Interference Reflection Microscopy

Valarie A. Barr1, Stephen C. Bunnell2

1 National Cancer Institute, National Institutes of Health, Bethesda, Maryland, 2 Sackler School of Graduate Biomedical Science, Tufts University, Boston, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.23
DOI:  10.1002/0471143030.cb0423s45
Online Posting Date:  December, 2009
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Interference reflection microscopy (IRM) is an optical technique used to study cell adhesion or cell mobility on a glass coverslip. The interference of reflected light waves generates images with high contrast and definition. IRM can be used to examine almost any cell that will rest upon a glass surface, although it is most useful in examining sites of close contact between a cell and substratum. This unit presents methods for obtaining IRM images of cells with particular emphasis on IRM imaging with a laser scanning confocal microscope (LSCM), as most LSCM are already capable of recording these images without any modification of the instrument. Techniques are presented for imaging fixed and live cells, as well as simultaneous multi‐channel capture of fluorescence and reflection images. Curr. Protoc. Cell Biol. 45:4.23.1‐4.23.19. © 2009 by John Wiley & Sons, Inc.

Keywords: reflection contrast microscopy; confocal microscopy; contrast enhancement; refractive index; cell adhesion

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Interference Reflection Imaging of Paraformaldehyde‐Fixed NIH 3T3 Cells with a Laser Scanning Confocal Microscope
  • Alternate Protocol 1: Time‐Lapse IRM Imaging of Live Migrating Dicytostelium Discoideum AMOEBA
  • Alternate Protocol 2: Multi‐Channel Fluorescence and IRM Imaging
  • Support Protocol 1: Preparing Coated Coverslips for IRM Imaging
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Interference Reflection Imaging of Paraformaldehyde‐Fixed NIH 3T3 Cells with a Laser Scanning Confocal Microscope

  • Samples (e.g., fixed NIH 3T3 cells)
  • DMEM cell culture medium (see recipe) or other suitable cell culture medium (with refraction index different from immersion oil)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 2.5% (w/v) paraformaldehyde (see recipe)
  • Culture flasks for maintaining cell cultures before imaging
  • Lab‐Tek II Chambered Cover Glasses, two‐well (Nalge Nunc, cat. no. 155379) or other no. 1 or no. 1.5 coverslips (glass‐bottom 96‐ or 384‐well plates may be used)
  • 37°C, 5% CO 2, humidified incubator
  • Laser Scanning Confocal Microscope equipped with oil immersion objective

Alternate Protocol 1: Time‐Lapse IRM Imaging of Live Migrating Dicytostelium Discoideum AMOEBA

  • PB buffer for Dicytostelium discoideum (see recipe) or culture medium without phenol red for other cells
  • Dicytostelium discoideum amoeba developed according to standard protocols (Parent, ) or other live cells
  • Lab‐Tek II one‐well chambers (Nalge Nunc, cat. no. 155360)

Alternate Protocol 2: Multi‐Channel Fluorescence and IRM Imaging

  • Live sample cells expressing a fluorescent marker [e.g., transfected Jurkat T cells, clone E6.1 (ATCC # TIB‐152) expressing a ZAP70 tagged with YFP and spreading on a stimulatory surface]
  • Imaging buffer (see recipe)
  • Stage incubator
  • Hot air blower (Nevtek) and objective heater (Bioptechs) for temperature control on the microscope stage for the Jurkat T cells
  • Lab‐Tek II four‐well chambered coverslips (Nalge Nunc, cat. No. 155382) coated with murine IgG1 to CDɛ clone Hit3a (BD Pharmingen) to activate the Jurkat T cells (see protocol 4)
  • Laser Scanning Confocal Microscope equipped with at least two photomultipliers

Support Protocol 1: Preparing Coated Coverslips for IRM Imaging

  • Coverslip cleaning solution (see recipe)
  • Poly‐L‐lysine solution (Sigma‐Aldrich, cat. No. P8920)
  • Distilled water
  • Fibronectin coating solution (see recipe)
  • Antibody coating solution (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Coverslips [e.g., Lab‐Tek II 4‐well chambered coverslips; Nalge Nunc, cat. no. 155382 (but any chamber with a no. 1 or no. 1.5 coverslip bottom can be used)]
  • Drying oven
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Literature Cited

   Abercrombie, M. and Dunn, G.A. 1975. Adhesions of fibroblasts to substratum during contact inhibition observed by interference reflection microscopy. Exp. Cell Res. 92:57‐62.
   Beck, K. and Bereiter‐Hahn, J. 1981. Evaluation of reflection interference contrast microscope images of living cells. Microsc. Acta 84:153‐178.
   Bereiter‐Hahn, J., Fox, C.H., and Thorell, B. 1979. Quantitative reflection contrast microscopy of living cells. J. Cell Biol. 82:767‐779.
   Csaderova, L., Riehle, M., and Curtis, A. 2002. Detection of cell forces by measuring deformation of polymer films using interference reflection microscopy. Eur. Cells Mater. 4:64‐65.
   Curtis, A.S. 1964. The mechanism of adhesion of cells to glass. A study by interference reflection microscopy. J. Cell Biol. 20:199‐215.
   Dorogi, P.L. and Keller, H.E. 1993. Antiflex microscopy of cell adhesion. J. N.I.H. Res. 5:79.
   Gingell, D. 1981. The interpretation of interference‐reflection images of spread cells: Significant contributions from thin peripheral cytoplasm. J. Cell Sci. 49:237‐247.
   Gingell, D. and Todd, I. 1979. Interference reflection microscopy. A quantitative theory for image interpretation and its application to cell‐substratum separation measurement. Biophys. J. 26:507‐526.
   Gingell, D. and Vince, S. 1982. Cell‐glass separation depends on salt concentration and valency: Measurements on Dictyostelium amoeba by finite apertrue interferometry. J. Cell Sci. 54:299‐310.
   Izzard, C.S. and Lochner, L.R. 1976. Cell‐to‐substrate contacts in living fibroblasts: An interference reflexion study with an evaluation of the technique. J. Cell Sci. 21:129‐159.
   Keller, H.E. 1997. Contrast enhancement in light microscopy. Curr. Protoc. Cytom. 0:2.1.1‐2.1.11.
   Opas, M. 1990. Biomedical applications of interference reflection microscopy. Proc. SPIE. 1121:351‐356.
   Parent, C.A. 2001. Dictyostelium cell dynamics. Curr. Protoc. Cell Biol. 9:12.5.1‐12.5.19.
   Ploem, J.S. 1975. Reflection‐contrast microscopy as a tool for investigation of the attachment of living cells to a glass surface. In Mononuclear Phagocytes in Immunity, Infection and Pathology. (R. von Furth, ed.) pp.405‐421. Blackwell Scientific Publications, London.
   Prins, F.A., Cornelese‐ten Velde, I., and Heer, E. 2005. Reflection contrast microscopy; The bridge between light and electron microscopy. In Cell Imaging Techniques: Methods and Protocols. Vol. 319. (B.T. Mossman, ed.) pp. 363‐401. Humana Press, Totowa, N.J.
   Toomre, D. and Manstein, D.J. 2001. Lighting up the cell surface with evanescent wave microscopy. Trends Cell Biol. 11:298‐303.
   Verschueren, H. 1985. Interference reflection microscopy in cell biology: Methodology and applications. J. Cell Sci. 75:279‐301.
Key References
   Beck and Bereiter‐Hahn, 1981. See above.
  Very thorough analysis of IRM images and the quantitative aspects of thin‐film optics
   Cornelese‐ten Velde, I., Bonnet, J., Tanke, H.J., and Ploem, J.S. 1990. Reflection contrast microscopy performed on epi‐illumination microscope stands: Comparison of reflection contrast‐ and epi‐polarization microscopy. J. Microsc. 159:1‐13.
  Descriptions of epi‐illumination microscopes configured for IRM imaging.
   Curtis, A.S. 1964. See above.
  First application of IRM to biological samples and quantitative analysis of the images.
   Gingell, D. 1981. See above.
  Analysis showing it is necessary to consider the thickness of the cell cytoplasm when analyzing an IRM image.
   Izzard and Lochner, 1976. See above.
  Description of the role of higher‐order interference fringes in IRM images.
   Ploem, 1975. See above.
  Classic work on applying IRM imaging to biological samples using epi‐illumination.
   Verschueren, 1985. See above.
  Very clear review of IRM imaging in biology that discusses many of the seminal papers.
Internet Resources
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