Interference reflection microscopy (IRM), also called Reflection Interference Contrast Microscopy (RICM) or Reflection Contrast Microscopy (RCM) depending on the specific optical elements used, is an optical microscopy technique that leverages thin-film interference effects 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 as is the case for TIRF microscopy.
History and name In 1964, Adam S. G. Curtis coined the term Interference Reflection Microscopy (IRM), using it in the field of cell biology 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. In 1975, Johan Sebastiaan Ploem introduced an improvement to IRM (published in a book chapter), which he called Reflection Contrast Microscopy (RCM). The improvement is to use a so-called anti-flex objective and crossed polarizers to further reduce stray light in the optical system. Today, this scheme is mainly referred to as Reflection Interference Contrast Microscopy (RICM), the name of which was introduced by Bareiter-Hahn and Konrad Beck in 1979. However, the term IRM is sometimes used to describe an RICM setup. The multiplicity of names used to describe the technique has caused some confusion, and was discussed as early as 1985 by Verschueren.
Theory 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. First, when the membrane is close to the glass, the reflected light from the glass is shifted half of a wavelength, so that light reflected from the membrane will have a phase shift compared to the reflected light from the glass phases and therefore cancel each other out (interference). This interference results in a dark pixel in the final image (the left case in the figure). Second, when the membrane is not attached to the glass, the reflection from the membrane has a smaller phase shift compared to the reflected light from the glass, and therefore they will not cancel each other out, resulting in a bright pixel in the image (the right case in the figure). Third, when there is no specimen, only the reflected light from the glass is detected and will appear as bright pixels in the final image. 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. The polarizers can increase the efficiency by reducing scattered light; however in a modern setup with a sensitive digital camera, they are not required.
Theory 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 r 12 {\displaystyle r_{12}\!} , according to the following rule: r 12 = n 1 − n 2 n 1 + n 2 {\displaystyle r_{12}={\frac {n_{1}-n_{2}}{n_{1}+n_{2}}}}
Reflectivity R {\displaystyle R\!} is a ratio of the reflected light intensity ( I r {\displaystyle I_{r}\!} ) and the incoming light intensity ( I i {\displaystyle I_{i}\!} ): R = I r I i = [ n 1 − n 2 n 1 + n 2 ] 2 = r 12 2 {\displaystyle R={\frac {I_{r}}{I_{i}}}=\left\lbrack {\frac {n_{1}-n_{2}}{n_{1}+n_{2}}}\right\rbrack ^{2}={r_{12}}^{2}}
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