INTERFERENCE MICROSCOPE

Recently, in microscopy, the phase-contrast method has become widely used, making it possible to obtain contrast images of transparent objects. A distinctive modification of this method is the interference microscope proposed by the author of the paper reviewed here*). The idea of the method consists in first obtaining an interference image of the object at natural size, with its subsequent examination in an ordinary microscope.

The principle of operation of the device is clear from the figure. The illuminating cone of rays from the condenser (not shown in the figure), which images the light source on the object, falls on a plane-parallel plate whose upper surface is coated with a semitransparent layer of silver. On the lower surface of the plate there is deposited an opaque reflecting silver “spot,” whose size is somewhat larger than the field of view of the microscope.

A light ray incident on the upper semitransparent face of the plate partly passes through it, reaching the object, and is partly reflected onto the “spot.” Then both parts of the ray reach a second plane-parallel plate (identical with the first), both faces of which are coated with semitransparent layers of silver; the ray reflected from the “spot” partly passes through it, while the ray that has passed through the object partly passes through it after first undergoing two reflections. As a result the rays recombine and then proceed in a common direction. (Thus, this part of the device acts analogously to a Jamin interferometer.) On emerging from the upper plane-parallel plate, the combined rays fall on a spherical reflecting surface (the center of the sphere coincides with the center of the “spot”; the upper face of the upper plane-parallel plate divides the radius of the sphere in half). Having undergone reflection from the spherical surface,

) J. Dyson, Proc. Roy. Soc. A204*, 107 (1950).

the rays return, undergo partial reflection at the upper face of the upper plane-parallel plate, and are collected in a small “window” made in the reflecting silver layer covering the sphere.

Thus, two coherent real images of the light source are obtained at the “window”: one produced by the rays that have passed through the object, the other by rays that have passed around it and have been reflected from the “spot.” As a result of interference in the plane of the “window,” an interference image of the object is obtained, which is then examined with an ordinary microscope.

Diagram of an interferometer for interference microscopy.

Since the cone of the “comparison rays” reflected from the “spot” is deprived of only its central part (occupied by the “spot” at the initial incidence on the system) and has, in the plane of the object, a diameter of several millimeters, the presence of the object does not introduce into it any appreciable distortions. In the absence of the object, the phase difference of the interfering rays arises only as a result of phase jumps upon reflection. The presence of the object introduces an additional phase difference, which leads to the formation of an interference image of the object—the phase modulation of one of the rays is thereby converted into amplitude modulation.

Since the dimensions of the “window” only slightly exceed the dimensions of the microscope field of view, and all the “multiple” secondary reflections give secondary images at a distance of several millimeters from the center of the “window” (i.e., they do not enter the microscope objective), the presence of secondary reflections does not lead to distortion of the image and affects only a reduction in its intensity. The author indicates that a distinct interference image of the object is obtained not only in monochromatic, but also in white light.

If the plates are not exactly plane-parallel, but constitute wedges with a small angle, oriented toward one another, then a slight displacement of them relative to one another makes it possible to change the path difference of the interfering rays and thus to change both the character of the field on which the image is obtained and the character of the image itself (the corresponding device is described in detail by the author).

The analysis carried out by the author shows that, in the case of phase contrast (transparent objects), the best results are achieved with a darkened field and equality of the intensities of both interfering rays. In the case of amplitude contrast (absorbing but not refracting objects), the contrast of the image is half as great as when an ordinary microscope is used.

To obtain an interference image of the object it is necessary that both images of the light source be exactly superposed on one another, i.e. that the corresponding points of both interfering images coincide, for different parts of the image of the light source are incoherent with one another. Therefore, to obtain a contrast image it is necessary that the displacement relative to one another of the two interfering images of the source, obtained on the “window,” be small in comparison with the limit of resolution for the numerical aperture of the illuminating cone of rays. This imposes strict requirements on the mutual arrangement of the plates of the interferometer.

The author describes in detail both the constructive design of the interference microscope and the method of working with it. Referring to the original work for details, we shall indicate only that the lower plate of the interferometer is mounted on the stage of the microscope with the aid of a simple device that makes it possible easily to regulate its position. The upper part of the interferometer (together with the cover glass) is rigidly attached directly to the objective of the microscope. The specimen slide with the object is placed on the lower plate; the gaps between the specimen slide and both plates of the interferometer are filled with immersion liquid. In the rough adjustment of the interferometer, fringes of equal thickness are observed; eliminating these fringes is essentially what its adjustment amounts to. The author notes that handling the interferometer does not require great skill.

One might expect that an interferometer of this kind would be suitable only for the investigation of very thin objects. However, a special calculation carried out by the author shows that these limitations are not so severe and do not lead to serious difficulties in microscopic investigations.

A series of microphotographs of various objects presented by the author illustrates the capabilities of such a device. The images obtained with its aid are highly contrasted, not inferior in this respect to images obtained by other methods. The interference microscope can also be used to measure the refractive index of microscopic objects. The technique of this application is likewise described by the author. In particular, measurement of the refractive index of the cytoplasm of epithelial cells of a frog’s tongue gave its value as \(n = 1.358 \pm 0.010\), which indicates a comparatively high accuracy of the measurements.

Among the merits of the instrument the author includes:

1. The possibility of using a standard microscope without any significant alterations.

2. The absence of restrictions in the aperture of the illuminator.

3. The possibility of regulating the character of the image obtained (by the relative displacement of the interferometer plates), which makes it possible easily to attain optimal conditions for visibility of the object.

1. The possibility of measuring the optical thickness and, consequently, the refractive index of the object.

2. The possibility of using white light.

The author considers the main drawback of the instrument to be the large losses of light (to obtain a microphotograph in monochromatic light requires about 0.2 sec.).

There is no doubt that interference microscopes, after suitable improvement, will find wide application.

V. Yuryev