Why is resolution dependent on wavelength

The limit at which two Airy discs can be resolved into separate entities is often called the Rayleigh criterion. This is when the first diffraction minimum of the image of one source point coincides with the maximum of another. Circular apertures produce diffraction patterns with circular symmetry. Mathematical analysis gives the equation,. The primary minimum sets a limit to the useful magnification of the objective lens.

A point source of light produced by the lens is always seen as a central spot, and second and higher order maxima, which is only avoided if the lens is of infinite diameter.

Previous Next Resolution and Imaging The limit of resolution or resolving power is a measure of the ability of the objective lens to separate in the image adjacent details that are present in the object. Numerical Aperture The numerical aperture of a microscope objective is a measure of its ability to resolve fine specimen detail.

Low numerical aperture Low value for a Low resolution High numerical aperture High value for a High resolution Airy Discs When light from the various points of a specimen passes through the objective and an image is created, the various points in the specimen appear as small patterns in the image.

The wavelength spectrum of light used to image a specimen is also a determining factor in the degree of resolution afforded by the microscope. Shorter wavelengths are capable of resolving details to a greater degree than are the longer wavelengths. There are several equations that have been derived to express the relationship between numerical aperture, wavelength, and resolution :. Notice that equation 1 and 2 differ by the multiplication factor, which is 0.

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.

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.

The following table Table 1 provides a list resolution r and numerical aperture NA values by objective magnification and correction.

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 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.

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. NAobj is the NA of the objective.

NAcond is the NA of the condenser. These are used for calculating problems in systems such as wave propagation. Taking the NA of the condenser into consideration, air with a refractive index of 1. If using a green light of nm, an oil immersion objective with an NA of 1. As stated above, the shorter the wavelength of light used to image a specimen, then the more detail will be resolved. So, if using the shortest visible wavelength of light of nm, with an oil immersion objective with an NA of 1.

To achieve the maximum theoretical resolution in a microscope system, each of the optical components should be of the highest NA available taking into consideration the angular aperture. In addition, using a shorter wavelength of light to view the specimen will increase the resolution.

Finally, the whole microscope system should be correctly aligned. Talk to our experts. We are happy to answer all your questions and concerns. Do you prefer personal consulting? You will find a more detailed list of local contacts here. December 02, Story. Despite writing in a different scientific field, these observations are relevant to other optical systems and indeed, the microscope An Airy Disc is the optimally focussed point of light which can be determined by a circular aperture in a perfectly aligned system limited by diffraction.

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