![]() ![]() ![]() When the light level is limiting, the highest numerical aperture objective should be employed, yet the magnifications of the both the objective and eyepiece should be kept at the lowest level compatible with the desired resolution. In practice, the image brightness numbers vary (see Table 1) due to objective rear aperture size differences. ![]() As the objective magnification increases, the light source image is reduced (demagnified) by an equivalent amount, resulting in a brightness level that is less dependent on objective magnification and more dependent on numerical aperture (brightness is governed by the fourth power of numerical aperture in epi-illumination). In the case of epi-illumination, the same considerations apply, except that the objective also acts as the condenser, and this must be taken into account when considering image brightness. The terms F(trans) and F(epi) refer to the light-gathering power of an objective and were calculated according to the following equations : Formula 2 - F(trans)į(trans) = 10 4 × NA 2/M 2 Formula 3 - F(epi) Utilization of a specific objective for epi-illumination produces increasingly brighter images as the magnification increases, whereas the reverse is often true for the same objective with transmitted light. It is evident from examining the data in Table 1 that when an objective is used in transillumination, image brightness decreases rapidly as the magnification increases. Thus, for the same magnification, higher numerical aperture objectives collect more light, produce a brighter and better-corrected image (see Table 1), and the overall image is better resolved. In general, objectives with high numerical apertures are also better corrected for aberrations. Examples of the light-gathering power of selected Nikon objectives having varying degrees of optical correction are listed in Table 1. The ratio given in the equation above expresses the light-gathering power of the objective in transillumination (note: the case with epi-illumination is somewhat different, as discussed below). Where NA is the objective numerical aperture and M is the magnification. ![]()
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