Interference Reflection Microscopy

Igor Weber, Ruder Bošković Institute, Zagreb, Croatia

Published online: January 2011

DOI: 10.1002/9780470015902.a0002636.pub2

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.
close

References

Abercrombie M and Dunn GA (1975) Adhesions of fibroblasts to substratum during contact inhibition observed by interference reflection microscopy. Experimental Cell Research 92: 57–62.

Bohnet S, Ananthakrishnan R, Mogilner A et al. (2006) Weak force stalls protrusion at the leading edge of the lamellipodium. Biophysical Journal 90: 1810–1820.

Choi CK, Margraves CH, English AE and Kihm KD (2008) Multicontrast microscopy technique to dynamically fingerprint live‐cell focal contacts during exposure and replacement of a cytotoxic medium. Journal of Biomedical Optics 13: 54069.

Cretel E, Touchard D, Benoliel AM et al. (2010) Early contacts between T lymphocytes and activating surfaces. Journal of Physics‐Condensed Matter 22: 94107.

Curtis ASG (1964) The mechanism of adhesion of cells to glass. A study by interference reflection microscopy. Journal of Cell Biology 20: 199–215.

Dubreuil F, Elsner N and Fery A (2003) Elastic properties of polyelectrolyte capsules studied by atomic‐force microscopy and RICM. European Physical Journal E 12: 215–221.

Evans E, Ritchie K and Merkel R (1995) Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces. Biophysical Journal 68: 2580–2587.

Fang N, Zhu AP, Chan‐Park MB and Chan V (2005) Adhesion contact dynamics of fibroblasts on biomacromolecular surfaces. Macromolecular Bioscience 5: 1022–1031.

Heinrich V, Wong WP, Halvorsen K and Evans E (2008) Imaging biomolecular interactions by fast three‐dimensional tracking of laser‐confined carrier particles. Langmuir 24: 1194–1203.

Holt MR, Calle Y, Sutton DH et al. (2008) Quantifying cell‐matrix adhesion dynamics in living cells using interference reflection microscopy. Journal of Microscopy 232: 73–81.

Huang ZH, Massiera G, Limozin L et al. (2010) Sensitive detection of ultra‐weak adhesion states of vesicles by interferometric microscopy. Soft Matter 6: 1948–1957.

Izzard CS and Lochner LR (1976) Cell‐to‐substrate contacts in living fibroblasts: an interference reflection study with an evaluation of the technique. Journal of Cell Science 21: 129–159.

Kim K and Saleh OA (2008) Stabilizing method for reflection interference contrast microscopy. Applied Optics 47: 2070–2075.

Kühner M and Sackmann E (1996) Ultrathin hydrated dextran films grafted on glass: preparation and characterization of structural, viscous, and elastic properties by quantitative microinterferometry. Langmuir 12: 4866–4876.

Limozin L and Sengupta K (2007) Modulation of vesicle adhesion and spreading kinetics by haluronan cushions. Biophysical Journal 93: 3300–3313.

Limozin L and Sengupta K (2009) Quantitative reflection interference contrast microscopy (RICM) in soft matter and cell adhesion. ChemPhysChem 10: 2752–2768.

Llobet A, Beaumont V and Lagnado L (2003) Real‐time measurement of exocytosis and endocytosis using interference of light. Neuron 40: 1075–1086.

Monzel C, Fenz SF, Merkel R and Sengupta K (2009) Probing biomembrane dynamics by dual‐wavelength reflection interference contrast microscopy. ChemPhysChem 10: 2828–2838.

Owens NF, Gingell D and Bailey J (1988) Contact‐mediated triggering of lamella formation by Dictyostelium amoebae on solid surfaces. Journal of Cell Science 91: 367–377.

Picart C, Sengupta K, Schilling J et al. (2004) Microinterferometric study of the structure, interfacial potential, and viscoelastic properties of polyelectrolyte multilayer films on a planar substrate. Journal of Physical Chemistry B 108: 7196–7205.

Pierres A, Benoliel AM, Touchard D and Bongrand P (2008) How cells tiptoe on adhesive surfaces before sticking. Biophysical Journal 94: 4114–4122.

Ploem JS (1975) Reflection‐contrast microscopy as a tool for investigation of the attachment of living cells to a glass surface. In: Van Furth R (ed.) Mononuclear Phagocytes in Immunity, Infection and Pathology, pp. 405–421. London: Blackwell Scientific.

Rädler J and Sackmann E (1993) Imaging optical thicknesses and separation distances of phospholipid vesicles at solid surfaces. Journal de Physique II France 3: 727–748.

Robert P, Sengupta K, Puech PH et al. (2008) Tuning the formation and rupture of single ligand‐receptor bonds by hyaluronan‐induced repulsion. Biophysical Journal 95: 3999–4012.

Schilling J, Sengupta K, Goennenwein S et al. (2004) Absolute interfacial distance measurements by dual‐wavelength reflection interference contrast microscopy. Physical Review E 69: 1901.

Schindl M, Wallraff E, Deubzer B et al. (1995) Cell–substrate interactions and locomotion of Dictyostelium wild‐type cells and mutants defective in three cytoskeletal proteins: a study using reflection interference contrast microscopy. Biophysical Journal 68: 1177–1190.

Sengupta K, Aranda‐Espinoza H, Smith L et al. (2006) Spreading of neutrophils: from activation to migration. Biophysical Journal 91: 4638–4648.

Simon SM (2009) Partial internal reflections on total internal reflection fluorescent microscopy. Trends in Cell Biology 19: 661–668.

Simson R, Wallraff E, Faix J et al. (1998) Membrane bending modulus and adhesion energy of wild‐type and mutant cells of Dictyostelium lacking talin or cortexillins. Biophysical Journal 74: 514–522.

Smith AS, Sengupta K, Goennenwein S et al. (2008) Force‐induced growth of adhesion domains is controlled by receptor mobility. Proceedings of the National Academy of Sciences of the USA 105: 6906–6911.

Smith AS, Fenz SF and Sengupta K (2010) Inferring spatial organization of bonds within adhesion clusters by exploiting fluctuations of soft interfaces. Europhysics Letters 10: 28003.

Stuart JK and Hlady V (1999) Reflection interference contrast microscopy combined with scanning force microscopy verifies the nature of protein‐ligand interaction force measurements. Biophysical Journal 76: 500–508.

Theodoly O, Huang ZH and Valignat MP (2010) New modeling of reflection interference contrast microscopy including polarization and numerical aperture effects: application to nanometric distance measurements and object profile reconstruction. Langmuir 26: 1940–1948.

Uchida KSK and Yumura S (2004) Dynamics of novel feet of Dictyostelium cells during migration. Journal of Cell Science 117: 1443–1455.

Weber I and Albrecht R (1997) Image processing for combined bright‐field and reflection interference contrast video microscopy. Computer Methods and Programs in Biomedicine 53: 113–118.

Weber I, Wallraff E, Albrecht R and Gerisch G (1995) Motility and substratum adhesion of Dictyostelium wild‐type and cytoskeletal mutant cells: a study by RICM/bright‐field double‐view image analysis. Journal of Cell Science 108: 1519–1530.

Zhu AP, Fang N, Chan‐Park MB and Chan V (2006) Adhesion contact dynamics of 3T3 fibroblasts on poly (lactide‐co‐glycolide acid) surface modified by photochemical immobilization of biomacromolecules. Biomaterials 27: 2566–2576.

Zilker A, Ziegler M and Sackmann E (1992) Spectral analysis of erythrocyte flickering in the 0.3–4 mm−1 regime by microinferometry combined with fast image processing. Physical Review A 46: 7998–8001.

Further Reading

Bereiter‐Hahn J and Vesely P (1998) Reflection interference microscopy. In: Celis JE (ed.) Cell Biology, vol. 3, pp. 54–63. San Diego: Academic Press.

Fenz SF, Merkel R and Sengupta K (2009) Diffusion and intermembrane distance: case study of avidin and E‐cadherin mediated adhesion. Langmuir 25: 1074–1085.

He T, Shi ZL, Fang N et al. (2009) The effect of adhesive ligands on bacterial and fibroblast adhesions to surfaces. Biomaterials 30: 317–326.

Smith AS and Sackmann E (2009) Progress in mimetic studies of cell adhesion and the mechanosensing. ChemPhysChem 10: 66–78.

Verschueren H (1985) Interference reflection microscopy in cell biology: methodology and applications. Journal of Cell Science 75: 279–301.

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.

Related Sub-Topics:

Cell Membranes | Optical Microscopy | Techniques in Cell Biology
Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

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