Interference Reflection Microscopy

Abstract

Interference reflection microscopy (IRM) utilises interference of light reflected from closely apposed surfaces to provide an image containing information about the separation of those surfaces. In cell biology, IRM is used to image structures at the base of adherent cells and to measure cell–substratum distances, as well as to investigate mechanisms of cell–substratum adhesion. IRM is also used to study topology and dynamics of biomimetic systems such as vesicles, supported membranes and other multilayered structures. Basic IRM optical configuration is relatively easy to set up, and image analysis can provide information about interfacial distances with nanometer precision and millisecond time resolution. IRM can be readily combined with other microscopic techniques, and with force transducing devices such as optical tweezers, micropipettes and microcantilevers. New advancements in the field include dual‐wavelength IRM and fluctuation contrast IRM.

Key Concepts:

  • Interference reflection microscopy measures the distance between close surfaces.

  • Cell adhesion areas such as focal contacts can be mapped by IRM.

  • IRM provides vertical resolution in the nanometer range.

  • Dual‐wavelength IRM removes ambiguity in measurements of vertical distances up to 800 nm.

  • IRM can determine amplitudes of local membrane fluctuations.

Keywords: IRM; RICM; reflection interference contrast; cell adhesion; cell–substratum contact; microinterferometry; interfacial distance measurements; membrane fluctuations; focal contacts

Figure 1.

The basic principle of IRM. Two beams of light, I1 and I2, reflected from the glass–liquid and liquid–cell interfaces, respectively, interfere with each other. Their optical path difference depends on the incidence angle θ, cell–substratum separation h, and the index of refraction of the liquid medium n1. Indices of refraction: n0=1.515; n1=1.34; n2 » 1.37.

Figure 2.

IRM image of a Dictyostelium cell moving on the glass surface from left to right. Contact areas appear dark and interference fringes are visible where the cell is not attached to the glass surface.

Figure 3.

Antiflex device used for contrast enhancemet in IRM. The light reflected from a specimen passes through the analyser (solid line), whereas the stray light is blocked off (dashed line). Light beams linearly polarised in mutually perpendicular directions are designated by encircled dots and crosses, respectively. See text for details.

Figure 4.

Dual‐wavelength RICM. (a) A single wavelength (green illumination, solid line) does not permit the unambiguous retrieval of the height h from a measurement of the intensity. Usage of a second wavelength (blue illumination, dashed line) lifts the ambiguity for h up to 800 nm. (b) Variation of intensity of the blue light as a function of intensity of the green light with h as a parameter is used to determine h. Reproduced from Limozin and Sengupta , by permission of Wiley‐VCH Verlag GmbH & Co. KGaA.

Figure 5.

Three‐dimensional reconstruction of a membrane profile obtained by fluctuation contrast IRM and ordinary IRM, showing a considerably better contrast in the former case. The membrane belongs to a vesicle adhering to a substratum by formation of small agglomerates of ligand‐receptor bonds (Smith et al., ). Numerous agglomerates remain undetected in the image of membrane topography due to their small size, but are visible by fluctuation contrast. Courtesy of K Sengupta and A‐S Smith.

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Further Reading

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Weber I (2003) Reflection interference contrast microscopy. In: Marriott J (ed.) Biophotonics, Part B, Methods in Enzymology, vol. 361, pp. 34–47. San Diego: Academic Press.

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Weber, Igor(Jan 2011) Interference Reflection Microscopy. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002636.pub2]