Abstract

A discussion of the performance characteristics of an infra-red microspectrometer is given in terms of the cross-sectional area and minimum volume, V, which can be observed with satisfactory signal-to-noise ratio. Methods of increasing the ratio of measured absorption to spectrometer noise are discussed and five ways are enumerated. It is pointed out that for a microscope objective of numerical aperture NAm associated with a spectrometer of numerical aperture NAs, the useful magnification is (NAm)/(NAs). The design of reflecting-type infra-red microscope objectives having numerical apertures up to 1.5 is described.

A description of an experimental infra-red microspectrometer is given. Use is made of a commercial spectrometer and conversion from a micro to a macro instrument can be made in a few minutes. In the experimental arrangement no changes were introduced that affected the operation of the spectrometer as a macro instrument. Calculations indicate that by making use of other components now available, such as a hotter source and a smaller, more sensitive thermal detector, it will be possible to obtain infra-red spectral measurements of specimens whose linear dimensions approximate those set by diffraction.

Examples of infra-red spectra of crystals, fibers, and tissues of microscopic area are shown. Comparisons of spectral data with those obtained for macro samples are made and an indication of the experimental limitations of the technique is given.

© 1950 Optical Society of America

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References

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  1. (a)H. W. Thompson, Ann. Repts. on Progress Chem. (Chem. Soc. London)42, 5 (1945), (b)E. A. Braude, Ann. Repts. on Progress Chem. (Chem. Soc. London)42, 105 (1945).
  2. T. Caspersson, J. Roy. Microscop. Soc. [3] 60, 8 (1940).
    [CrossRef]
  3. E. E. Jelley, Ind. Eng. Chem. Anal. Ed. 13, 196 (1941).
    [CrossRef]
  4. Barer, Cole, and Thompson, Nature 163, 198 (1949).
    [CrossRef] [PubMed]
  5. V. Z. Williams, Rev. Sci. Inst. 19, 135 (1948).
    [CrossRef]
  6. H. W. Thompson, J. Chem. Soc.1948, 328.
    [CrossRef]
  7. Barnes, Gore, Stafford, and Williams, Anal. Chem. 20, 402 (1948).
    [CrossRef]
  8. R. B. Barnes (private communication).
  9. D. D. Maksutov, U.S.S.R. Patent No. 40,859 (December13, 1932); for references to the Russian work, see E. M. Brumberg, Nature 152, 357 (1943).
    [CrossRef]
  10. C. R. Burch, Proc. Phys. Soc. (London) 59, 41 (1947).
    [CrossRef]
  11. Land, Blout, Grey, Flower, Husek, Jones, Matz, and Merrill, Science 109, 371 (1949).
    [CrossRef] [PubMed]
  12. D. S. Grey, J. Opt. Soc. Am. 39, 723 (1949).
    [CrossRef] [PubMed]
  13. C. S. Rupert and J. Strong, J. Opt. Soc. Am. 39, 1061A (1949).
  14. I. S. Bowen, Astrophys. J. 88, 113 (1938).
    [CrossRef]
  15. R. Clark Jones, J. Opt. Soc. Am. 39, 344 (1949).
    [CrossRef]
  16. E. R. Blout and R. C. Mellors, Science 110, 137 (1949).
    [CrossRef] [PubMed]
  17. N. Wright, J. Opt. Soc. Am. 38, 69 (1948).
    [CrossRef]
  18. R. Newman and R. S. Halford, Rev. Sci. Inst. 19, 270 (1948).
    [CrossRef]

1949 (6)

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

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

D. S. Grey, J. Opt. Soc. Am. 39, 723 (1949).
[CrossRef] [PubMed]

C. S. Rupert and J. Strong, J. Opt. Soc. Am. 39, 1061A (1949).

R. Clark Jones, J. Opt. Soc. Am. 39, 344 (1949).
[CrossRef]

E. R. Blout and R. C. Mellors, Science 110, 137 (1949).
[CrossRef] [PubMed]

1948 (5)

N. Wright, J. Opt. Soc. Am. 38, 69 (1948).
[CrossRef]

R. Newman and R. S. Halford, Rev. Sci. Inst. 19, 270 (1948).
[CrossRef]

V. Z. Williams, Rev. Sci. Inst. 19, 135 (1948).
[CrossRef]

H. W. Thompson, J. Chem. Soc.1948, 328.
[CrossRef]

Barnes, Gore, Stafford, and Williams, Anal. Chem. 20, 402 (1948).
[CrossRef]

1947 (1)

C. R. Burch, Proc. Phys. Soc. (London) 59, 41 (1947).
[CrossRef]

1941 (1)

E. E. Jelley, Ind. Eng. Chem. Anal. Ed. 13, 196 (1941).
[CrossRef]

1940 (1)

T. Caspersson, J. Roy. Microscop. Soc. [3] 60, 8 (1940).
[CrossRef]

1938 (1)

I. S. Bowen, Astrophys. J. 88, 113 (1938).
[CrossRef]

Barer,

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

Barnes,

Barnes, Gore, Stafford, and Williams, Anal. Chem. 20, 402 (1948).
[CrossRef]

Barnes, R. B.

R. B. Barnes (private communication).

Blout,

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

Blout, E. R.

E. R. Blout and R. C. Mellors, Science 110, 137 (1949).
[CrossRef] [PubMed]

Bowen, I. S.

I. S. Bowen, Astrophys. J. 88, 113 (1938).
[CrossRef]

Burch, C. R.

C. R. Burch, Proc. Phys. Soc. (London) 59, 41 (1947).
[CrossRef]

Caspersson, T.

T. Caspersson, J. Roy. Microscop. Soc. [3] 60, 8 (1940).
[CrossRef]

Clark Jones, R.

Cole,

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

Flower,

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

Gore,

Barnes, Gore, Stafford, and Williams, Anal. Chem. 20, 402 (1948).
[CrossRef]

Grey,

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

Grey, D. S.

Halford, R. S.

R. Newman and R. S. Halford, Rev. Sci. Inst. 19, 270 (1948).
[CrossRef]

Husek,

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

Jelley, E. E.

E. E. Jelley, Ind. Eng. Chem. Anal. Ed. 13, 196 (1941).
[CrossRef]

Jones,

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

Land,

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

Maksutov, D. D.

D. D. Maksutov, U.S.S.R. Patent No. 40,859 (December13, 1932); for references to the Russian work, see E. M. Brumberg, Nature 152, 357 (1943).
[CrossRef]

Matz,

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

Mellors, R. C.

E. R. Blout and R. C. Mellors, Science 110, 137 (1949).
[CrossRef] [PubMed]

Merrill,

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

Newman, R.

R. Newman and R. S. Halford, Rev. Sci. Inst. 19, 270 (1948).
[CrossRef]

Rupert, C. S.

C. S. Rupert and J. Strong, J. Opt. Soc. Am. 39, 1061A (1949).

Stafford,

Barnes, Gore, Stafford, and Williams, Anal. Chem. 20, 402 (1948).
[CrossRef]

Strong, J.

C. S. Rupert and J. Strong, J. Opt. Soc. Am. 39, 1061A (1949).

Thompson,

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

Thompson, H. W.

H. W. Thompson, J. Chem. Soc.1948, 328.
[CrossRef]

(a)H. W. Thompson, Ann. Repts. on Progress Chem. (Chem. Soc. London)42, 5 (1945), (b)E. A. Braude, Ann. Repts. on Progress Chem. (Chem. Soc. London)42, 105 (1945).

Williams,

Barnes, Gore, Stafford, and Williams, Anal. Chem. 20, 402 (1948).
[CrossRef]

Williams, V. Z.

V. Z. Williams, Rev. Sci. Inst. 19, 135 (1948).
[CrossRef]

Wright, N.

Anal. Chem. (1)

Barnes, Gore, Stafford, and Williams, Anal. Chem. 20, 402 (1948).
[CrossRef]

Astrophys. J. (1)

I. S. Bowen, Astrophys. J. 88, 113 (1938).
[CrossRef]

Ind. Eng. Chem. Anal. Ed. (1)

E. E. Jelley, Ind. Eng. Chem. Anal. Ed. 13, 196 (1941).
[CrossRef]

J. Chem. Soc. (1)

H. W. Thompson, J. Chem. Soc.1948, 328.
[CrossRef]

J. Opt. Soc. Am. (4)

J. Roy. Microscop. Soc. [3] (1)

T. Caspersson, J. Roy. Microscop. Soc. [3] 60, 8 (1940).
[CrossRef]

Nature (1)

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

Proc. Phys. Soc. (London) (1)

C. R. Burch, Proc. Phys. Soc. (London) 59, 41 (1947).
[CrossRef]

Rev. Sci. Inst. (2)

V. Z. Williams, Rev. Sci. Inst. 19, 135 (1948).
[CrossRef]

R. Newman and R. S. Halford, Rev. Sci. Inst. 19, 270 (1948).
[CrossRef]

Science (2)

E. R. Blout and R. C. Mellors, Science 110, 137 (1949).
[CrossRef] [PubMed]

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

Other (3)

R. B. Barnes (private communication).

D. D. Maksutov, U.S.S.R. Patent No. 40,859 (December13, 1932); for references to the Russian work, see E. M. Brumberg, Nature 152, 357 (1943).
[CrossRef]

(a)H. W. Thompson, Ann. Repts. on Progress Chem. (Chem. Soc. London)42, 5 (1945), (b)E. A. Braude, Ann. Repts. on Progress Chem. (Chem. Soc. London)42, 105 (1945).

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Figures (13)

Fig. 1
Fig. 1

When a microscope objective is used to image the specimen onto the spectrometer slit, the path length through the sample increases with the obliquity, θ, of rays to the microscope axis. If t is the specimen thickness and θ is the inclination in the specimen of a ray to the optic axis of the microscope, the path length for that ray is t/(cosθ).

Fig. 2
Fig. 2

The percent of incident energy which is absorbed by a sample depends on the absorption coefficient k multiplied by the thickness t along the optic axis of the microscope and on the numerical aperture NAm of the microscope divided by the refractive index n of the specimen. The ratio of absorbed energy to incident energy is (1−ekt)−δ, where δ is the ordinate in the above figure. The subtractive correction δ is plotted against percent absorption read for various values of (NAm)/n.

Fig. 3
Fig. 3

Convertible infra-red microscope objective. Two spherical mirrors may form a microscope objective suitable for use infra-red at numerical apertures of the order of 0.65. A single hemisphere of silver chloride or thallium bromide-iodide may added to the objective (as indicated by dotted lines) to multiply the numerical aperture by the index of the refracting medium. The hemisphere introduces no aberration; this objective is thus convertible for use at numerical aperture 0.63 (no hemisphere), 1.25 (silver chloride hemisphere), or 1.5 (thallium bromide-iodide hemisphere). The numerical aperture cannot, of course, exceed the refractive index of the material in which the specimen is embedded. A meniscus lens of either of these materials or rocksalt may be inserted to give numerical aperture between 0.63 and 0.9 in a “dry” system.

Fig. 4
Fig. 4

The energy lost in viewing a very small area of the specimen may not be completely compensated by the increased numerical aperture over which radiation from the specimen is accepted. A further gain in energy may be realized by increasing the spectral band width passed by the monochromator. This may be done by opening the exit slit independently of the entrance slit (giving the spectral pass band B) or by increasing the magnification provided by the microscope and opening both slits (giving the spectral pass band A).

Fig. 5
Fig. 5

Diagrammatic drawing of infra-red microspectrometer assembly.

Fig. 6
Fig. 6

Photograph of infra-red microspectrometer showing reflecting microscope assembly.

Fig. 7
Fig. 7

Infra-red spectra of two organic compounds ((a) thymine and (b) d,l-β-phenylalanine) in crystalline state showing comparison of results obtained by micro and macro techniques. Dashed lines indicate places where absorption is caused by mineral oil.

Fig. 8
Fig. 8

(a) Photomicrograph of ramie fibers. (b) Infra-red spectrum of single fiber of ramie.

Fig. 9
Fig. 9

Infra-red spectrum of polyvinyl alcohol film compared with that obtained from a single fiber.

Fig. 10
Fig. 10

(a) Photomicrograph of bundle of nucleohistone fibers. Single fibers have diameters <5μ. (b) Infra-red spectrum of a bundle of nucleohistone fibers (micro) compared with that obtained from a film of nucleohistone.

Fig. 11
Fig. 11

(a) Comparison of infra-red spectra obtained with same tissue section using macro and micro techniques. (b) Similar data for a peripheral blood smear. Broken lines indicate region of numeral oil absorption.

Fig. 12
Fig. 12

Comparison of infra-red data obtained from a single crystal of a male hormone, androsterone, with that obtained from a carbon disulfide solution of the same material. The latter data is from K. Dobriner et al., J. Biol. Chem. 172, 297 (1948).

Fig. 13
Fig. 13

Points showing wave-lengths and separation in doublets in experimentally observed spectra of micro samples with present instrument.

Equations (13)

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E = h s w s π N A s 2 I ( λ 0 ) Δ λ .
h s w s E m I ( λ 0 ) Δ λ π N A s 2
I ( λ 0 ) Δ λ 2 A n 2 π sin θ cos θ d θ ,
I ( λ 0 ) Δ λ 2 A n 2 π sin θ cos θ [ 1 - exp ( - k t / cos θ ) ] d θ ,
I ( λ 0 ) Δ λ 2 A n 2 π sin θ cos θ × { ( 1 - exp ( - k t / cos θ ) - [ 1 - exp ( - k t ) ] } d θ .
sin θ cos θ [ exp ( - k t ) - exp ( - k t sec θ ) ] d θ exp ( - k t ) sin θ cos θ d θ ,
sin θ cos θ [ 1 - exp ( k t - k t sec θ ) ] d θ sin θ cos θ d θ .
P = E θ - E 0 E - E 0 , E 0 E = ( P E E 0 + 1 - P ) - 1 = B , E 0 = E θ B - E θ - C , E θ - E θ B = C ( ordinate of Fig . 2 ) , E θ E = E 0 E 1 B = 1 - exp ( - k t ) B ( abscissa of Fig . 2 ) .
E m I 0 ( λ ) Δ λ ( N A m ) 2 .
E m I 0 ( λ ) Δ λ ( N A m ) 2 + 2 ( h s * + w s * ) N A s N A m [ tan ( sin - 1 N A m n ) ] z + π [ tan ( sin - 1 N A m n ) ] 2 z 2 .
V = t { E m I 0 ( λ ) Δ λ π ( N A m ) 2 + ( h s * + w s * ) N A s N A m [ tan ( sin - 1 N A m n ) ] t + π [ tan ( sin - 1 N A m n ) ] 2 t 2 12 } .
t { E m I 0 ( λ ) Δ λ π ( N A m ) 2 + ( h s * + w s * ) N A s t n + π ( N A m ) 2 t 2 12 n 2 } .
V t π ( N A m ) { h s * w s * ( N A s ) 2 + ( h s * + w s * ) N A s π ( N A m ) 2 t n + π 2 12 n 2 ( N A m ) 4 t 2 + } .