Modern optical microscopes use Köhaler illumination. The Köhaler illumination system treats the light path with the same precision as the optical path. A lens in front of the source of light places an image of the light source at a position that is not in the plane containing the specimen. A second lens at the condenser puts an image of the surface of the first lens onto the specimen to be examined with as short a focal length as possible to achieve as wide a cone of light as possible illuminating the specimen.

NA Numerical Aperture

Using the figure at the left it can be seen that there is a limited angle of acceptance (a) of light that a lens is capable of taking from the specimen. For dry lenses the top value of NA is limited by the critical angle of the light ray in the glass coverslip covering the specimen. A top working value of NA = .95 is normal for a dry lens. If however, an oil is placed between the objective lens and the specimen, a much wider angle of light can be accepted by the lens. The NA is now the product of the value of the refractive index of the immersion liquid and sina. A good immersion lens has an NA of around 1.3. Condensers in the Köhler illumination system also have an NA. A high NA condenser will produce a wide angled cone of light.

NA, lack of spherical and chromatic aberration and flatness of field are the goals of lens manufacturers. High NA dry lenses are very finely corrected for aberrations and they need to be used with precise coverslip thickness. The coverslip thickness tends to be of less importance with oil immersion objectives as the change in the index of refraction between the oil and coverslip is much less than that of air and the coverslip.

An excellent immersion oil is Resolve from Stephens Scientific, Riverdale, NJ 07457 (201) 831-9800.

The limit of magnification for ight microscopy is approximately 2,000 diameters. A reasonably clear image at this power may be achieved with Köhler illumination, oil imersion and plan optics.