Interference reflection microscopy

Interference reflection microscopy (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. r 12 = n 1 − n 2 n 1 + n 2 {displaystyle r_{12}={frac {n_{1}-n_{2}}{n_{1}+n_{2}}}} R = I r I i = [ n 1 − n 2 n 1 + n 2 ] 2 = r 12 2 {displaystyle R={frac {I_{r}}{I_{i}}}=leftlbrack {frac {n_{1}-n_{2}}{n_{1}+n_{2}}} ight brack ^{2}={r_{12}}^{2}} Interference reflection microscopy (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. 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. 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 r 12 {displaystyle r_{12}!} , according to the following rule:.mw-parser-output .templatequote{overflow:hidden;margin:1em 0;padding:0 40px}.mw-parser-output .templatequote .templatequotecite{line-height:1.5em;text-align:left;padding-left:1.6em;margin-top:0} 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}!} ): Using typical refractive indices for glass (1.50-1.54, see list), water (1.31, see list), the cell membrane (1.48) and the cytosol (1.35), one can calculate the fraction of light being reflected by each interface. The amount of reflection increases as the difference between refractive indices increases, resulting in a large reflection from the interface between the glass surface and the culture medium (about equal to water: 1.31-1.33). This means that without a cell the image will be bright, whereas when the cell is attached, the difference between medium and the membrane causes a large reflection that is slightly shifted in phase, causing interference with the light reflected by the glass. Because the amplitude of the light reflected from the medium-membrane interface is decreased due to scattering, the attached area will appear darker but not completely black. Because the cone of light focused on the sample gives rise to different angles of incident light, there is a broad range of interference patterns. When the patterns differ by less than 1 wavelength (the zero-order fringe), the patterns converge, resulting in increased intensity. This can be obtained by using an objective with a numerical aperture greater than 1. In order to image cells using IRM, a microscope needs at least the following elements: 1) a light source, such as a halogen lamp, 2) an optical filter (which passes a small range of wavelengths), and 3) a beam splitter (which reflects 50% and transmits 50% of the chosen wavelength)