![]() The visible light spectrum is centered at about 550 nanometers, the dominant wavelength for green light (our eyes are most sensitive to green light). Under most circumstances, microscopists use white light generated by a tungsten-halogen bulb to illuminate the specimen. Near-ultraviolet light is followed by blue, then green, and finally red light in the ability to resolve specimen detail. The greatest resolving power in optical microscopy is realized with near-ultraviolet light, the shortest effective imaging wavelength. Shorter wavelengths yield higher resolution (lower values for r) and visa versa. An important fact 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.Īs we have mentioned (and can be seen in the equations) the wavelength of light is an important factor in the resolution of a microscope. ![]() 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). Table 1 - Resolution and Numerical Aperture by Objective Correction The following table (Table 1) provides a list resolution ( r) and numerical aperture ( NA) values by objective magnification and correction. 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 0.25 µm using a mid-spectrum wavelength of 550 nanometers) and a numerical aperture of 1.35 to 1.40 are not realized in practice. In some instances, such as confocal and fluorescence microscopy, the resolution may actually exceed the limits placed by any one of these three equations. 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. Notice that equation (1) and (2) differ by the multiplication factor, which is 0.5 for equation (1) and 0.61 for equation (2). Where r is resolution (the smallest resolvable distance between two objects), NA is a general term for the microscope numerical aperture, λ is the imaging wavelength, NA(obj) equals the objective numerical aperture, and NA(cond) is the condenser numerical aperture.
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