Microspectrophotometry: The Technique and Some of Its Pitfalls

  • Ferenc I. Hárosi
Part of the NATO Advanced Study Institutes Series book series (NSSA, volume 1)


Caspersson, by combining the procedures of microscopy and spectrophotometry, founded the technique of microspectro-photometry (MSP) nearly forty years ago. Whereas the light microscope affords the visual identification of subcellular elements, spectroscopy, by virtue of the fact that many substances possess characteristic absorption spectra, allows the determination of the presence and concentration of known substances by the measure of light fluxes transmitted at various wavelengths through structures of known thickness. Thus, he reasoned, the absorption of light by cellular components may be used as a tool for their qualitative and quantitative chemical analyses. Caspersson (1940, 1950) succeeded not only in designing the first microspectrophotometer and setting down the principles of the method but also in identifying nucleic acids (λmax ≃ 260 nm) in the nucleus and proteins with cyclic amino acids (λmax ≃ 280 nm) in the cytoplasm of isolated cells.


Numerical Aperture Outer Segment Visual Pigment Linear Dichroism Characteristic Absorption Spectrum 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Brown, P.K. (1961). A system for microspectrophotometry employing a commercial recording spectrophotometer. J. Opt. Soc. Am. 51: 1000–1008.CrossRefGoogle Scholar
  2. Brown, P.K. and G. Wald (1963). Visual pigments in human and monkey retinas. Nature 200: 37–43.PubMedCrossRefGoogle Scholar
  3. Brown, P.K. and G. Wald (1964). Visual pigments in single rods and cones of the human retina. Science 144: 45–52.PubMedCrossRefGoogle Scholar
  4. Caspersson, T.O. (1940). Methods for the determination of the absorption spectra of cell structures. J.R. Microsc. Soc. 60: 8–25.CrossRefGoogle Scholar
  5. Caspersson, T.O. (1950). Cell growth and cell function. A cytochemical study. Norton, New York.Google Scholar
  6. Chance, B. (1951). Rapid and sensitive spectrophotometry. I. The accelerated and stopped-flow methods for the measurement of the Reaction Kinetics and spectra of unstable compounds in the visible region of the spectrum. Rev. Sci. Instr. 22: 619–627.CrossRefGoogle Scholar
  7. Chance, B., R. Perry, L. Akerman and B. Thorell (1959). Highly sensitive recording microspectrophotometer. Rev. Sci. Instr. 30: 735–741.CrossRefGoogle Scholar
  8. Dartnall, H.J.A. (1953). The interpretation of spectral sensitivity curves. Br. Med. Bull. 9: 24–30.PubMedGoogle Scholar
  9. Dartnall, H.J.A. (1957). The visual pigments. Methuen & Co., Ltd., London; John Wiley & Sons, Inc., New York.Google Scholar
  10. Dartnall, H.J.A. (1962). The photobiology of visual processes. In: The Eye, Vol. 2, p. 323–533, edited by H. Davson. Academic Press, New York.Google Scholar
  11. Denton, E.J. (1959). The contributions of the orientated photosensitive and other molecules to the absorption of whole retina. Proc. R. Soc. Lond. Ser. B. 150: 78–94.CrossRefGoogle Scholar
  12. Denton, E.J. and M.A. Walker (1958). The visual pigment of the conger eel. Proc. R. Lond. Sér. Soc. B. 148: 257–269.CrossRefGoogle Scholar
  13. Denton, E.J. and J.H. Wyllie (1955). Study of the photosensitive pigments in the pink and green rods of the frog. J. Physiol. 127: 81–89.PubMedGoogle Scholar
  14. Dobelle, W.H., W.B. Marks, and E.F. MacNichol, Jr. (1969). Visual pigment density in single primate foveal cones. Science 166: 1508–1510.PubMedCrossRefGoogle Scholar
  15. Goldsmith, T.H. (1972). The natural history of invertebrate visual pigments. In: Handbook of Sensory Physiology, Vol. VII/1. Photochemistry of Vision. Chapter 17, p. 685–719, edited by H.J.A. Dartnall, Springer-Verlag, New York.Google Scholar
  16. Hanaoka, T. and K. Fujimoto (1957). Absorption spectrum of a single cone in carp retina. Jap. J. Physiol. 7: 276–285.CrossRefGoogle Scholar
  17. Harosi, F.I. (1971). Frog rhodopsin in situ: orientational and spectral changes in the chromophores of isolated retinal rod cells. Ph.D. Thesis. The Johns Hopkins University, Baltimore, Md.Google Scholar
  18. Harosi, F.I. (1974). Absorption spectra and linear dichroism of some amphibian photoreceptors. (In preparation).Google Scholar
  19. Harosi, F.I. and E.F. MacNichol, Jr. (1974a). Visual pigments of goldfish cones. Spectral properties and dichroism. J. Gen. Physiol. 63: 279–304.PubMedCrossRefGoogle Scholar
  20. Harosi, F.I. and E.F. MacNichol, Jr. (1974b). Dichroic micro-spectrophotometer: A computer-assisted, rapid, wavelength-scanning photometer for measuring linear dichroism in single cells. J. Opt. Soc. Am. 64: 903–918.PubMedCrossRefGoogle Scholar
  21. Harosi, F.I. and F.E. Malerba (1974). Plane-polarised light in microspectrophotometry. Vision Res. 14:(in press).Google Scholar
  22. Laufer, M. and E. Millan (1970). Spectral analysis of L-type S-potentials and their relation to photopigment absorption in a fish (Eugerres plumieri) Retina. Vision Res. 10: 237–251.PubMedCrossRefGoogle Scholar
  23. Liebman, P.A. (1962). In situ microspectrophotometric studies on the pigments of single retinal rods. Biophys. J. 2: 161–178.PubMedCrossRefGoogle Scholar
  24. Liebman, P.A. (1969). Microspectrophotometry of retinal cells. Ann. N.Y. Acad. Sci. 157: 250–264.CrossRefGoogle Scholar
  25. Liebman, P.A. (1972). Microspectrophotometry of photoreceptors. In: Handbook os Sensory Physiology, Vol. VII/1. Photochemistry of Vision, Chapter 12, p. 481–528, edited by H.J.A. Dartnall, Springer-Verlag, New York.Google Scholar
  26. Liebman, P.A. (1973). Microspectrophotometry of visual receptors. In: Biochemistry and Physiology of Visual Pigments, p. 299–305, edited by H. Langer, Springer-Verlag, New York.CrossRefGoogle Scholar
  27. Liebman, P.A. and G. Entine (1964). Sensitive low-light-level microspectrophotometer: detection of photosensitive pigments of retinal cones. J. Opt. Soc. Am. 54: 1451–1459.PubMedCrossRefGoogle Scholar
  28. MacNichol, E.F., Jr. (1964). Three-pigment colour vision. Scientific American 211: 48–56.PubMedCrossRefGoogle Scholar
  29. MacNichol, E.F., Jr., R. Feinberg and F.I. Harosi (1973). Colour discrimination processes in the retina. In: Colour 73 Proceedings for the Second Congress of the International Colour Association, p. 191–251. Adam Hilger, Rank Precision Industries Ltd., London.Google Scholar
  30. Marks, W.B. (1963). Difference spectra of the visual pigments in single goldfish cones. Ph.D. Thesis. The Johns Hopkins University, Baltimore, Maryland.Google Scholar
  31. Marks, W.B. (1965). Visual pigments of single goldfish cones. J. Physiol. 178: 14–32.PubMedGoogle Scholar
  32. Marks, W.B., W.H. Dobelle and E.F. MacNichol, Jr. (1964). Visual pigments of single primate cones. Science 143: 1181–1183.PubMedCrossRefGoogle Scholar
  33. Muntz, W.R.A. (1973). Yellow filters and the absorption of light by the visual pigments of some Amazonian fishes. Vision Res. 13: 2235–2254.PubMedCrossRefGoogle Scholar
  34. Murray, G.C. (1968). Visual pigment multiplicity in cones of the primate fovea. Ph.D. Thesis. The Johns Hopkins University, Baltimore, Maryland.Google Scholar
  35. Svaetichin, G., K. Negishi, and R. Fatehchand (1965). Cellular mechanisms of a Young-Hering visual system. In: Ciba Foundation Symposium on Colour Vision: Physiology and Experimental Psychology, p. 178–207, edited by A.V.S. DeReuck and J. Knight. Little, Brown, Boston.Google Scholar
  36. Yang, C.C. and V. Legallais (1954). A rapid and sensitive recording spectrophotometer for the visible and ultraviolet region. I. Description and performance. Rev. Sci. Instr. 25: 801–807.CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1975

Authors and Affiliations

  • Ferenc I. Hárosi
    • 1
  1. 1.NINDSNational Institutes of HealthBethesdaUSA

Personalised recommendations