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Digital Cameras for Microscopy
Nikon DXM 1200 vs Optronics MagnaFire
CCD Specifications
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Table 1
The photomicrograph in Figure 1 illustrates a thin section of human cerebellum brain tissue impregnated with a solution of silver nitrate, which stains selected portions of the neurons black. A gold-yellowish background color pervades the entire section, but this cast is an artifact of the method and not due to a counterstain. The image was captured digitally using an Olympus DP-11 digital camera operating in high resolution mode using a Nikon Eclipse E-600 microscope and a 40x fluorite objective having a numerical aperture of 0.75 with a 1x C-mount adapter lacking a projection lens. Assuming an average wavelength of 580 nanometers (green light), the resolution for this objective is 0.39 microns, which yields a projected size of 7.7 microns on the CCD surface. The DP-11 CCD is a progressive scan, interline transfer device having a Bayer RGB mosaic filter complement and a pixel size of 6.7 microns. Calculations (described below) of the minimum number of pixels required to capture the image at the maximum spatial resolution produced by the microscope optics indicate that a CCD array with 3.9 million pixels is required. DP-11 has an effective array of 2.4 million pixels arranged at 1784 (horizontal) x 1382 (vertical).
The wavelength spectrum of visible light used to create an image of a specimen is one of the determining factors in the performance of the microscope with respect to optical resolution. Shorter wavelengths (375-500 nanometers) are capable of resolving details to a greater degree than are the longer wavelengths (greater than 500 nanometers). The limits of spatial resolution are also dictated by the diffraction of light through the optical system, a term that is generally referred to as "diffraction limited" resolution. Investigators have derived several equations that have been used to express the relationship between numerical aperture, wavelength, and optical resolution:
R =  /(2NA)
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(1) |
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R = 0.61/NA
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(2) |
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R = 1.22/(NA(obj) + NA(cond))
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(3) |
Where R is resolution (the smallest resolvable distance between two objects), NA equals numerical aperture, equals wavelength, NA(obj) equals the objective numerical aperture, and NA(Cond) is the condenser numerical aperture. Notice that equation (1) and (2) differ by the multiplication factor, which is 0.5 for equation (1) and 0.61 for equation (2). These equations are based upon a number of factors, including a variety of theoretical calculations made by optical physicists to account for the behavior of objectives and condensers, and should not be considered an absolute value of any one general physical law. The assumption is that two point light sources can be resolved (separately imaged) when the center of the Airy disk generated by one of the sources overlaps with the first order reflection in the diffraction pattern of the second Airy disk, a condition known as the Rayleigh Criterion. In some instances, such as confocal and fluorescence microscopy, the resolution may actually exceed the limits placed by any one of these three equations. Other factors, such as low specimen contrast and improper illumination may serve to lower resolution and, more often than not, the real-world maximum value of R (about 0.22 microns using a mid-spectrum wavelength of 580 nanometers) and a numerical aperture of 1.35 to 1.40 are not realized in practice.
When the microscope is in perfect alignment and has the objectives appropriately matched with the substage condenser, then we can substitute the numerical aperture of the objective into equations (1) and (2), with the added result that equation (3) reduces to equation (2). An important concept to note is that magnification does not appear as a factor in any of these equations, because only numerical aperture and wavelength of the illuminating light determine specimen resolution. As we have mentioned (and can be seen in the equations) the wavelength of light is an important factor in the resolution of a microscope. Shorter wavelengths yield higher resolution (lower values for R) and visa versa. The greatest resolving power in optical microscopy is realized with near-ultraviolet light, the shortest effective imaging wavelength. Near-ultraviolet light is followed by blue, then green, and finally red light in the ability to resolve specimen detail. Under most circumstances, microscopists use white light generated by a tungsten-halogen bulb to illuminate the specimen. The visible light spectrum is centered at about 550 nanometers, the dominant wavelength for green light (our eyes are most sensitive to green light). It is this wavelength that was used to calculate resolution values in Table 2. The numerical aperture value is also important in these equations and higher numerical apertures will also produce higher resolution (see Table 2). The effect of the wavelength of light on resolution, at a fixed numerical aperture (0.95), is listed in Table 2.
Comparison of Camera Specifications
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Table 2
Contributing Authors
Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.
Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
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