![]() ![]() Image Brightness (Fluorescence) λ NA 4/M 2 Because the light gathering power of the objective is also proportional to the numerical aperture squared, image brightness will vary as the fourth power of the objective numerical aperture according to the equation : 4 A high numerical aperture objective acting as a condenser will increase the signal (light) intensity in a manner that is proportional to the square of the numerical aperture. Secondary fluorescence emitted by fluorophores attached to the specimen is then gathered by the same objective lens system and passed back through the dichromatic mirror and barrier filter before being projected into the eyepieces or the imaging system. Light passing through the excitation filter and reflected from the dichromatic mirror surface in the filter cube is first passed through the objective to form a cone of illumination necessary to excite the specimen. As discussed above, overall image brightness decreases rapidly as magnification increases, so the components of a fluorescence microscope should be carefully chosen to maximize the amount of light passing through the optical train.įluorescence microscopes that utilize epi-illumination are equipped with objectives that serve the dual purpose of both condenser and objective. In situations where high resolution fluorescence imaging requires high magnifications with a minimal loss of image brightness, the highest numerical aperture objectives having the greatest degree of light transmission should be employed. The amount of light transmitted through the optical components of the microscope, as a function of incident intensity, is especially critical in fluorescence microscopy. These objectives yield a 16-fold difference in image brightness under epi-fluorescence illumination, with the high numerical aperture oil immersion version producing the brightest images. For example, the 40x plan apochromatic objective in Table 1 has twice the numerical aperture of the plan achromat 40x dry objective, and produces four times the image brightness in transmitted light. In many cases, manufacturers are now providing oil immersion objectives with higher numerical apertures, and correspondingly higher image brightness values, than high-dry counterparts of similar magnification. 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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