科學問題20分

For microscope objectives having high numerical apertures, the optical properties and thickness of the medium lying between the front lens element and the specimen critically affect the calculations necessary to satisfy the aplanatic and sine conditions and otherwise to correct for image aberrations.


If homogeneous immersion objectives are designed to be used with the refractive indices and dispersion of the immersion oil, coverslip, and medium imbibing the specimen matching that of the objective front lens element, then the calculation is straightforward because all of the media can be considered an extension of the front lens element.

However, with non-immersion objectives, the coverslip can become a source of chromatic aberration, which increases with coverslip thickness and dispersion. Another optical artifact, spherical aberration, is also proportional to the thickness of the coverslip. In designing objectives that are not to be used with homogeneous immersion, one assumes the presence of a standard coverslip and other specific optical media between the front lens element and the specimen. As conditions depart from these designated specifications, spherical aberration and coma increase with the numerical aperture of the objective. This occurs because the difference between the tangent and the sine of the incident angle (which is responsible for departure from the sine condition needed to correct for these aberrations) becomes greater with longer numerical apertures.

When using oil immersion objectives, it may appear that coverslip thickness is of only limited concern, because its refractive index approximately matches that of the immersion oil. That is true when the specimen is mounted in Canada balsam or other mounting media with refractive indexes similar to that of the coverslip. However, it is no longer true when the specimen is mounted, for example, in physiological saline or other aqueous media whose refractive index is significantly different from that of the coverslip. In these conditions, even focusing through a thin layer of water only 10 microns thick can lead to significant aberrations and a point spread function (PSF) that is no longer symmetrical above and below the focal plane. This is because oil immersion objectives are designed assuming that the specimen slide is homogeneously immersed. Unless the specimen region is adjacent to the coverslip (even in an aqueous environment), optical assumptions employed to calculate the aberration corrections are no longer valid.