Abstract

Microspectroscopy involves the combination of microscope and spectroscope optics for the purpose of investigating the spectra of small samples. The general methods of microspectroscopy are described as applied to absorption measurements in the ultraviolet, visible, and infra-red regions, emission spectroscopy, and other applications. Recently developed components and systems for microspectroscopy are emphasized, and some of the important requirements for their effective design and use are considered briefly.

© 1950 Optical Society of America

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References

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  1. T. Caspersson, Skand. Arch. f. Physiol., Supplement 73 (1936).
  2. J. F. Scott and R. L. Sinsheimer, “Ultraviolet microscopy and microspectroscopy,” in Medical Physics (Year Book Publishers, Inc., Chicago, in press), Vol. 2.
  3. A. Kohler, Zeits. f. wiss. Mik. 21, 129, 273 (1904).
  4. B. K. Johnson, Proc. Phys. Soc., London 51, 1034 (1939).
    [Crossref]
  5. L. V. Foster and E. M. Thiel, J. Opt. Soc. Am. 38, 689 (1948); L. V. Foster, Analyt. Chem. 21, 432 (1949).
    [Crossref] [PubMed]
  6. E. M. Brumberg, Bull. Acad. Sci. U.S.S.R. 23, 486 (1941); Bull. Acad. Sci. U.S.S.R. 24, 33 (1942); Nature 152, 357 (1943).
  7. D. S. Grey and P. Lee, J. Opt. Soc. Am. 39, 719 (1949); D. S. Grey, J. Opt. Soc. Am. 39, 723 (1949).
    [Crossref] [PubMed]
  8. A. J. Kavanaugh (personal communication).
  9. W. E. Leeds and M. H. F. Wilkins, Nature 164, 228 (1949).
    [Crossref]
  10. C. R. Burch, Nature 152, 748 (1943); Proc. Phys. Soc., London 59, 41 (1947).
    [Crossref]
  11. T. Dunham, “The effect of central stops on the resolution of a microscope, I,” (July, 1948).
  12. F. Sjostrand, “Über die eigenfluoreszenz tierischer gewebe mit besonderer Berucksichtigung der sangetierniere,” Stockholm (1944).
  13. H. W. Wyckoff, Quarterly Progress Report, M.I.T. Research Laboratory of Electronics (July, 1949).
  14. T. Dunham, see J. R. Loofbourow, Growth Symposium 12, 75 (1948).
  15. Harrison, Lord, and Loofbourow, Practical Spectroscopy (Prentice-Hall, Inc., New York, 1948).
  16. P. A. Cole and E. Sutton, Cold Spring Harbor Symposia on Quantitative Biology 9, 66 (1941).
    [Crossref]
  17. E. W. Dempsey and et al., Anat. Rev. 98, 417 (1947); see also R. E. Stowell, J. Nat. Cancer Inst. 3, 111 (1942); M. A. Lessler and H. A. Charipper, Science 110, 429 (1949).
    [Crossref] [PubMed]
  18. A. W. Pollister and H. Ris, Cold Spring Harbor Symposia on Quantitative Biology 12, 147 (1947).
    [Crossref]
  19. R. C. Mellors, Science (to be published); Mellors, Keane, and Streim, Nature (to be published).
  20. Land, Blout, Grey, Flower, Husek, Jones, Matz, and Merrill, Science 109, 371 (1949).
    [Crossref] [PubMed]
  21. R. L. Sinsheimer, Ph.D. thesis, M.I.T. (1948).
  22. W. E. Forsythe, editor, Measurement of Radiant Energy (McGraw-Hill Book Company, Inc., New York, 1937).
  23. P. A. Cole and F. S. Brackett, J. Opt. Soc. Am. 11, 419 (1940).
    [Crossref]
  24. Barer, Cole, and Thompson, Nature 163, 198 (1949).
    [Crossref] [PubMed]
  25. Blout, Bird, and Grey, J. Opt. Soc. Am. 39, 1052 (1949).
  26. R. C. Gore, Science 110, 710 (1949).
    [Crossref]

1949 (6)

D. S. Grey and P. Lee, J. Opt. Soc. Am. 39, 719 (1949); D. S. Grey, J. Opt. Soc. Am. 39, 723 (1949).
[Crossref] [PubMed]

W. E. Leeds and M. H. F. Wilkins, Nature 164, 228 (1949).
[Crossref]

Land, Blout, Grey, Flower, Husek, Jones, Matz, and Merrill, Science 109, 371 (1949).
[Crossref] [PubMed]

Barer, Cole, and Thompson, Nature 163, 198 (1949).
[Crossref] [PubMed]

Blout, Bird, and Grey, J. Opt. Soc. Am. 39, 1052 (1949).

R. C. Gore, Science 110, 710 (1949).
[Crossref]

1948 (2)

T. Dunham, see J. R. Loofbourow, Growth Symposium 12, 75 (1948).

T. Dunham, see J. R. Loofbourow, Growth Symposium 12, 75 (1948).

L. V. Foster and E. M. Thiel, J. Opt. Soc. Am. 38, 689 (1948); L. V. Foster, Analyt. Chem. 21, 432 (1949).
[Crossref] [PubMed]

1947 (2)

E. W. Dempsey and et al., Anat. Rev. 98, 417 (1947); see also R. E. Stowell, J. Nat. Cancer Inst. 3, 111 (1942); M. A. Lessler and H. A. Charipper, Science 110, 429 (1949).
[Crossref] [PubMed]

A. W. Pollister and H. Ris, Cold Spring Harbor Symposia on Quantitative Biology 12, 147 (1947).
[Crossref]

1943 (1)

C. R. Burch, Nature 152, 748 (1943); Proc. Phys. Soc., London 59, 41 (1947).
[Crossref]

1941 (2)

P. A. Cole and E. Sutton, Cold Spring Harbor Symposia on Quantitative Biology 9, 66 (1941).
[Crossref]

E. M. Brumberg, Bull. Acad. Sci. U.S.S.R. 23, 486 (1941); Bull. Acad. Sci. U.S.S.R. 24, 33 (1942); Nature 152, 357 (1943).

1940 (1)

1939 (1)

B. K. Johnson, Proc. Phys. Soc., London 51, 1034 (1939).
[Crossref]

1936 (1)

T. Caspersson, Skand. Arch. f. Physiol., Supplement 73 (1936).

1904 (1)

A. Kohler, Zeits. f. wiss. Mik. 21, 129, 273 (1904).

Barer,

Barer, Cole, and Thompson, Nature 163, 198 (1949).
[Crossref] [PubMed]

Bird,

Blout,

Blout, Bird, and Grey, J. Opt. Soc. Am. 39, 1052 (1949).

Land, Blout, Grey, Flower, Husek, Jones, Matz, and Merrill, Science 109, 371 (1949).
[Crossref] [PubMed]

Brackett, F. S.

Brumberg, E. M.

E. M. Brumberg, Bull. Acad. Sci. U.S.S.R. 23, 486 (1941); Bull. Acad. Sci. U.S.S.R. 24, 33 (1942); Nature 152, 357 (1943).

Burch, C. R.

C. R. Burch, Nature 152, 748 (1943); Proc. Phys. Soc., London 59, 41 (1947).
[Crossref]

Caspersson, T.

T. Caspersson, Skand. Arch. f. Physiol., Supplement 73 (1936).

Cole,

Barer, Cole, and Thompson, Nature 163, 198 (1949).
[Crossref] [PubMed]

Cole, P. A.

P. A. Cole and E. Sutton, Cold Spring Harbor Symposia on Quantitative Biology 9, 66 (1941).
[Crossref]

P. A. Cole and F. S. Brackett, J. Opt. Soc. Am. 11, 419 (1940).
[Crossref]

Dempsey, E. W.

E. W. Dempsey and et al., Anat. Rev. 98, 417 (1947); see also R. E. Stowell, J. Nat. Cancer Inst. 3, 111 (1942); M. A. Lessler and H. A. Charipper, Science 110, 429 (1949).
[Crossref] [PubMed]

Dunham, T.

T. Dunham, see J. R. Loofbourow, Growth Symposium 12, 75 (1948).

T. Dunham, “The effect of central stops on the resolution of a microscope, I,” (July, 1948).

Flower,

Land, Blout, Grey, Flower, Husek, Jones, Matz, and Merrill, Science 109, 371 (1949).
[Crossref] [PubMed]

Foster, L. V.

Gore, R. C.

R. C. Gore, Science 110, 710 (1949).
[Crossref]

Grey,

Land, Blout, Grey, Flower, Husek, Jones, Matz, and Merrill, Science 109, 371 (1949).
[Crossref] [PubMed]

Blout, Bird, and Grey, J. Opt. Soc. Am. 39, 1052 (1949).

Grey, D. S.

Harrison,

Harrison, Lord, and Loofbourow, Practical Spectroscopy (Prentice-Hall, Inc., New York, 1948).

Husek,

Land, Blout, Grey, Flower, Husek, Jones, Matz, and Merrill, Science 109, 371 (1949).
[Crossref] [PubMed]

Johnson, B. K.

B. K. Johnson, Proc. Phys. Soc., London 51, 1034 (1939).
[Crossref]

Jones,

Land, Blout, Grey, Flower, Husek, Jones, Matz, and Merrill, Science 109, 371 (1949).
[Crossref] [PubMed]

Kavanaugh, A. J.

A. J. Kavanaugh (personal communication).

Kohler, A.

A. Kohler, Zeits. f. wiss. Mik. 21, 129, 273 (1904).

Land,

Land, Blout, Grey, Flower, Husek, Jones, Matz, and Merrill, Science 109, 371 (1949).
[Crossref] [PubMed]

Lee, P.

Leeds, W. E.

W. E. Leeds and M. H. F. Wilkins, Nature 164, 228 (1949).
[Crossref]

Loofbourow,

Harrison, Lord, and Loofbourow, Practical Spectroscopy (Prentice-Hall, Inc., New York, 1948).

Loofbourow, J. R.

T. Dunham, see J. R. Loofbourow, Growth Symposium 12, 75 (1948).

Lord,

Harrison, Lord, and Loofbourow, Practical Spectroscopy (Prentice-Hall, Inc., New York, 1948).

Matz,

Land, Blout, Grey, Flower, Husek, Jones, Matz, and Merrill, Science 109, 371 (1949).
[Crossref] [PubMed]

Mellors, R. C.

R. C. Mellors, Science (to be published); Mellors, Keane, and Streim, Nature (to be published).

Merrill,

Land, Blout, Grey, Flower, Husek, Jones, Matz, and Merrill, Science 109, 371 (1949).
[Crossref] [PubMed]

Pollister, A. W.

A. W. Pollister and H. Ris, Cold Spring Harbor Symposia on Quantitative Biology 12, 147 (1947).
[Crossref]

Ris, H.

A. W. Pollister and H. Ris, Cold Spring Harbor Symposia on Quantitative Biology 12, 147 (1947).
[Crossref]

Scott, J. F.

J. F. Scott and R. L. Sinsheimer, “Ultraviolet microscopy and microspectroscopy,” in Medical Physics (Year Book Publishers, Inc., Chicago, in press), Vol. 2.

Sinsheimer, R. L.

J. F. Scott and R. L. Sinsheimer, “Ultraviolet microscopy and microspectroscopy,” in Medical Physics (Year Book Publishers, Inc., Chicago, in press), Vol. 2.

R. L. Sinsheimer, Ph.D. thesis, M.I.T. (1948).

Sjostrand, F.

F. Sjostrand, “Über die eigenfluoreszenz tierischer gewebe mit besonderer Berucksichtigung der sangetierniere,” Stockholm (1944).

Sutton, E.

P. A. Cole and E. Sutton, Cold Spring Harbor Symposia on Quantitative Biology 9, 66 (1941).
[Crossref]

Thiel, E. M.

Thompson,

Barer, Cole, and Thompson, Nature 163, 198 (1949).
[Crossref] [PubMed]

Wilkins, M. H. F.

W. E. Leeds and M. H. F. Wilkins, Nature 164, 228 (1949).
[Crossref]

Wyckoff, H. W.

H. W. Wyckoff, Quarterly Progress Report, M.I.T. Research Laboratory of Electronics (July, 1949).

Anat. Rev. (1)

E. W. Dempsey and et al., Anat. Rev. 98, 417 (1947); see also R. E. Stowell, J. Nat. Cancer Inst. 3, 111 (1942); M. A. Lessler and H. A. Charipper, Science 110, 429 (1949).
[Crossref] [PubMed]

Bull. Acad. Sci. U.S.S.R. (1)

E. M. Brumberg, Bull. Acad. Sci. U.S.S.R. 23, 486 (1941); Bull. Acad. Sci. U.S.S.R. 24, 33 (1942); Nature 152, 357 (1943).

Cold Spring Harbor Symposia on Quantitative Biology (2)

A. W. Pollister and H. Ris, Cold Spring Harbor Symposia on Quantitative Biology 12, 147 (1947).
[Crossref]

P. A. Cole and E. Sutton, Cold Spring Harbor Symposia on Quantitative Biology 9, 66 (1941).
[Crossref]

Growth Symposium (1)

T. Dunham, see J. R. Loofbourow, Growth Symposium 12, 75 (1948).

J. Opt. Soc. Am. (4)

Nature (3)

Barer, Cole, and Thompson, Nature 163, 198 (1949).
[Crossref] [PubMed]

W. E. Leeds and M. H. F. Wilkins, Nature 164, 228 (1949).
[Crossref]

C. R. Burch, Nature 152, 748 (1943); Proc. Phys. Soc., London 59, 41 (1947).
[Crossref]

Proc. Phys. Soc., London (1)

B. K. Johnson, Proc. Phys. Soc., London 51, 1034 (1939).
[Crossref]

Science (2)

Land, Blout, Grey, Flower, Husek, Jones, Matz, and Merrill, Science 109, 371 (1949).
[Crossref] [PubMed]

R. C. Gore, Science 110, 710 (1949).
[Crossref]

Skand. Arch. f. Physiol., Supplement (1)

T. Caspersson, Skand. Arch. f. Physiol., Supplement 73 (1936).

Zeits. f. wiss. Mik. (1)

A. Kohler, Zeits. f. wiss. Mik. 21, 129, 273 (1904).

Other (9)

J. F. Scott and R. L. Sinsheimer, “Ultraviolet microscopy and microspectroscopy,” in Medical Physics (Year Book Publishers, Inc., Chicago, in press), Vol. 2.

T. Dunham, “The effect of central stops on the resolution of a microscope, I,” (July, 1948).

F. Sjostrand, “Über die eigenfluoreszenz tierischer gewebe mit besonderer Berucksichtigung der sangetierniere,” Stockholm (1944).

H. W. Wyckoff, Quarterly Progress Report, M.I.T. Research Laboratory of Electronics (July, 1949).

R. C. Mellors, Science (to be published); Mellors, Keane, and Streim, Nature (to be published).

A. J. Kavanaugh (personal communication).

Harrison, Lord, and Loofbourow, Practical Spectroscopy (Prentice-Hall, Inc., New York, 1948).

R. L. Sinsheimer, Ph.D. thesis, M.I.T. (1948).

W. E. Forsythe, editor, Measurement of Radiant Energy (McGraw-Hill Book Company, Inc., New York, 1937).

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Figures (15)

Fig. 1
Fig. 1

Recently designed elements for microspectroscopy. a, A.O. quartz ocular; b, B. & L. quartz projection ocular; c, e, f, i, n, A.O. two-mirror reflection objectives; g and k, B. & L. two-mirror reflection-refraction objectives; h and j, M.I.T.—Polaroid reflection-refraction objectives; l, m, o, and p, elements of single-spherical-mirror systems. “B. & L.” refers to designs by Grey fabricated by Bausch and Lomb Optical Company, “A.O.” refers to designs by Kavanaugh fabricated by American Optical Company.

Fig. 2
Fig. 2

Diagram of simple system for microabsorption spectroscopy of single crystals, thin solid films, and solutions in microcuvettes, by Method I.

Fig. 3
Fig. 3

Photograph of the system shown in Fig. 1. a, hydrogen arc; b, quartz condensing lens; c, diaphragm; d, microscope condenser; e, microscope objective; f, spectrograph. A telescope is used for focusing the microscope image on the spectrograph slit.

Fig. 4
Fig. 4

Densitometer traces of the ultraviolet absorption spectra of a single crystal of phenylalanine together with calibration and wave-length-scale traces. Spectra taken with the system illustrated in Figs. 2 and 3.

Fig. 5
Fig. 5

System similar to that in Fig. 3, but with microscope optics of higher N.A. a, hydrogen arc; b, quartz, fluorite, rocksalt condenser; c, reflection-refraction objective, 2.8 mm E.F.L., 0.72 N.A.; d, spectrograph.

Fig. 6
Fig. 6

Absorption spectra of evaporated film of tyrosine obtained with the system shown in Fig. 5 (see text).

Fig. 7
Fig. 7

Microcuvette for liquids and solutions. A second hole (not shown, since it is in a different cross section) is provided to be used for the solvent when solution spectra are determined.

Fig. 8
Fig. 8

Simple set-ups for microemission spectroscopy.

Fig. 9
Fig. 9

Iron arc emission spectra photographed with macro- and microcondensers. Top: Microcondenser set-up.—Arc magnified 19 times. Area of source used: 166μ2. Exposure: 6 sec. E.F.L., 2.5 cm. Effective f:No. 1.0. Bottom: Macrocondenser set-up.—Arc magnified 5+ times. Area of source used: 2400μ2. Exposure: 6 sec. E.F.L., 8.3 cm. Effective f:No. 3.1.

Fig. 10
Fig. 10

System for ultraviolet and visible microabsorption spectroscopy, polarized light investigations, etc., by Method II. a, light source; b, quartz monochromator; c, fluorescent eyepiece that can be moved quickly into or out of the light path from the objective; d, photo-multiplier mount; e, regulated supply for photo-multiplier.

Fig. 11
Fig. 11

Absorption spectrum of evaporated adenine film determined with the system illustrated in Fig. 10.

Fig. 12
Fig. 12

Block diagram of a system for continuous recording of ultraviolet and visible microabsorption spectra by Method II.

Fig. 13
Fig. 13

Wadsworth-mounted grating monochromator, showing reflection microscope in background, a, approximate location of entrance and exit apertures, back of grating mount; b, collimating mirror and mount; c, concave diffraction grating and mount; d, double mirror and slit for isolating spectral region and returning the beam through the system.

Fig. 14
Fig. 14

Electronic components for the system illustrated in Fig. 12.

Fig. 15
Fig. 15

Ultraviolet transmission measurements of various combinations of experimental reflection objectives and condensers. “BG5” refers to the Grey reflection-refraction design listed in Table I, “AO9” to the Kavanaugh, dry, two-mirror system listed in the same table, and “achromatic condenser” to the quartz, fluorite, rock salt condenser referred to in the text.

Tables (1)

Tables Icon

Table I Examples of special objectives suitable for microspectroscopy.

Equations (6)

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log 10 / ( I 0 / I x ) = k c x ,
M w = w / l 1 ;             M h = h / l 2 ,
M + 1 < ¯ ¯ ( f 1 / d 1 ) ( f 2 / d 2 ) ,
( f 1 / d 1 ) / ( f 2 / d 2 ) = ( d s / d f ) [ 1 + ( 1 / M ) ]
f 1 / d 1 f 2 / d 2 = Δ λ ( d l / d λ ) d f ( 1 + 1 M )
d f = Δ λ d 1 ( d θ / d λ ) ( f 2 / d 2 ) [ 1 + ( 1 / M ) ] ,