Abstract

Microspectrophotometers, manual and recording, have been constructed with cadmium selenide (CdSe) photoconductive cells and are in use in our laboratory as a spectroscopic tool in the study of the pigment chemistry of photoreceptors in living cells. The basic design and operation of these microspectrophotometers for obtaining absorption spectra are described. The absorption spectrum from the ultraviolet, visible, and near infrared can be obtained in a single sweep for specimen areas as small as 2 μ2. Examples of absorption spectra of the chloroplast of plant cells, the retinal rods of the visual cells of the eye, and the red blood cell are illustrated. The applications of microspectrophotometry to a variety of biological, biochemical, and chemical problems are indicated.

© 1963 Optical Society of America

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  1. T. Caspersson, Cell Growth and Cell Function (Norton, New York, 1950).
  2. T. Caspersson, G. Lomakka, G. Svensson, R. Säfström, Exp. Cell Res. Suppl. 3, 40 (1955).
  3. T. Caspersson, Fed. Proc. 20, 858 (1961).
  4. A. W. Pollister, L. Ornstein, in Analytical Cytology, 2nd ed., R. C. Mellors, ed. (McGraw-Hill, New York, 1959), p. 431.
  5. P. M. M. Wallace, in General Cytochemical Methods, J. F. Danielli, ed. (Academic Press, New York, 1958), Vol. 1, p. 163.
  6. H. Swift, E. Rasch, in Physical Techniques in Biological Research, G. Oster, A. W. Pollister, eds. (Academic Press, New York, 1956), Vol. III, p. 353.
  7. Cytochemical Methods With Quantitative Aims, Exp. Cell Res. Suppl.4 (1957).
  8. Scanning Techniques in Biology and Medicine, Ann. N.Y. Acad. Sci. 97, 331 (1962).
  9. E. J. Denton, Proc. Roy. Soc. B, 150, 78 (1959).
    [CrossRef]
  10. B. Chance, R. Perry, L. Akerman, B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
    [CrossRef]
  11. G. K. Strother, J. J. Wolken, Science, 130, 1084 (1959).
    [CrossRef] [PubMed]
  12. B. Henry, H. Cole, Rev. Sci. Instr. 30, 90 (1959).
    [CrossRef]
  13. J. J. Wolken, V. Jackman, R. Kerr, G. Ludwig, (unpublished research).
  14. J. J. Wolken, J. Theoret. Biol. 3, 192 (1962).
    [CrossRef]
  15. J. J. Wolken, J. Opt. Soc. Am. 53, 1 (1963).
    [CrossRef]
  16. J. J. Wolken, Euglena: An Experimental Organism for Biochemical and Biophysical Studies (Rutgers University Press, New Brunswick, New Jersey, 1961).
  17. G. K. Strother, J. J. Wolken, J. Protozool. 8, 261 (1961).
  18. J. J. Wolken, F. A. Schwertz, J. Gen. Physiol. 37, 111 (1953).
    [CrossRef] [PubMed]
  19. J. J. Wolken, in Macromolecular Complexes, M. V. Edds, ed. (Ronald Press, New York, 1961), p. 85.
  20. J. J. Wolken, Inves. Ophth. 1, 327 (1962).
  21. H. J. A. Dartnall, in The Visual Pigments (Wiley, New York, 1957).
  22. T. Hanaoka, K. Fujimoto, Jap. J. Physiol. 7, 276 (1957).
    [CrossRef]
  23. W. B. Marks, E. F. MacNichol, Abstracts of the Sixth Biophysical Society Meeting, TE-2, Washington, D.C.and W. B. Marks, “Difference Spectra of Visual Pigments in Single Goldfish Cones”, Thesis, Johns Hopkins Univ., Baltimore, Md., 1963.
  24. G. K. Strother, J. J. Wolken, Exp. Cell Res. 21, 504 (1960).
    [CrossRef]
  25. G. K. Strother, Exp. Cell Res. 29, 349 (1963).
    [CrossRef]
  26. J. J. Wolken, J. A. Gross, J. Protozool. (in press, 1963).
  27. C. L. Wells, J. J. Wolken, Nature 193, 977 (1962).
    [CrossRef]
  28. P. K. Brown, J. Opt. Soc. Am. 51, 1000 (1961).
    [CrossRef]
  29. P. A. Liebman, Biophys. J. 2, 161 (1962).
    [CrossRef]
  30. Carl Zeiss, Inc., 485 Fifth Avenue, New York, N.Y.
  31. Canal Industrial Corporation, Bethesda, Maryland.
  32. J. J. D. van der Gon, J. J. Wolken (unpublished research) and 9th Annual Report Biophysical Research Lab., Univ. of Pittsburgh School of Medicine, 1962–1963.

1963

G. K. Strother, Exp. Cell Res. 29, 349 (1963).
[CrossRef]

J. J. Wolken, J. Opt. Soc. Am. 53, 1 (1963).
[CrossRef]

1962

C. L. Wells, J. J. Wolken, Nature 193, 977 (1962).
[CrossRef]

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

J. J. Wolken, Inves. Ophth. 1, 327 (1962).

Scanning Techniques in Biology and Medicine, Ann. N.Y. Acad. Sci. 97, 331 (1962).

J. J. Wolken, J. Theoret. Biol. 3, 192 (1962).
[CrossRef]

1961

G. K. Strother, J. J. Wolken, J. Protozool. 8, 261 (1961).

T. Caspersson, Fed. Proc. 20, 858 (1961).

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

1960

G. K. Strother, J. J. Wolken, Exp. Cell Res. 21, 504 (1960).
[CrossRef]

1959

E. J. Denton, Proc. Roy. Soc. B, 150, 78 (1959).
[CrossRef]

B. Chance, R. Perry, L. Akerman, B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[CrossRef]

G. K. Strother, J. J. Wolken, Science, 130, 1084 (1959).
[CrossRef] [PubMed]

B. Henry, H. Cole, Rev. Sci. Instr. 30, 90 (1959).
[CrossRef]

1957

Cytochemical Methods With Quantitative Aims, Exp. Cell Res. Suppl.4 (1957).

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

1955

T. Caspersson, G. Lomakka, G. Svensson, R. Säfström, Exp. Cell Res. Suppl. 3, 40 (1955).

1953

J. J. Wolken, F. A. Schwertz, J. Gen. Physiol. 37, 111 (1953).
[CrossRef] [PubMed]

Akerman, L.

B. Chance, R. Perry, L. Akerman, B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[CrossRef]

Brown, P. K.

Caspersson, T.

T. Caspersson, Fed. Proc. 20, 858 (1961).

T. Caspersson, G. Lomakka, G. Svensson, R. Säfström, Exp. Cell Res. Suppl. 3, 40 (1955).

T. Caspersson, Cell Growth and Cell Function (Norton, New York, 1950).

Chance, B.

B. Chance, R. Perry, L. Akerman, B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[CrossRef]

Cole, H.

B. Henry, H. Cole, Rev. Sci. Instr. 30, 90 (1959).
[CrossRef]

Dartnall, H. J. A.

H. J. A. Dartnall, in The Visual Pigments (Wiley, New York, 1957).

Denton, E. J.

E. J. Denton, Proc. Roy. Soc. B, 150, 78 (1959).
[CrossRef]

Fujimoto, K.

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

Gross, J. A.

J. J. Wolken, J. A. Gross, J. Protozool. (in press, 1963).

Hanaoka, T.

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

Henry, B.

B. Henry, H. Cole, Rev. Sci. Instr. 30, 90 (1959).
[CrossRef]

Jackman, V.

J. J. Wolken, V. Jackman, R. Kerr, G. Ludwig, (unpublished research).

Kerr, R.

J. J. Wolken, V. Jackman, R. Kerr, G. Ludwig, (unpublished research).

Liebman, P. A.

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

Lomakka, G.

T. Caspersson, G. Lomakka, G. Svensson, R. Säfström, Exp. Cell Res. Suppl. 3, 40 (1955).

Ludwig, G.

J. J. Wolken, V. Jackman, R. Kerr, G. Ludwig, (unpublished research).

MacNichol, E. F.

W. B. Marks, E. F. MacNichol, Abstracts of the Sixth Biophysical Society Meeting, TE-2, Washington, D.C.and W. B. Marks, “Difference Spectra of Visual Pigments in Single Goldfish Cones”, Thesis, Johns Hopkins Univ., Baltimore, Md., 1963.

Marks, W. B.

W. B. Marks, E. F. MacNichol, Abstracts of the Sixth Biophysical Society Meeting, TE-2, Washington, D.C.and W. B. Marks, “Difference Spectra of Visual Pigments in Single Goldfish Cones”, Thesis, Johns Hopkins Univ., Baltimore, Md., 1963.

Ornstein, L.

A. W. Pollister, L. Ornstein, in Analytical Cytology, 2nd ed., R. C. Mellors, ed. (McGraw-Hill, New York, 1959), p. 431.

Perry, R.

B. Chance, R. Perry, L. Akerman, B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[CrossRef]

Pollister, A. W.

A. W. Pollister, L. Ornstein, in Analytical Cytology, 2nd ed., R. C. Mellors, ed. (McGraw-Hill, New York, 1959), p. 431.

Rasch, E.

H. Swift, E. Rasch, in Physical Techniques in Biological Research, G. Oster, A. W. Pollister, eds. (Academic Press, New York, 1956), Vol. III, p. 353.

Säfström, R.

T. Caspersson, G. Lomakka, G. Svensson, R. Säfström, Exp. Cell Res. Suppl. 3, 40 (1955).

Schwertz, F. A.

J. J. Wolken, F. A. Schwertz, J. Gen. Physiol. 37, 111 (1953).
[CrossRef] [PubMed]

Strother, G. K.

G. K. Strother, Exp. Cell Res. 29, 349 (1963).
[CrossRef]

G. K. Strother, J. J. Wolken, J. Protozool. 8, 261 (1961).

G. K. Strother, J. J. Wolken, Exp. Cell Res. 21, 504 (1960).
[CrossRef]

G. K. Strother, J. J. Wolken, Science, 130, 1084 (1959).
[CrossRef] [PubMed]

Svensson, G.

T. Caspersson, G. Lomakka, G. Svensson, R. Säfström, Exp. Cell Res. Suppl. 3, 40 (1955).

Swift, H.

H. Swift, E. Rasch, in Physical Techniques in Biological Research, G. Oster, A. W. Pollister, eds. (Academic Press, New York, 1956), Vol. III, p. 353.

Thorell, B.

B. Chance, R. Perry, L. Akerman, B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[CrossRef]

van der Gon, J. J. D.

J. J. D. van der Gon, J. J. Wolken (unpublished research) and 9th Annual Report Biophysical Research Lab., Univ. of Pittsburgh School of Medicine, 1962–1963.

Wallace, P. M. M.

P. M. M. Wallace, in General Cytochemical Methods, J. F. Danielli, ed. (Academic Press, New York, 1958), Vol. 1, p. 163.

Wells, C. L.

C. L. Wells, J. J. Wolken, Nature 193, 977 (1962).
[CrossRef]

Wolken, J. J.

J. J. Wolken, J. Opt. Soc. Am. 53, 1 (1963).
[CrossRef]

C. L. Wells, J. J. Wolken, Nature 193, 977 (1962).
[CrossRef]

J. J. Wolken, J. Theoret. Biol. 3, 192 (1962).
[CrossRef]

J. J. Wolken, Inves. Ophth. 1, 327 (1962).

G. K. Strother, J. J. Wolken, J. Protozool. 8, 261 (1961).

G. K. Strother, J. J. Wolken, Exp. Cell Res. 21, 504 (1960).
[CrossRef]

G. K. Strother, J. J. Wolken, Science, 130, 1084 (1959).
[CrossRef] [PubMed]

J. J. Wolken, F. A. Schwertz, J. Gen. Physiol. 37, 111 (1953).
[CrossRef] [PubMed]

J. J. D. van der Gon, J. J. Wolken (unpublished research) and 9th Annual Report Biophysical Research Lab., Univ. of Pittsburgh School of Medicine, 1962–1963.

J. J. Wolken, J. A. Gross, J. Protozool. (in press, 1963).

J. J. Wolken, Euglena: An Experimental Organism for Biochemical and Biophysical Studies (Rutgers University Press, New Brunswick, New Jersey, 1961).

J. J. Wolken, V. Jackman, R. Kerr, G. Ludwig, (unpublished research).

J. J. Wolken, in Macromolecular Complexes, M. V. Edds, ed. (Ronald Press, New York, 1961), p. 85.

Ann. N.Y. Acad. Sci.

Scanning Techniques in Biology and Medicine, Ann. N.Y. Acad. Sci. 97, 331 (1962).

Biophys. J.

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

Exp. Cell Res.

G. K. Strother, J. J. Wolken, Exp. Cell Res. 21, 504 (1960).
[CrossRef]

G. K. Strother, Exp. Cell Res. 29, 349 (1963).
[CrossRef]

Exp. Cell Res. Suppl.

T. Caspersson, G. Lomakka, G. Svensson, R. Säfström, Exp. Cell Res. Suppl. 3, 40 (1955).

Cytochemical Methods With Quantitative Aims, Exp. Cell Res. Suppl.4 (1957).

Fed. Proc.

T. Caspersson, Fed. Proc. 20, 858 (1961).

Inves. Ophth.

J. J. Wolken, Inves. Ophth. 1, 327 (1962).

J. Gen. Physiol.

J. J. Wolken, F. A. Schwertz, J. Gen. Physiol. 37, 111 (1953).
[CrossRef] [PubMed]

J. Opt. Soc. Am.

J. Protozool.

G. K. Strother, J. J. Wolken, J. Protozool. 8, 261 (1961).

J. Theoret. Biol.

J. J. Wolken, J. Theoret. Biol. 3, 192 (1962).
[CrossRef]

Jap. J. Physiol.

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

Nature

C. L. Wells, J. J. Wolken, Nature 193, 977 (1962).
[CrossRef]

Proc. Roy. Soc. B

E. J. Denton, Proc. Roy. Soc. B, 150, 78 (1959).
[CrossRef]

Rev. Sci. Instr.

B. Chance, R. Perry, L. Akerman, B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[CrossRef]

B. Henry, H. Cole, Rev. Sci. Instr. 30, 90 (1959).
[CrossRef]

Science

G. K. Strother, J. J. Wolken, Science, 130, 1084 (1959).
[CrossRef] [PubMed]

Other

A. W. Pollister, L. Ornstein, in Analytical Cytology, 2nd ed., R. C. Mellors, ed. (McGraw-Hill, New York, 1959), p. 431.

P. M. M. Wallace, in General Cytochemical Methods, J. F. Danielli, ed. (Academic Press, New York, 1958), Vol. 1, p. 163.

H. Swift, E. Rasch, in Physical Techniques in Biological Research, G. Oster, A. W. Pollister, eds. (Academic Press, New York, 1956), Vol. III, p. 353.

J. J. Wolken, V. Jackman, R. Kerr, G. Ludwig, (unpublished research).

T. Caspersson, Cell Growth and Cell Function (Norton, New York, 1950).

J. J. Wolken, Euglena: An Experimental Organism for Biochemical and Biophysical Studies (Rutgers University Press, New Brunswick, New Jersey, 1961).

J. J. Wolken, in Macromolecular Complexes, M. V. Edds, ed. (Ronald Press, New York, 1961), p. 85.

J. J. Wolken, J. A. Gross, J. Protozool. (in press, 1963).

W. B. Marks, E. F. MacNichol, Abstracts of the Sixth Biophysical Society Meeting, TE-2, Washington, D.C.and W. B. Marks, “Difference Spectra of Visual Pigments in Single Goldfish Cones”, Thesis, Johns Hopkins Univ., Baltimore, Md., 1963.

H. J. A. Dartnall, in The Visual Pigments (Wiley, New York, 1957).

Carl Zeiss, Inc., 485 Fifth Avenue, New York, N.Y.

Canal Industrial Corporation, Bethesda, Maryland.

J. J. D. van der Gon, J. J. Wolken (unpublished research) and 9th Annual Report Biophysical Research Lab., Univ. of Pittsburgh School of Medicine, 1962–1963.

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Figures (7)

Fig. 1
Fig. 1

Diagram of simplified microspectrophotometer (M-1). L, light source; G, monochromator; M, microscope with reflecting optics; C, photoconductive cell; A, de amplifier; D, oscilloscope. (In place of A and D, a dc voltmeter, Hewlett-Packard type 412 A, is used.) The line drawing shows housing for photocell, viewer, and mounting on sliding track.

Fig. 2
Fig. 2

Diagram of recording microspectrophotometer (M-2) showing mounting of photocell and rotating sampling disk.

Fig. 3
Fig. 3

Recording microspectrophotometer (M-3) adapted from M-2; light chopper incorporated in sampling disk.

Fig. 4
Fig. 4

Section of Euglena gracilis, electron micrograph (fixed in 1% osmium tetroxide); note size of chloroplasts (cl) and other cellular organelles, e.g., mitochondria (m), nucleus (n). Curve (a)—chloroplast absorption spectrum in the uv. Curve (b)—chloroplast absorption spectrum in the visible. Curve (c)—chloroplast absorption spectrum of young culture at low light levels from 675 to 710 mμ. Curve (d)—scan across chloroplast (0.5-μ intervals) at a chlorophyll major absorption peak, 675 mμ. (Figure 4 is taken from refs. 16, 17, and 19.)

Fig. 5
Fig. 5

Euglena chloroplast absorption spectrum obtained with recording microspectrophotometer (M-2). Peak positions and peak heights compare very closely with chlorophyll a. Entering half-bandwidth, 6 mμ; time of scan, 4 min.

Fig. 6
Fig. 6

Frog retinal rod, freshly isolated and photographed in red light. (a) Absorption spectrum of frog retinal rod (curve a), compared to bleached rod, in light and NH2OH (curve b). (b) Composite absorption spectrum (curve a) of frog rods, in which only one wavelength was used for each rod, total average of 100 retinal rods. Curve b, frog rhodopsin (extract of rods in 2% aqueous digitonin). (c) Absorption at 500 mμ for scan across (0.5-μ intervals) length and diameter of frog retinal rod. (Figure 6 is taken from refs. 15 and 20.)

Fig. 7
Fig. 7

Frog red blood cell, 4 μ diam, entering half-bandwidth, 5 mμ; time of scan, 2 min.

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