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 equals numerical aperture, λ equals wavelength, NA(obj) equals the objective numerical aperture, and NA(Cond) is the condenser numerical aperture. There are several equations that have been derived to express the relationship between numerical aperture, wavelength, and resolution : Shorter wavelengths are capable of resolving details to a greater degree than are the longer wavelengths. The wavelength spectrum of light used to image a specimen is also a determining factor in resolution. The substage condenser must be matched to the objective with respect to numerical aperture and adjustment of the aperture iris diaphragm for accurate light cone formation. The higher the numerical aperture of the total system, the better the resolution.Ĭorrect alignment of the microscope optical system is also of paramount importance to ensure maximum resolution. Numerical aperture determines the resolving power of an objective, but the total resolution of a microscope system is also dependent upon the numerical aperture of the substage condenser. Resolution is a somewhat subjective value in microscopy because at high magnification, an image may appear unsharp but still be resolved to the maximum ability of the objective. The resolution of a microscope objective is defined as the smallest distance between two points on a specimen that can still be distinguished as two separate entities. Most manufacturers strive to ensure that their objectives have the highest correction and numerical aperture that is possible for each class of objective.
#ANGULAR RESOLUTION CALCULATOR SERIES#
This feature of increasing numerical aperture across an increasing optical correction factor in a series of objectives of similar magnification holds true throughout the range of magnifications as shown in Table 1. Objective Numerical Apertures Magnification In practice, however, most oil immersion objectives have a maximum numerical aperture of 1.4, with the most common numerical apertures ranging from 1.0 to 1.35. By examining the numerical aperture equation above, we find that the highest theoretical numerical aperture obtainable with immersion oil is 1.51 (when sin ( µ) = 1). Most objectives in the magnification range between 60x and 100x (and higher) are designed for use with immersion oil. You should check with the manufacturer if there are any doubts. We suggest that microscopists never use objectives designed for oil immersion with either glycerin or water, although several newer objectives have recently been introduced that will work with multiple media. Care should be used with these objectives to prevent unwanted artifacts that will arise when an objective is used with a different immersion medium than it was designed for. Microscope objectives are now available that allow imaging in alternative media such as water (refractive index = 1.33), glycerin (refractive index = 1.47), and immersion oil (refractive index = 1.51). Therefore, in order to obtain higher working numerical apertures, the refractive index of the medium between the front lens of the objective and the specimen must be increased.
By examining the numerical aperture equation, it is apparent that refractive index is the limiting factor in achieving numerical apertures greater than 1.0.
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