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Journal of the Optical Society of America

Journal of the Optical Society of America

  • Vol. 34, Iss. 12 — Dec. 1, 1944
  • pp: 695–709

Principles of Microscope Illumination and the Problem of Glare

WILFRID TAYLOR DEMPSTER  »View Author Affiliations


JOSA, Vol. 34, Issue 12, pp. 695-709 (1944)
http://dx.doi.org/10.1364/JOSA.34.000695


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Abstract

The manipulations in using the microscope, other than the selection of lenses, focusing, and placement of slides, relate to the illumination. When the illumination is not handled with judgment, glare and lack of contrast result, and crisp images are not obtained. Effective illuminations depend equally upon three factors, the condenser, the field stop, and the illuminant proper. The adjustments for superior glare-free illumination are discussed and the term “controlled” illumination is suggested as an appropriate designation for such illumination. Aspects of the lighting requirements of the microscope are analyzed, and it is shown that suitable regulation of the apertures of the field and condenser stops are the basic requirements for controlled illumination. Various types of lighting may be used in conjunction with these adjustments. The paper is presented against a background of the pertinent historical developments and the various factors relating to illumination are reviewed in a broader fashion than is common in texts on microscopy. Explanations of the principles and the defects of illumination are presented with the general biological user of the microscope in mind. The shapes and proportions of the various illuminating beams that play on specimens are illustrated. Aberrations and the origin of glare are explained. In general, there are two extremes in the practical use of illumination—illumination for high resolution and illumination for maximum contrast. The microscopist should ordinarily vary his illumination as he works to obtain the advantages of each type of illumination. The conditions of each are fully treated.

Citation
WILFRID TAYLOR DEMPSTER, "Principles of Microscope Illumination and the Problem of Glare," J. Opt. Soc. Am. 34, 695-709 (1944)
http://www.opticsinfobase.org/josa/abstract.cfm?URI=josa-34-12-695


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References

  1. John Belling, The Use of the Microscope (McGraw-Hill Book Company, Inc., New York, 1930). S. H. Gage, The Microscope (Comstock Publishing Company, Ithaca, New York, 1932). Conrad Beck, The Microscope, Theory and Practice (R. and J. Beck, London, 1938). F. J. Muñoz and H. A. Charipper, The Microscope and Its Use (Chemical Publishing Company, Brooklyn, New York, 1943). J. E. Barnard and F. V. Welch, Practical Photomicrography (E. Arnold and Company, London, 1936). G. G. Reinert, Praktische Mikrofotografie (W. Knapp, Halle, 1937). R. M. Allen, Photomicrography (D. Van Nostrand Company, New York, 1941). C. P. Shillaber, Photomicrography in Theory and Practice (John Wiley & Sons, Inc., New York, 1944).
  2. I am greatly indebted to Dr. O. W. Richards, Spencer Lens Company, and Dr. G. L. Walls, Bausch and Lomb Optical Company, for their critical comments on a preliminary draft of the manuscript.
  3. Hugo von Mohl, Mikrographie, oder Anleitung zur Kenntniss und zum Gebrauche des Mikroskops (L. F. Fues, Tübingen, 1846). E. M. Nelson, J. Roy. MIicroscop. Soc. 7, 90–105 (1891).
  4. E. Abbe, Arch. f. mikroskop. Anat. 9, 469–480 (1873). Reprinted in Gesammelte Abhandlungen von Ernst Abbe (G. Fischer, Jena, 1904), Vol. 1, and condensed translation in E. Abbe, Monthly Microscop. J. 13, 77–82 (1875).
  5. Nelson, reference 3. Focused illumination is but one of the possible techniques for securing effective illumination; it lends itself well to control. Control is the sine qua non of good illumination and, as will be shown, control is possible only when the plane of condenser focus is adjusted so that its falls at or near the level of objects (on a slide) in the field of view.
  6. Note in J. Roy. Microscop. Soc., p. 609 (1889).
  7. E. Abbe, Arch. f. mikroskop. Anat. 9, 413–468 (1873). Also in Gesammelte Abhandlungen von Ernst Abbe, Vol. 1, pp. 45–100.
  8. E. Abbe, J. Roy. Microscop. Soc., pp. 721–724 (1889).
  9. The distinction between wide and narrow cones is shown in Fig. 6.
  10. It should be stated emphatically at the outset that illumination intensity is a most important variable in microscopy; and where serious microscopy is attempted, a rheostat, neutral wedges, or filters should be employed to regulate intensity. The condenser is not for this purpose.
  11. E. M. Nelson, Eng. Mech. 40, 68, 157–158, 263, 282 (1884). E. M. Nelson, J. Roy. Microscop. Soc., pp. 282–289 (1910). Nelson, reference 3.
  12. Lord Rayleigh, Phil. Mag. 42, [5], 167–195 (1896).
  13. L. C. Martin, An Introduction to Applied Optics (Isaac Pitman and Sons, London, Vol. 1, 1930; Vol. 2, 1932).
  14. N. A. is a numerical designation of the wideness of cone angle for the largest light cone that may be transmitted by a given objective. Similarly, it refers to the proportions of a beam emerging from a condenser and is correlated with the size of the condenser stop. N. A. = (the sine of the half-angle—in air—of the marginal rays of the light cone) × (the refractive index of the medium into which the cone is projected). Cf. Fig. 6, E. The lower angle of the triangle is the half-angle of the light cone.
  15. For instance, in: C. R. Marshall and H. D. Griffith, An Introduction to the Theory and Use of the Microscope (G. Routledge and Sons, London, 1928), and Beck, reference 1. Shillaber, reference 1 shows photographs of starch grains with different illumination.
  16. The general method of examining the luminous appearance of an objective through the open draw tube (or by means of a suitable lens, through the ocular) was described by Abbe, reference 7.
  17. J. W. Gordon, J. Roy. Microscop. Soc., pp. 425–429 (1908). F. Welch, J. Roy. Microscop. Soc., pp. 34–37 (1930). R. E. Fitzpatrick, Stain Tech. 16, 107–109 (1941).
  18. M. H. Knisely, Anat. Rec. 64, 499–524 (1936).
  19. A. Köhler, Zeits. f. wiss. Mikrosk. 10, 433–440 (1893). A. Köhler, "Mikrophotographie," Handb. der biol. Arbeitsmethoden (Abderhalden), Abt. 2, Part 2, No. 2, 1691–1978 (1031).
  20. Distributed for a time by the Zeiss Company. The more elaborate photomicrographic outfits are provided with lenses which can be combined to produce projecting lantern illumination.
  21. M. Berek, J. Roy. Microscop. Soc. 49, 240–244 (1929).
  22. Allen, reference 1.
  23. The pancratic condenser of the Zeiss Company (advertised about 1937 et seq.) for illumination of objectives between N.A. 0.16 and N.A. 1.40 should meet illumination requirements most satisfactorily.
  24. A. E. Wright, Principles of Microscopy, being a Handbook to the Microscope (The Macmillan Company, New York, 1917).
  25. A Spencer and a Leitz microscope, provided with apochromatic objectives, compensating type oculars, and corrected condensers, formed the basis for the various observations made. Occasionally, points were checked on student type microscopes of the same and other makers.
  26. The shape of the condenser beam is visualized in a dark room if a vertically held cigarette paper or a lightly exposed photographic film is moved back and forth through the beam. The proportions of the beam may be determined also by placing paper or a film horizontally on the microscope stage and racking the condenser up and down; the condenser beam is intercepted and a circle of measurable diameter may be noted for each level of the condenser. The circles are measured while they are being viewed by the microscope provided with lowest power lenses. An inch cube of uranium glass on the stage and in a darkened room shows a similar double cone from the condenser, but such a cone is a little less wide angled than one projected into air. Cf. Marshall and Griffith, reference 15; Muñoz and Charipper, reference 1. The cone within a slide in oil-immersion work is visualized this way.
  27. The effect of loss of contrast on visual resolution and the significance of other factors on visual performance in microscopy have been dealt with in a separate paper. J. Opt. Soc. Am. 34, 711 (1944).
  28. C. Beck, J. Roy. Microscop. Soc., pp. 399–405 (1922). C. Beck, J. Roy. Microscop. Soc., pp. 1–8 (1933). C. Fabry, Proc. Phys. Soc. London 48, 747–762 (1936).
  29. K. B. Blodgett, Phys. Rev. 55, 391–404 (1939). C. H. Cartwright, Phys. Rev. 57, 1060 (1940). F. L. Jones and H. J. Homer, J. Opt. Soc. Am. 31, 34–37 (1941).
  30. R. W. Wood, Physical Optics (The Macmillan Company, New York, 1934), third edition.
  31. The similarity of spherical aberration and glare in obscuring an image may be illustrated by looking through a microscope and condenser so adjusted that objects at a distance (i.e., out of a window) are seen as through a telescope. The degree of image fuzziness is largely owing to residual aberrations of the condenser. Opening the condenser increases spherical aberration and an increased haziness appears.
  32. Several slides, coated by the Cartwright method (reference 29), were prepared for me through the kindness of Professor R. C. Williams. When light of an appropriate color was used, a slight but perceptible decrease in glare was evident. Since coated slides do not alter the amount of internal reflection, the major problem remains.

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