Interference Microscopy

Cross section of bone in interference contrast (300x enlarged in A3 format). Processed at the Reference Laboratory of Optical Microscopy of Olympus C&S s.r.o., based at the Biological Institute of the Faculty of Medicine of the UK in Pilsen.

Interference microscopy is an imaging method of light microscopy, which is mainly used to increase the contrast when observing transparent or colorless phase objects (non-contrast structures under normal conditions, i.e. practically invisible or only very poorly visible structures in a light microscope). Microscopes working on the basis of phase-contrast methods are also used for the same purpose, but nowadays it prevails due to the higher efficiency of the use of interference microscopy procedures, even though it is a much more complicated method and more demanding on the technical equipment of the device. It provides very reliable information, for example, about the surface of observed objects, especially thanks to its high resolution when studying surface irregularities in the direction of the optical axis of the microscope.

Interference Microscope

An interference microscope is a device that combines a light microscope and some type of interferometer(a device that measures various physical quantities, especially length, on the basis of radiation interference). It uses two-beam interference, multi-beam interference, or the already mentioned interference phase contrast method. The main advantage of multi-beam interference microscopes compared to microscopes working on the principle of phase contrast methods is the suppression of the so-called halo phenomenon, which makes observation very unpleasant. This allows their use to observe relatively thick specimens. The two-beam interference microscope is most widely used in metallography. An essential part of the device is, for example, a Wollaston or Norman prism. The Wollaston prism consists of two wedges of birefringent Icelandic limestone glued together with Canada balsam in a subtractive position. Their optical axis lies perpendicular to the direction of the incident light, which must first,

Principle of Interference Microscopy

In microscopes with two-beam interference, the beam arriving at the specimen is divided, for example, by a Wollaston prism into two coherent bundles of light beams. One of them passes through the preparation, the other passes outside it. Both beams rejoin and interfere with each other after passing through different optical paths. The passage of the beam through the phase object (preparation) causes a phase difference between the two beams (this is therefore caused by the structure of the preparation), as a result of which interference of light waves occurs in the combined beam. This results in changes in the amplitude of the waves, which cause contrasts. The originally transparent observed specimen (phase object) is thus made visible. The fact that the optical path depends both on the geometric path and one of the refractive index of the medium through which the light beam passes allows the use of interference microscopy to examine and measure:

1. The difference in the geometric path assuming that both parts of the split beam pass through the same medium, i.e., a medium with the same refractive index.
2. The difference in the values ​​of the refractive indices of individual environments, if the geometric path of both rays is the same.

Applications Of Interference Microscope

It is a very useful method in the study of surface topography and in the quantitative assessment of surface relief. The principle of this technique is the evaluation of the interference between the beam reflected from the selected comparison surface and the surface under investigation. It also falls under diagnostic procedures in microbiology and pathology, for example, interference microscopy is used to make living cells and their components visible (or other transparent, colorless samples that are normally difficult to observe). The method of interference microscopy is also used in film imaging, e.g. of dividing and moving cells, and is also used for additional contrast enhancement in weakly stained tissue sections.

Links

Related Articles

• Phase microscopy
• Inverted microscope
• Polarization microscopy
• Fluorescence microscope
• Confocal microscope

References

• NAVRÁTIL, Leoš – ROSINA, Jozef. Medicínská biofyzika. 1. edition. Praha : Grada, 2005. 524 pp. ISBN 80-247-1152-4.

Source

• Optoteam, s.r.o.. Základní metody světelné mikroskopie [online]. Nikon, ©2004. [cit. 2015-11-29]. <http://www.are.cz/documents/ZAKLADNI_METODY_SVETELNE_MIKROSKOPIE.pdf>.

• PLACHÝ, Radek. Interferenční mikroskopie [online]. [cit. 2015-11-29]. <http://biologie.upol.cz/mikroskopie/interferencni%20mikroskopie.htm>.

• http://www.mtfdca.szm.com/subory/defektoskopia2.pdf

Kategorie:Histologie Kategorie:Biophysics Kategorie:Exam Questions from Biophysics (2. LF UK)/Light Microscopy