Interference reflection microscopy
Interference reflection microscopy or IRM is a 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.
History
The method was first used for the studying of thin films of oil[1][2]. In 1964, the first application of the technique in cell biology was introduced by Curtis to study embryonic chick heart fibroblasts[3]. The technique was refined and later described by Hendrik Verschueren in 1985[4].
Theory
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:
- When the membrane is directly attached to the glass, the reflected light from the glass itself and that from the membrane will have (almost) identical phases and therefore cancel each other out (interference). This interference results in a dark pixel in the final image.
- When the membrane is not attached to the glass, the reflection from the membrane will be out of phase with the reflected light from the glass, and therefore they will not cancel each other out, resulting in a bright pixel in the image.
- When there is no specimen, only the reflected light 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 elliminates scattered light, before reaching the detector (usually a CCD camera) in order to form the final picture.
Refinements
There have been many refinements to the basic theory of IRM, most of which increase the efficiency and yield of the image formation. By placing a quarter wave plate between the beam splitter and the specimen, the linear polarized light can be converted into circular polarized light and afterwards be converted back to linear polarized light, which increases the efficiency of the system.
Biological applications
There are several ways IRM can be used to study biological samples. Early examples of uses of the technique focused on cell adhesion[3] and cell migration[5]. More recently, the technique has been used to study exocytosis in chromaffin cells[6]
References
- ^ Van den Tempel, M (1958): Distance between emulsified oil globules upon coalescence. J Coll Sci 13(2): 125-133. PMID: 10727991
- ^ Vašíček, A (1960): Theory of Light Reflection From a Thin Absorbing Film Deposited on a Metal. Optics and Spectroscopy 11: 128
- ^ a b Curtis, AS (1964): The mechanism of adhesion of cells to glass. A study by interference reflection microscopy. J Cell Biol 20: 199-215. PMID: 14126869
- ^ Verschueren, H (1985): Interference reflection microscopy in cell biology: methodology and applications. J Cell Sci 75: 279-301. PMID: 3900106
- ^ Godwin, SL et al (1989): Interference reflection microscopic study of sites of association between gliding bacteria and glass substrata. J Bacteriol 171(9):4589-4594. PMID: 2768185
- ^ Wu, MM et al (2009): Loose coupling between calcium channels and sites of exocytosis in chromaffin cells. J Physiol 587(Pt 22):5377-5391. PMID: 19752110