Abstract

The present paper describes the design and performance of an attachment for the Cary model 14 recording spectrophotometer, which permits the accurate recording of absorption spectra in small areas. A special compartment built into the light path of the spectrophotometer holds a low-magnification microscope ("macroscope") consisting of two opposed quartz condensers, with which measurements can be made between 300 and 700 mµ in fields 0.1 to 1 mm in diameter. This can be replaced by a conventional microscope with which spectra can be measured from about 350–650 mµ in fields as small as 4 µ in diameter. These arrangements have been used to measure the absorption spectra of visual pigments in situ. With the macroscope, such measurements have been made in small areas of surviving retinas; and with the microscope, they have been made in single isolated outer segments of rods. The present paper contains examples of each type of measurement.

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  1. T. Caspersson, Skand. Arch. Physiol. 73, Suppl. 8, 1 (1936); Chromosoma 1, 147 (1939); J. Roy. Microscop. Soc. 60, 8 (1940).
  2. Recent general discussions of microspectrophotometry will be found in "Optical Methods of Investigating Cell Structure," Discussions Faraday Soc. No. 9, (1950); E. R. Blout, Advances in Biol. and Med. Phys. 3, 285 (1953); H. Swift and E. Rasch, in Physical Techniques in Biological Research, edited by G. Oster and A. W. Pollister (Academic Press, Inc., New York, 1956), Vol. 3, p. 353; and P. M. B. Walker ibid., Vol. 3, p. 401. For recent contributions to the art, see B. Chance, R. Perry, L. Åkerman, and B. Thorell, Rev. Sci. Instr. 30, 735 (1959); and G. K. Strother and J. J. Wolken, Science 130, 1084 (1959).
  3. We wish to thank Mr. Frank White of the Biological Laboratories of Harvard University for making the photographs shown as Figs. 1 and 2.
  4. This attachment and the stands for the macroscope and microscope were built in the machine shop of the Biological Laboratories by Mr. Robert Chapman and his assistant, Mr. Bernard Dillon. We wish to thank Mr. Chapman for many helpful suggestions regarding the design and machining of the apparatus.
  5. The quartz condenser lenses, which are identical, were made by the Jones Optical Company of Cambridge, Massachusetts and were mounted by Mr. Kenneth A. Dawson of Belmont, Massachusetts.
  6. It should be said at once that we were encouraged to prepare to make measurements of this type by the pioneer work of E. J. Denton at the Marine Biological Station in Plymouth, England. Denton showed that spectrophotometric measurements on intact retinas can yield spectra of visual pigments that rival in accuracy the best work done in solution [cf. E. J. Denton, in Visual Problems of Colour, National Physical Laboratory, Teddington, England, Symposium No. 8 (Her Majesty's Stationary Office, London, 1958), p. 177; also Proc. Roy. Soc. (London) B150, 78 (1959)].
  7. G. Wald, J. Opt. Soc. Am. 41, 949 (1951).
  8. G. Wald and P. K. Brown, J. Gen. Physiol. 37, 189 (1953–54).

Brown, P. K.

G. Wald and P. K. Brown, J. Gen. Physiol. 37, 189 (1953–54).

Caspersson, T.

T. Caspersson, Skand. Arch. Physiol. 73, Suppl. 8, 1 (1936); Chromosoma 1, 147 (1939); J. Roy. Microscop. Soc. 60, 8 (1940).

Denton, E. J.

It should be said at once that we were encouraged to prepare to make measurements of this type by the pioneer work of E. J. Denton at the Marine Biological Station in Plymouth, England. Denton showed that spectrophotometric measurements on intact retinas can yield spectra of visual pigments that rival in accuracy the best work done in solution [cf. E. J. Denton, in Visual Problems of Colour, National Physical Laboratory, Teddington, England, Symposium No. 8 (Her Majesty's Stationary Office, London, 1958), p. 177; also Proc. Roy. Soc. (London) B150, 78 (1959)].

Wald, G.

G. Wald and P. K. Brown, J. Gen. Physiol. 37, 189 (1953–54).

G. Wald, J. Opt. Soc. Am. 41, 949 (1951).

Other (8)

T. Caspersson, Skand. Arch. Physiol. 73, Suppl. 8, 1 (1936); Chromosoma 1, 147 (1939); J. Roy. Microscop. Soc. 60, 8 (1940).

Recent general discussions of microspectrophotometry will be found in "Optical Methods of Investigating Cell Structure," Discussions Faraday Soc. No. 9, (1950); E. R. Blout, Advances in Biol. and Med. Phys. 3, 285 (1953); H. Swift and E. Rasch, in Physical Techniques in Biological Research, edited by G. Oster and A. W. Pollister (Academic Press, Inc., New York, 1956), Vol. 3, p. 353; and P. M. B. Walker ibid., Vol. 3, p. 401. For recent contributions to the art, see B. Chance, R. Perry, L. Åkerman, and B. Thorell, Rev. Sci. Instr. 30, 735 (1959); and G. K. Strother and J. J. Wolken, Science 130, 1084 (1959).

We wish to thank Mr. Frank White of the Biological Laboratories of Harvard University for making the photographs shown as Figs. 1 and 2.

This attachment and the stands for the macroscope and microscope were built in the machine shop of the Biological Laboratories by Mr. Robert Chapman and his assistant, Mr. Bernard Dillon. We wish to thank Mr. Chapman for many helpful suggestions regarding the design and machining of the apparatus.

The quartz condenser lenses, which are identical, were made by the Jones Optical Company of Cambridge, Massachusetts and were mounted by Mr. Kenneth A. Dawson of Belmont, Massachusetts.

It should be said at once that we were encouraged to prepare to make measurements of this type by the pioneer work of E. J. Denton at the Marine Biological Station in Plymouth, England. Denton showed that spectrophotometric measurements on intact retinas can yield spectra of visual pigments that rival in accuracy the best work done in solution [cf. E. J. Denton, in Visual Problems of Colour, National Physical Laboratory, Teddington, England, Symposium No. 8 (Her Majesty's Stationary Office, London, 1958), p. 177; also Proc. Roy. Soc. (London) B150, 78 (1959)].

G. Wald, J. Opt. Soc. Am. 41, 949 (1951).

G. Wald and P. K. Brown, J. Gen. Physiol. 37, 189 (1953–54).

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