Classical interference microscopy

Classical interference microscopy utilizes two separate light beams with much greater lateral separation than that used in differential interference microscopy (DIC). This can be as much as 60 mm (as in the double optical path Mach-Zehnder system of Leitz).

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In variants of the interference microscope where object and reference beam pass through the same objective, two images are produced of every object (one being the "ghost image"). The two images are separated either laterally within the visual field or at different focal planes, as determined by the optical principles employed. These two images can be a nuisance when they overlap, since they can severely affect the accuracy of mass thickness measurements. Rotation of the preparation may thus be necessary, as in the case of DIC.

The two-image problem is completely avoided in the Mach-Zehnder design implemented by Horn, which employs precisely matched duplicated objectives and condensers, but here the new difficulty arises of balancing optical thicknesses of two separate microscope slide preparations (sample and dummy) during longer observations (e.g. of living cells at 37°C) where a gradual change in background interference colour can occur over time.

The main advantage offered by interference microscopy measurements is the possibility of measuring the projected dry mass of living cells, which was first effectively exploited by Andrew Huxley in studies of striated muscle cell structure and function, leading to the sliding filament model of muscle contraction. Huxley, A. F. & Niedergerke, R. Nature 173, 971−973 (1954).

Interference microscopy became relatively popular in the 1940-1970 decades but fell into disuse because of the complexity of the instrument and difficulties in both its use and in the interpretation of image data. In recent years, however, the classical interference microscope (in particular the Mach-Zehnder instrument) has been "rediscovered" because its main original disadvantage (difficult interpretation of translated interference bands or complex coloured images) can now be easily surmounted by means of digital camera image recording, followed by the application of computer algorithms which rapidly deliver the processed data as false-colour images of projected dry mass.