This chapter defines and discusses criteria for optical resolution and indicates those factors that reduce it from its theoretical limits. Many of the definitions of resolution were originally derived for two-point resolution in telescopes used in astronomy and adopted for optical microscopy. Stars observed in an optical telescope can be considered to be point sources of light. Biological specimens observed in a light microscope are not point sources of light; however, the detection of fluorescence from single molecules is within the capability of a modern fluorescence microscope. Single fluorescent beads of submicron diameter can approximate point sources of light and therefore be used to experimentally determine the optical performance or resolving power of a microscope objective.
4.1 Criteria for Two-Point Resolution
A discussion of the criteria for two-point resolution begins with the definition of two related terms. Resolving power denotes the smallest detail that a microscope can resolve when imaging a specimen; it is a function of the design of the instrument and the properties of the light used in image formation. Resolution indicates the level of detail actually observed in the specimen. It depends on the resolving power of the microscope, the contrast generated in the microscope, the contrast in the specimen, and the noise in the detector.
Abbe's theory yields a limited far-field spatial resolution for the light microscope. The lateral resolution is approximately 180 nm, and the axial resolution approximately 500 nm. The Abbe theory of the role of diffraction and interference in image formation in the optical microscope leads to this summary of several important points and their consequences: First, the resolving power of a microscope objective is measured by its ability to differentiate two points. The smaller the distance between the two points that can be distinguished, the higher the resolving power. Second, as the wavelength of the light used to illuminate the source is decreased (shorter-wavelength illumination), two points can be resolved at a smaller distance of separation. Third, as we increase the NA, then two points closer together can be resolved.
Abbe's theory explains this in terms of higher orders of diffracted light from the specimen entering the collection angle of the objective.
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