. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. D. W. PISTON. Figure 1. An objective lens with typical iiKiikmgs. Although this is a Zeiss lens, most manufacturers use similar markings. itication, the working distance is not marked on most lenses. Nikon has now started writing the working dis- tance on their CFI60 optics, and it is hoped that this trend will be followed by the other manufacturers. This short article describes the relative importance of magnification and numerical aperture for digital optical mi- croscopy. Traditionally, observations made with optical mi-
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. D. W. PISTON. Figure 1. An objective lens with typical iiKiikmgs. Although this is a Zeiss lens, most manufacturers use similar markings. itication, the working distance is not marked on most lenses. Nikon has now started writing the working dis- tance on their CFI60 optics, and it is hoped that this trend will be followed by the other manufacturers. This short article describes the relative importance of magnification and numerical aperture for digital optical mi- croscopy. Traditionally, observations made with optical mi- croscopes were detected by eye. and in this case, the size of the detector pixels—given by physiological factors in the human eye—is not optimal, so the magnification was increased so the sample could be "seen" better. In digital imaging, however, the magnification can be determined by the combination of resolution and detector pixel size. To understand the relative importance of NA and magnifica- tion, we must consider the basics of image formation and the effect of lens parameters on the resolution and informa- tion content in optical microscopy. Because the resolving power of an optical microscope is dependent only on the numerical aperture, magnification should be thought of as a secondary parameter whose optimal value can be deter- mined by the NA, detector pixel size, and other instrument- independent imaging parameters. Thus, NA is a more im- portant parameter than magnification in digital imaging. The practical implications of this conclusion are described tor two commonly used modes of fluorescence imasiii": widefield epi-fluorescence microscopy with a CCD camera as the detector, and laser scanning confocal microscopy with photomultiplier tube detectors. Basics of Image Formation As might be guessed by looking at the markings on any objective lens, the magnification and numerical aper- ture are important for the image-formation properties of an optic
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