H. Jennings, Discuss. Faraday Soc. 27, 180 (1959).
[Crossref]

W. A. H. Rushton, F. W. Campbell, W. A. Hagins, and G. S. Brindley, Opt. Acta 1, 183 (1955).
[Crossref]

Hanaoka, T.

T. Hanaoka and K. Fujimoto, Jap. J. Physiol. 7, 276 (1957).
[Crossref]

Hardy, A. C.

Hárosi, F. I.

F. I. Hárosi and E. F. MacNichol, J. Gen. Physiol. 63, 279 (1974).
[Crossref]

E. F. MacNichol, R. Feinberg, and F. I. Hárosi, in Colour 73 (A. Hilger, Rank Precision Industries, London, 1973), p. 191.

F. I. Hárosi, Ph.D. dissertation (The Johns Hopkins University, Baltimore, Md., 1971).

Hubbard, R.

G. Wald and R. Hubbard, Proc. Natl. Acad. Sci. USA 36, 92 (1950); G. Wald and P. K. Brown, Proc. Natl. Acad. Sci. USA 36, 84 (1950); H. J. A. Dartnall, Vision Res. 8, 339 (1968).
[Crossref] [PubMed]

Jennings, W. H.

W. A. Hagins and W. H. Jennings, Discuss. Faraday Soc. 27, 180 (1959).
[Crossref]

Legallais, V.

C. C. Yang and V. Legallais, Rev. Sci. Instr. 25, 801 (1954); B. Chance, R. Perry, L. Åkerman, and B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[Crossref]

Liebman, P. A.

P. A. Liebman and G. Entine, Vision Res. 8, 761 (1968).
[Crossref] [PubMed]

P. A. Liebman and G. Entine, J. Opt. Soc. Am. 54, 1451 (1964).
[Crossref] [PubMed]

P. A. Liebman, Biophys. J. 2, 161 (1962).

P. A. Liebman, in Handbook of Sensory Physiology, Vol. VII/1. Photochemistry of Vision, edited by H. J. A. Dartnall (Springer, Berlin, Heidelberg, New York, 1972), Ch. 12, p. 481.
[Crossref]

MacNichol, E. F.

F. I. Hárosi and E. F. MacNichol, J. Gen. Physiol. 63, 279 (1974).
[Crossref]

W. H. Dobelle, W. B. Marks, and E. F. MacNichol, Science 166, 1508 (1969).
[Crossref] [PubMed]

W. B. Marks, W. H. Dobelle, and E. F. MacNichol, Science 143, 1181 (1964).
[Crossref] [PubMed]

E. F. MacNichol, R. Feinberg, and F. I. Hárosi, in Colour 73 (A. Hilger, Rank Precision Industries, London, 1973), p. 191.

Marks, W. B.

W. H. Dobelle, W. B. Marks, and E. F. MacNichol, Science 166, 1508 (1969).
[Crossref] [PubMed]

W. B. Marks, W. H. Dobelle, and E. F. MacNichol, Science 143, 1181 (1964).
[Crossref] [PubMed]

W. B. Marks, Ph.D. dissertation (The Johns Hopkins University, Baltimore, Md., 1963); W. B. Marks, J. Physiol. (Lond.) 178, 14 (1965).

Maxfield, M. W.

See, for example, E. L. Crow, F. A. Davis, and M. W. Maxfield, Statistics Manual (Dover, New York, 1960).

Michaelson, J. L.

Moody, M. F.

M. F. Moody, Biol. Rev. 39, 43 (1964).
[Crossref]

Morton, R. A.

See, for example, the recent review by R. A. Morton, in Handbook of Sensory Physiology, Vol VII/1. Photochemistry of Vision, edited by H. J. A. Dartnall (Springer, Berlin, Heidelberg, New York, 1972), Ch. 2, p. 33.
[Crossref]

Negishi, K.

G. Svaetichin, K. Negishi, and R. Fatehchand, in Ciba Foundation Symposium on Colour Vision: Physiology and Experimental Psychology, edited by A. V. S. DeReuck and J. Knight (Little, Brown, Boston, 1965), p. 178.

Ostroy, S. E.

See, for example, the following reviews: E. W. Abrahamson and S. E. Ostroy, Progr. Biophys. Molec. Biol. 17, 181 (1967); G. Wald, Nature 219, 800 (1968); R. A. Morton and G. A. J. Pitt, Adv. Enzymol. 32, 97 (1969).
[Crossref] [PubMed]

Rushton, W. A. H.

W. A. H. Rushton, F. W. Campbell, W. A. Hagins, and G. S. Brindley, Opt. Acta 1, 183 (1955).
[Crossref]

Schmidt, W. J.

W. J. Schmidt, Kolloid-Z. 85, 137 (1938).
[Crossref]

Shichi, H.

According to H. Shichi, Biochem. 9, 1973 (1970), the density ratio of the β band peak to the α band peak is 0.29 for extracted, purified cattle rhodopsin. In the publication of H. Shichi, M. S. Lewis, F. Irreverre, and A. L. Stone, J. Biol. Chem. 244, 529 (1969), the ratio of molar extinction coefficients of the β band and the α band is 0.266 for pure cattle rhodopsin. On the other hand, the same ratio obtained from the same preparation by H. Shichi appears to be close to 0.21, in one of his more-recent records that he provided for us. Our choice of 0.25 for this figure represents not only an average to the above numbers, but also our best estimate of the density ratio of the, β and α band peaks for in situ frog rhodopsin.
[Crossref]

Sjöstrand, F. S.

F. S. Sjöstrand, J. Cellular Comp. Physiol. 42, 15 (1953); S. E. G. Nilsson, J. Ultrastr. Res. 12, 207 (1965).
[Crossref]

Svaetichin, G.

G. Svaetichin, K. Negishi, and R. Fatehchand, in Ciba Foundation Symposium on Colour Vision: Physiology and Experimental Psychology, edited by A. V. S. DeReuck and J. Knight (Little, Brown, Boston, 1965), p. 178.

Wald, G.

P. K. Brown and G. Wald, Science 144, 45 (1964); G. Wald and P. K. Brown, Cold Spring Harbor Symp. Quant. Biol. 30, 345 (1965).
[Crossref] [PubMed]

G. Wald, P. K. Brown, and I. R. Gibbons, J. Opt. Soc. Am. 53, 20 (1963).
[Crossref] [PubMed]

G. Wald and P. K. Brown, J. Gen. Physiol. 37, 189 (1953).

G. Wald and R. Hubbard, Proc. Natl. Acad. Sci. USA 36, 92 (1950); G. Wald and P. K. Brown, Proc. Natl. Acad. Sci. USA 36, 84 (1950); H. J. A. Dartnall, Vision Res. 8, 339 (1968).
[Crossref] [PubMed]

See, for example, Fig. 1 of G. Wald, Doc. Ophthalmol. 3, 94 (1949).
[Crossref]

Wolken, J. J.

J. J. Wolken, R. Forsberg, G. Gallik, and R. Florida, Rev. Sci. Instr. 39, 1734 (1968).
[Crossref]

Yang, C. C.

C. C. Yang and V. Legallais, Rev. Sci. Instr. 25, 801 (1954); B. Chance, R. Perry, L. Åkerman, and B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[Crossref]

Biochem. (1)

According to H. Shichi, Biochem. 9, 1973 (1970), the density ratio of the β band peak to the α band peak is 0.29 for extracted, purified cattle rhodopsin. In the publication of H. Shichi, M. S. Lewis, F. Irreverre, and A. L. Stone, J. Biol. Chem. 244, 529 (1969), the ratio of molar extinction coefficients of the β band and the α band is 0.266 for pure cattle rhodopsin. On the other hand, the same ratio obtained from the same preparation by H. Shichi appears to be close to 0.21, in one of his more-recent records that he provided for us. Our choice of 0.25 for this figure represents not only an average to the above numbers, but also our best estimate of the density ratio of the, β and α band peaks for in situ frog rhodopsin.
[Crossref]

Biol. Rev. (1)

M. F. Moody, Biol. Rev. 39, 43 (1964).
[Crossref]

Biophys. J. (1)

P. A. Liebman, Biophys. J. 2, 161 (1962).

Br. Med. Bull. (1)

H. J. A. Dartnall, Br. Med. Bull. 9, 24 (1953).

Discuss. Faraday Soc. (1)

W. A. Hagins and W. H. Jennings, Discuss. Faraday Soc. 27, 180 (1959).
[Crossref]

Doc. Ophthalmol. (1)

See, for example, Fig. 1 of G. Wald, Doc. Ophthalmol. 3, 94 (1949).
[Crossref]

J. Cellular Comp. Physiol. (1)

F. S. Sjöstrand, J. Cellular Comp. Physiol. 42, 15 (1953); S. E. G. Nilsson, J. Ultrastr. Res. 12, 207 (1965).
[Crossref]

J. Gen. Physiol. (2)

F. I. Hárosi and E. F. MacNichol, J. Gen. Physiol. 63, 279 (1974).
[Crossref]

G. Wald and P. K. Brown, J. Gen. Physiol. 37, 189 (1953).

J. Opt. Soc. Am. (5)

J. Physiol. (Lond.) (1)

E. J. Denton, J. Physiol. (Lond.) 124, 16P and 17P (1954); E. J. Denton, Proc. R. Soc. (Lond.) B150, 78 (1959).

J. Roy. Microscop. Soc. (1)

T. Caspersson, J. Roy. Microscop. Soc. 60, 8 (1940).
[Crossref]

Jap. J. Physiol. (1)

T. Hanaoka and K. Fujimoto, Jap. J. Physiol. 7, 276 (1957).
[Crossref]

Kolloid-Z. (1)

W. J. Schmidt, Kolloid-Z. 85, 137 (1938).
[Crossref]

Opt. Acta (1)

W. A. H. Rushton, F. W. Campbell, W. A. Hagins, and G. S. Brindley, Opt. Acta 1, 183 (1955).
[Crossref]

Proc. Natl. Acad. Sci. USA (1)

G. Wald and R. Hubbard, Proc. Natl. Acad. Sci. USA 36, 92 (1950); G. Wald and P. K. Brown, Proc. Natl. Acad. Sci. USA 36, 84 (1950); H. J. A. Dartnall, Vision Res. 8, 339 (1968).
[Crossref] [PubMed]

Progr. Biophys. Molec. Biol. (1)

See, for example, the following reviews: E. W. Abrahamson and S. E. Ostroy, Progr. Biophys. Molec. Biol. 17, 181 (1967); G. Wald, Nature 219, 800 (1968); R. A. Morton and G. A. J. Pitt, Adv. Enzymol. 32, 97 (1969).
[Crossref] [PubMed]

Rev. Sci. Instr. (2)

J. J. Wolken, R. Forsberg, G. Gallik, and R. Florida, Rev. Sci. Instr. 39, 1734 (1968).
[Crossref]

C. C. Yang and V. Legallais, Rev. Sci. Instr. 25, 801 (1954); B. Chance, R. Perry, L. Åkerman, and B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[Crossref]

Science (3)

W. B. Marks, W. H. Dobelle, and E. F. MacNichol, Science 143, 1181 (1964).
[Crossref] [PubMed]

P. K. Brown and G. Wald, Science 144, 45 (1964); G. Wald and P. K. Brown, Cold Spring Harbor Symp. Quant. Biol. 30, 345 (1965).
[Crossref] [PubMed]

W. H. Dobelle, W. B. Marks, and E. F. MacNichol, Science 166, 1508 (1969).
[Crossref] [PubMed]

Vision Res. (1)

P. A. Liebman and G. Entine, Vision Res. 8, 761 (1968).
[Crossref] [PubMed]

Other (13)

P. A. Liebman, in Handbook of Sensory Physiology, Vol. VII/1. Photochemistry of Vision, edited by H. J. A. Dartnall (Springer, Berlin, Heidelberg, New York, 1972), Ch. 12, p. 481.
[Crossref]

G. Svaetichin, K. Negishi, and R. Fatehchand, in Ciba Foundation Symposium on Colour Vision: Physiology and Experimental Psychology, edited by A. V. S. DeReuck and J. Knight (Little, Brown, Boston, 1965), p. 178.

W. B. Marks, Ph.D. dissertation (The Johns Hopkins University, Baltimore, Md., 1963); W. B. Marks, J. Physiol. (Lond.) 178, 14 (1965).

See, for example, the recent review by R. A. Morton, in Handbook of Sensory Physiology, Vol VII/1. Photochemistry of Vision, edited by H. J. A. Dartnall (Springer, Berlin, Heidelberg, New York, 1972), Ch. 2, p. 33.
[Crossref]

It has been shown that the quantum efficiency of bleaching is about 23 for all visual pigments; e.g., H. J. A. Dartnall, in Handbook of Sensory Physiology, Vol. VII/1. Photochemistry of Vision, edited by H. J. A. Dartnall (Springer, Berlin, Heidelberg, New York, 1972), Ch. 4, p. 122.
[Crossref]

With this statement, we imply that long-term fluctuations are either non-existent in the DMSP, or that their effects are detectable so that all records subject to drift may be discarded. Because of its permanent memory, the DMSP is well suited to test for slow drifts arising from any source in the instrument by computer comparison of reference transmittances recorded at different times during an experiment. The acceptance or rejection of records we decide by our selection rule (Ref. 33), which states that a spectral recording may be regarded free of distortions if the absorptance trace falls within about ±1% of the zero line, before as well as after photolysis, in spectral regions where no absorption is expected. This procedure sets also the limit on the error that may result from the reference being a clear area in the preparation instead of the cell when devoid of pigment. These issues are discussed in more detail in Ref. 33.

F. I. Hárosi, Ph.D. dissertation (The Johns Hopkins University, Baltimore, Md., 1971).

E. F. MacNichol, R. Feinberg, and F. I. Hárosi, in Colour 73 (A. Hilger, Rank Precision Industries, London, 1973), p. 191.

The assumption, that the excess density measured at 370 nm over the expected density of rhodopsin is due to retinal oxime, should be valid only if other photoproducts do not persist long enough to interfere with the measurement. Although we can detect the formation of some Meta III, and the brief presence of some Meta II, at 5- and 10-mM hydroxylamine concentrations, at 75-mM concentration of this reagent the oxime formation appears to be faster than the time resolution of the DMSP.

On the basis of a theoretical analysis by Hárosi and Malerba we derived an equation that relates the different absorbing states of the same cell in terms of molar extinction coefficients (∊′, ∊″), molecular extinctions (M′, M″), cellular optical density components (D||′, D||″), and dichroic ratios (R′ = D⊥′/D||′, R″ = D⊥″/D||″), as ∊′∊″=M′M″=D‖′[1+R′(2-3b)]D‖″[1+R″(2-3b)].Coefficient b is related to the numerical aperture (NA) of the microscope condenser (with aperture cone 2α) as b= tan2(α/2). For an oil-immersion-type condenser (n= 1.455) of (NA) = 0.4, b= 0.0197, and thus ∊′∊″=D‖′(1+1.941R′)D‖″(1+1.941R″).We use this equation to compute photoproduct densities from presumed molar extinction coefficients, or vice versa, and assume unchanging pigment concentrations and path lengths of measuring light within the cell.

The effective bandwidth of 18 Hz is obtained with reference to a single-time-constant (τ) integrator (low-pass filter) for which the upper corner frequency (fc) is given by the relationship 2πfo= 1/τ. The DMSP is operated such that a 5-nm-wide spectral segment is swept in 10 ms. Since about 90% of this time is spent for summing repeated samples of the same signal (120 times on each channel), the integration time is about 9 ms. If τ= 9 × 10−3 s, fc≅ 18 Hz. Note that a simple low-pass filter with τ= RC has a slow roll-off characteristic (20 dB/decade) and hence a broader bandwidth than the fc= 1/2 πτ formula indicates. Digital summation, on the other hand, approaches ideal integration by virtue of its high speed and the lack of phase distortion. Because of these, it is possible to process in the DMSP the separated fast modulation and the slowly varying average signal with essentially no phase shift between them (except for a fixed lag of about 35 μ s due to multiplexing the two analog channels).

The time required for punching the data of one measurement is 12–13 s (on the high-speed paper-tape punch).

See, for example, E. L. Crow, F. A. Davis, and M. W. Maxfield, Statistics Manual (Dover, New York, 1960).

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Figures (23)

Fig. 1
Fig. 1

Optical schematic of the dichroic microspectrophotometer. S1 quartz-iodine lamp. S2 Hg–Cd spectral lamp. M1, M2, M3, M4 first surface mirrors. D1 adjustable iris diaphragm. G diffraction grating. SH1, SH2 electromagnetic shutter. QP quartz plate. D2, D3 adjustable rectangular diaphragms. P, (P2) polarizing prism. QB quartz beam splitter. SM swinging mirror. F1 F2, F3 filters. L1, L3 achromatic lenses. S3 tungsten lamp. S4 replaceable flashbulb. OB1, OB2 microscope objectives. SP specimen’s plane. EP (IRIC) eyepiece or infrared image converter. L2 quartz lens. SH3 manual shutter. C cathode of photomultiplier tube.

Fig. 2
Fig. 2

Signal-flow block diagram of the instrument. MON monochromator, driven by a two-pole, 60-Hz synchronous motor. P polarizer. MIC microscope with specimen. PM photomultiplier tube. CP control package for monochromator driving motor, shutters, etc. ROT rotor for polarizer, driven by a two-pole 60-Hz synchronous motor. Speed of rotation is approximately 6700 rpm. I(φ) phase information. PSD phase-sensitive detector. COM commands. I(λ) wavelength information. I(S) information on direction of scanning. IE interface equipment, containing A/D and D/A converters, control logic, etc. I(G) information on ac gain. OSC display oscilloscope. DP 4096-word, stored-program, digital processor. HPTR/P high-speed paper-tape reader and punch. LPTR/P mechanical paper-tape reader and punch. TTY Teletype. A1 amplifier connected as current-to-voltage converter. A2 variable-gain, direct-coupled amplifier. A3 variable-gain, capacitance-coupled amplifier. A4, A5 fixed-gain preamplifier of the interface equipment.

Fig. 3
Fig. 3

Relationships between signal waveforms and polarization of light in the measuring beam. Vd average or dc component of photocurrent. Vp local peak value of modulation. A, B, C instantaneous value of modulated wave at half-cycle center of carrier. tφ time lag between modulated signal and carrier. φ phase shift corresponding to tφ,. Vp cosφ excursion of modulated photocurrent from average (Vd) at points A, B, and C.

Fig. 4
Fig. 4

Normalized spectral response of the instrument to a Hg–Cd source (S2). It is a dc recording of light flux, normalized to its highest peak at 435 nm. The measurement consisted of 32 bidirectional scans of the spectrum. Simulating experimental usage, a small slit-shaped opening (D3) was focused in the plane of the specimen by the condenser. The seven major peaks in the record are marked with arrows. The corresponding spectral emission lines are designated with numbers representing their wavelengths rounded to the nearest nanometer.

Fig. 5
Fig. 5

Absorption spectrum of a turtle (Pseudemys scripta) cone oil droplet as measured with the DMSP. The record, consisting of two traces, is the average of 96 bidirectional scans. Although the circles (A||) and triangles (A) show distinguishable absorptance values at most wavelengths, no systematic difference can be observed between the two components.

Fig. 6
Fig. 6

Pre-bleach absorption spectrum of a frog (Rana pipiens) rod outer segment as measured with transversely penetrating, polarized light in the DMSP. The visually estimated cellular dimensions were 6-μm diameter and 60-μm length. The measuring light was stopped down (at D2) so that its image extended about 3 × 20 μm in the specimen’s plane. The aqueous suspending medium for the cells contained 110-mM NaCl, 2-mM KCl, 2-mM CaCl2, and 10-mM HEPES (N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer at pH 7.5. The temperature of the microscope stage was 22.0 °C. The recording shown is the average of 32 bidirectional scans of the spectrum. It was obtained 105 min after decapitation of the frog.

Fig. 7
Fig. 7

Same as Fig. 6 but this is the first post-bleach recording obtained following a 150-ms white flash. It is the result of a single spectral scan performed within 1 s. The direction of scanning was from 325 to 695 nm.

Fig. 8
Fig. 8

Second post-bleach recording, obtained (from the same cell as of Figs. 67) during a period approximately 15–55 s following the bleach. It is the average of 32 bidirectional scans.

Fig. 9
Fig. 9

Pre-bleach absorption spectrum of a frog-rod outer segment in a 4-hour-old preparation. The aqueous mounting medium contained 110-mM NaCl, 4.6-mM KCI, 2-mM CaCl2, at pH 6.4 (adjusted by addition of HCl). The depicted result was obtained at 22.0 °C as the first 32-scan recording.

Fig. 10
Fig. 10

Post-bleach recording (32-scan averaged) obtained from the same cell as Fig. 9 about 1 h following a strong orange-green bleach of one minute duration.

Fig. 11
Fig. 11

Pre-bleach absorption spectrum of a freshly isolated frog-rod outer segment, recorded at 21.0 °C. The suspending medium contained 110-mM NaCl, 2-mM KC1 and 1-mM EGTA [ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetate], at pH 7.4. The spectrum was obtained 20 min post-mortem, with transversely polarized light, as the average of 32 bidirectional spectral scans.

Fig. 12
Fig. 12

Same as Fig. 11, except for axially polarized light.

Fig. 13
Fig. 13

Post-bleach recording (32-scan average) for transversely polarized light obtained from the same cell as Fig. 11 about 50 min after the first of two white flashes.

Fig. 14
Fig. 14

Same as Fig. 13, except for axially polarized light.

Fig. 15
Fig. 15

Photomicrograph of a rod outer segment obtained from a frog retina. The photo was taken in the DMSP subsequent to all measurements. The length of the calibration mark represents 10 μm.

Fig. 16
Fig. 16

The second 32-scan pre-bleach recording obtained from the cell, depicted in Fig. 15, about 30 min post-mortem (the first such recording did not meet the acceptance criteria) in the presence of hydroxylamine at 21.5 °C. The aqueous suspending medium contained 75-mM NaCl and 75-mM NH2OH at pH 7.1. The measuring light beam extended about 3 × 30 μm in a plane transecting the cylindrical specimen’s center. This was achieved by focusing the objective to yield the sharpest contour of the cell, and then by adjusting the condenser for sharpest image of the field stop, D3. The auxiliary scales in the record are set to the peaks of the two components at 500 nm.

Fig. 17
Fig. 17

Post-bleach recording obtained from the same cell as Figs. 1516 about 40 min later, following exhaustive bleaching. The auxiliary scales in the record are set to the peaks at 370 and 500 nm.

Fig. 18
Fig. 18

Photomicrograph of another rod cell in the same preparation as of Figs. 1517. The essential difference here with respect to the conditions described in the caption of Fig. 16 is that D2 was used as the field stop. The length of the calibration mark represents 10 μm.

Fig. 19
Fig. 19

The first 32-scan pre-bleach recording obtained from the cell depicted in Fig. 18. The preparation was then about 2 h old, the temperature 22.0 °C. The auxiliary scales in the record are set to the peaks at 500 nm.

Fig. 20
Fig. 20

Post-bleach recording obtained from the same cell as Figs. 1819 about 15 min after a white flash. The auxiliary scales in the record are set to the peaks at 370 and 500 nm.

Fig. 21
Fig. 21

The average pre-bleach absorption spectra of 38 rhodopsin-containing rods of the frog as measured with plane-polarized light in the DMSP. Each single-cell recording consisted of the average of the first 32 bidirectional scans. The 38 cells were derived from nearly as many retinas; they were suspended in near-physiological solutions of various composition. The majority of the preparations were still fresh when measured (at room temperature). The 38 separate 32-scan spectra were averaged with equal weight at 5-nm increments of wavelength.

Fig. 22
Fig. 22

Average absorption spectrum for transversely polarized light reproduced from Fig. 21, and the same ±σ (three traces).

Fig. 23
Fig. 23

Same as Fig. 22, except for axially polarized light.

Tables (1)

Tables Icon

Table I Summary of measured and computed data concerning the specific density of rhodopsin-containing frog-rod outer segments suspended in hydroxylamine.

Equations (26)

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d ( t ) = 1 / n T t 0 t 0 + n T ( V d + V ac ) d t = V d .
a ( t ) = 1 / n T t 0 t 0 + n T G V ac V c d t = G V p 2 n / n T 0 T / 2 sin ω ( t - t φ ) d t = ( 2 G / T ) V p cos φ 0 T / 2 sin ω t d t = ( 2 G / π ) V p cos φ
d ( λ ) = N V d ,
a ( λ ) = N ( 2 G / π ) V p cos φ .
V d = ( 1 / N ) d ( λ ) ,
V p cos φ = ( 1 / N ) ( π / 2 G ) a ( λ ) .
Φ ( λ ) ( 1 / N ) [ d ( λ ) + ( π / 2 G ) a ( λ ) ] ,
Φ ( λ ) ( 1 / N ) [ d ( λ ) - ( π / 2 G ) a ( λ ) ] .
A ( λ ) = 1 - Φ ( λ ) / Φ ( λ ) ,
A ( λ ) = 1 - Φ ( λ ) / Φ ( λ ) .
A , ( λ ) = 1 - ( N / N ) ( 2 G / π ) d ( λ ) ± a ( λ ) ( 2 G / π ) d ( λ ) ± a ( λ ) ,
μ n = ( 1 / n ) ( x 1 + x 2 + + x n - 1 + x n ) = ( 1 / n ) i = 1 n x i .
μ n - 1 = [ 1 / ( n - 1 ) ] ( x 1 + x 2 + + x n - 1 ) = [ 1 / ( n - 1 ) ] i = 1 n - 1 x i .
n μ n = ( n - 1 ) μ n - 1 + x n .
μ n = μ n - 1 + ( 1 / n ) ( x n - μ n - 1 ) .
σ n = { [ 1 / ( n - 1 ) ] i = 1 n ( x i - μ n ) 2 } 1 2 ,
σ n 2 = [ 1 / ( n - 1 ) ] i = 1 n [ x i 2 - 2 x i μ n + μ n 2 ] .
i = 1 n 2 x i μ n = 2 n μ n 2             and             i = 1 n μ n 2 = n μ n 2 ,
σ n 2 = [ 1 / ( n - 1 ) ] i = 1 n [ x i 2 - n μ n 2 ] .
σ n - 1 2 = [ 1 / ( n - 2 ) ] i = 1 n - 1 [ x i 2 - ( n - 1 ) μ n - 1 2 ] .
i = 1 n x i 2 = i = 1 n - 1 x i 2 + x n 2 ,
i = 1 n - 1 x i 2 = ( n - 1 ) σ n 2 - x n 2 + n μ n 2 ,
σ n - 1 2 = [ 1 / ( n - 2 ) ] [ ( n - 1 ) σ n 2 - x n 2 + n μ n 2 - ( n - 1 ) μ n - 1 2 ] .
σ n 2 = [ ( n - 2 ) / ( n - 1 ) ] σ n - 1 2 + ( 1 / n ) [ x n - μ n - 1 ] 2 .
=MM=D[1+R(2-3b)]D[1+R(2-3b)].
=D(1+1.941R)D(1+1.941R).