In the optical microscope, image formation occurs at the intermediate image plane through interference between direct light that has passed through the specimen unaltered and light diffracted by minute features present in the specimen. The image produced by an objective lens is conjugate with the specimen, meaning that each image point is geometrically related to a corresponding point in the specimen. It follows that each point in the specimen is therefore represented by a corresponding point in the image.
Image resolution and contrast in the microscope can only be fully understood by considering light as a train of waves. Light emitted by a particular point on a specimen is not actually focused to an infinitely small point in the conjugate image plane, but instead light waves converge and interfere near the focal plane to produce a three-dimensional diffraction pattern. The ensemble of individual diffraction patterns spatially oriented in two dimensions, often termed Airy patterns, is what constitutes the image observed when viewing specimens through the eyepieces of a microscope. These and related concepts are discussed more fully in the sections listed below.
When direct or undeviated light from a specimen is projected by the objective, it is spread evenly across the entire image plane at the diaphragm of the eyepiece. The light diffracted by the specimen is brought to focus at various localized sites on the same image plane, and there the diffracted light causes destructive interference. A consequence is the reduction in light intensity resulting in more or less dark areas. These patterns of light and dark are what we recognize as an image of the specimen. Since our eyes are sensitive to variations in brightness, the image then becomes a more or less faithful reconstitution of the original specimen.
Explore the origin of Airy diffraction patterns formed by the rear aperture of the microscope objective and observed at the intermediate image plane in this featured interactive tutorial.
Discover how Airy pattern size changes with objective numerical aperture and the wavelength of illumination; it also simulates the close approach of two Airy patterns in this tutorial.
In this interactive tutorial, the visitor will be able to explore the mechanics of periodic diffraction gratings when utilized to interpret the Abbe theory of image formation in the optical microscope.
Examine the effects of objective numerical aperture on the resolution of the central bright disks present in the diffraction pattern, commonly known as Airy disks in this interactive tutorial.
The purpose of this tutorial is to explore the reciprocal relationship between line spacing in a periodic grid (simulating a specimen) and the separation of the conoscopic image at the objective aperture plane.
Explore the relationship between the distance separating these iris opening images and the periodic spacing (spatial frequency) of lines in the grating in this featured tutorial.
Examine how Airy disk sizes, at the limit of optical resolution, vary with changes in objective numerical aperture and illumination wavelength and how these changes affect the resolution of the objective.
Explore the structure of cross sections taken along the optical axis of the microscope near the focal plane using a virtual high numerical aperture objective free from spherical aberration.
In the featured tutorial, the visitor will examine diffraction images produced by a periodic object at several focal depths. The periodic object used in this tutorial is a Siemens test star.
Selected Literature References
An understanding of the distribution of light intensity throughout images observed in the optical microscope involves the laws of physical optics. Of primary consideration is the diffraction pattern exhibited by the specimen, which is composed of an array of elementary constituents known as the Airy disk. These and related concepts are reviewed in the reference materials listed in this section.