Interference reflection microscopy
Interference reflection microscopy or IRM is an optical microscopy technique that utilizes polarized light to form an image of an object on a glass surface. The intensity of the signal is a measure of proximity of the object to the glass surface. This technique can be used to study events at the cell membrane without the use of a (fluorescent) label in contrast to TIRF microscopy.
The method was first used for the studying of thin films of oil. In 1964, the first application of the technique in cell biology was introduced by Curtis to study embryonic chick heart fibroblasts. He used IRM to look at adhesion sites and distances of fibroblasts, noting that contact with the glass was mostly limited to the cell periphery and the pseudopodia.
The technique was refined and the qualitative and quantitative aspects of the technique were later described by several researchers in the 70s and 80s: Bereiter-Hahn and his colleagues correlated the technique with electron microscopy, showing that different mammalian cell lines adhere to the glass substrate in specific focal adhesion sites.
In order to form an image of the attached cell, light of a specific wavelength is passed through a polarizer. This linear polarized light is reflected by a beam splitter towards the objective, which focuses the light on the specimen. The glass surface is reflective to a certain degree and will reflect the polarized light. Light that is not reflected by the glass will travel into the cell and be reflected by the cell membrane. Three situations can occur:
The reflected light will travel back to the beam splitter and pass through a second polarizer, which eliminates scattered light, before reaching the detector (usually a CCD camera) in order to form the final picture. Note that the polarizers can increase the efficiency by reducing scattered light, however in a modern setup with a sensitive digital camera, they're not required.
Reflection is caused by a change in the refraction index, so on every boundary a part of the light will be reflected. The amount of reflection is given by the reflection coefficient , according to the following rule:
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