Classical interference microscopy
Encyclopedia
Classical interference microscopy (also referred to as quantitative interference microscopy) uses two separate light beams with much greater lateral separation than that used in phase contrast microscopy
or in differential interference microscopy
(DIC).
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.
One of the first usable interference microscopes was designed by Dyson and manufactured by Cooke Troughton & Simms (later Vickers Instruments), York England. This ingenious optical system achieved interference imaging without requiring polarizing elements in the beam path.
A later popular design involving polarizing elements was designed by Smith and marketed first by C. Baker, London, and subsequently by the American Optical Company in USA.
The double-image problem commonly encountered with all the above-mentioned designs was completely avoided in the Mach-Zehnder interferometer
design implemented by Horn, a most expensive instrument, not employing polarized light, but requiring precisely-matched duplicated objectives and condensers. With this design (marketed by E. Leitz) 60 mm beam separation was achieved in microscopy but here the new difficulty has arisen of balancing optical thicknesses of two separate microscope slide
preparations (sample and dummy) and maintaining this critical balance during longer observations (e.g. time-lapse
studies of living cells maintained at 37°C), otherwise a gradual change in background interference colour occurs 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.
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" by biologists 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. Examples of computer-assisted developments of the technique are found in the application of "DRIMAPS" from the laboratory of Graham Dunn and other recent developments of the methodology are described by Mahlmann et al .
Phase contrast microscopy
Phase contrast microscopy is an optical microscopy illumination technique of great importance to biologists in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image.A phase contrast microscope does not require...
or in differential interference microscopy
Differential interference contrast microscopy
Differential interference contrast microscopy , also known as Nomarski Interference Contrast or Nomarski microscopy, is an optical microscopy illumination technique used to enhance the contrast in unstained, transparent samples...
(DIC).
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.
One of the first usable interference microscopes was designed by Dyson and manufactured by Cooke Troughton & Simms (later Vickers Instruments), York England. This ingenious optical system achieved interference imaging without requiring polarizing elements in the beam path.
A later popular design involving polarizing elements was designed by Smith and marketed first by C. Baker, London, and subsequently by the American Optical Company in USA.
The double-image problem commonly encountered with all the above-mentioned designs was completely avoided in the Mach-Zehnder interferometer
Mach-Zehnder interferometer
The Mach–Zehnder interferometer is a device used to determine the relative phase shift between two collimated beams from a coherent light source. The interferometer has been used, amongst other things, to measure small phase shifts in one of the two beams caused by a small sample or the change in...
design implemented by Horn, a most expensive instrument, not employing polarized light, but requiring precisely-matched duplicated objectives and condensers. With this design (marketed by E. Leitz) 60 mm beam separation was achieved in microscopy but here the new difficulty has arisen of balancing optical thicknesses of two separate microscope slide
Microscope slide
A microscope slide is a thin flat piece of glass, typically 75 by 25 mm and about 1 mm thick, used to hold objects for examination under a microscope. Typically the object is placed or secured on the slide, and then both are inserted together in the microscope for viewing...
preparations (sample and dummy) and maintaining this critical balance during longer observations (e.g. time-lapse
Time-lapse
Time-lapse photography is a cinematography technique whereby the frequency at which film frames are captured is much lower than that which will be used to play the sequence back. When replayed at normal speed, time appears to be moving faster and thus lapsing...
studies of living cells maintained at 37°C), otherwise a gradual change in background interference colour occurs 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.
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" by biologists 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. Examples of computer-assisted developments of the technique are found in the application of "DRIMAPS" from the laboratory of Graham Dunn and other recent developments of the methodology are described by Mahlmann et al .
Instrumentation History & Makers' Names
- Smith system (C. Baker, London, England)
- Dyson (Cooke Troughton & Simms, York, England)
- Jamin-Lebedeff (E. Leitz, Wetzlar, & Zeiss, Germany)
- Mach-Zehnder (E. Leitz, Wetzlar, Germany)