Human chromosome methodology / edited by Jorge J. Yunis.

Date:
1965
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Human chromosome methodology / edited by Jorge J. Yunis. Source: Wellcome Collection.

    134/280 (page 116)
    116 Walter J. Runge every point in the intermediate image plane. All these wave motions generate, by interference with each other, a real image in this plane. This intermediate image is further magnified for visual observation by the eye lens of the eyepiece in the manner of a magnifying glass, without any contribution to detail or resolution.® For microscopic work requiring very high resolution (chromosome structure), it should be remembered that in regard to resolving power the wavelength of light is of the same importance as the numerical aper¬ ture of the optics.^ The shorter the wavelength (UV;blue), the smaller the angular deviation of orders of maxima which can enter a lens with a given numerical aperture. The resolving power of an objective can be doubled with illumination of the proper degree of obliquity. According to the diffraction theory a grating can be resolved when the 0-order and one of the diffracted orders are admitted. By use of the oblique critical illumination (Abbe, 1904; Köhler, 1928; Langeron, 1925; Romeis, 1943; Spitta, 1920; Martin, 1955), these two orders may be brought into the objective, which otherwise would gather only the nondeviated axial 0-order. E. Microscopic Depth of Focus The depth of focus to be expected from a chosen combination of eyepiece and objective is a term of practical importance as it relates to microscopic objects, which like chromosomes have a substantial thickness. The focal depth of a microscope is independent of the quality of lens systems and is determined solely by natural optical laws. The following formula is valid only for visual microscopic images seen at the standard (least distance of distinct vision) of 250 mm: n 250 = 4- ,. mm NA-M-0.25 ^ M2 (n refractive index of mounting medium) This formula shows that depends on the total magnification M and the numerical aperture (NA) of the objective. The depth of focus is smaller, the higher the total magnification. The of oil-immersion sys¬ tems ranges from 0.5 to 1.0 ¡я. 3 Abbe's theory has been questioned by others, but it still forms the basis for correlation of the principles and performances of lenses in microscopes and the reliable interpretation of the images. 4 Obtaining a shorter wavelength illumination with filters is less expensive than high NA objectives.