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Immunolabeling for Correlative Light and Electron Microscopy on Ultrathin Cryosections

Published online by Cambridge University Press:  03 March 2008

Irawati K. Kandela ,
Reiner Bleher  and
Ralph M. Albrecht
Irawati K. Kandela
Affiliation:
Department of Pharmaceutical Sciences, University of Wisconsin, Madison, WI 53705, USA
Reiner Bleher
Affiliation:
Department of Animal Sciences, University of Wisconsin, Madison, WI 53706, USA
Ralph M. Albrecht
Affiliation:
Department of Pharmaceutical Sciences, University of Wisconsin, Madison, WI 53705, USA Department of Animal Sciences, University of Wisconsin, Madison, WI 53706, USA Department of Pediatrics, University of Wisconsin, Madison, WI 53706, USA
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Abstract

Correlative labeling permits colocalization of molecular species for observation of the same sample in light (LM) and electron microscopy (EM). Myosin bands in ultrathin cryosections were labeled using both fluorophore conjugated to secondary antibody (IgG) and colloidal gold (cAu) particles conjugated to primary IgG as reporters for LM and transmission electron microscopy (TEM), respectively. This technique allows rapid evaluation of labeling via LM, prior to more time-consuming observations with TEM and also yields two complementary data sets in one labeling procedure. Quenching of the fluorescent signal was inversely related to the distance between fluorophore and cAu particles. The signal from fluorophore conjugated to secondary antibody was inversely proportional to the size of cAu conjugated to primary antibody. Where fluorophore and cAu were bound to the same antibody, the fluorescence signal was nearly completely quenched regardless of fluorophore excitation or emission wavelength and regardless of particle size, 3 nm and larger. Colloidal metal particles conjugated to primary antibody provide high spatial resolution for EM applications. Fluorophore conjugated to secondary antibody provides spatial resolution well within that of conventional fluorescence microscopy. Use of fluorescent secondary antibody moved the fluorophore a sufficient distance from the cAu particles on the primary antibody to limit quenching of fluorescence.

Keywords

correlative labelingimmunofluorescenceimmuno-EMcolloidal gold particlesfluorophorescryosectionsnanoparticles
Type
Research Article
Information
Microscopy and Microanalysis , Volume 14 , Issue 2 , April 2008 , pp. 159 - 165
Copyright
© 2008 Microscopy Society of America

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References

REFERENCES

Albrecht, R.M. & Meyer, D.A. (2007). Correlative labeling for LVSEM. In Low Voltage Field Emission Scanning Electron Microscopy, Schatten, H. & Pawley, J.B. (Eds.), Chapter 6, pp. 173–208. New York: Springer Science.
Albrecht, R.M., Simmons, S.R. & Pawley, J.B. (1993). Correlative video-enhanced light microscopy, high voltage transmission electron microscopy, and field emission scanning electron microscopy for the localization of colloidal gold labels. In Immunocytochemistry, A Practical Approach, Beesley, J.E. (Ed.), pp. 151–176. Oxford, UK: Oxford University Press.
Alivisatos, A.P., Gu, W. & Larabell, C. (2005). Quantum dots as cellular probes. Ann Rev Biomed Eng 7, 55–76.Google Scholar
Birrell, G.B., Habliston, D.L., Nadakavukaren, K.K. & Griffith, O.H. (1985). Immunophotoelectron microscopy: The electron optical analog of immunofluorescence microscopy. Proc Natl Acad Sci, USA 82, 109–113.Google Scholar
De Brabander, M., Geuens, G., Nuydens, R., Moeremans, M. & De May, J. (1985). Probing microtubule-dependent intracellular motility with nanometer particle video ultramicroscopy (nanovid ultramicroscopy). Cytobios 43, 273.Google Scholar
Deerinck, T.J., Giepmans, B.N.G. & Ellisman, M.H. (2005). Quantum dots as cellular probes for light and electron microscopy. Microsc Microanal 11(Suppl. 2), 914–915.Google Scholar
Dulkeith, E., Morteani, A.C., Niedereichhloz, T., Klar, T.A. & Feldmann, J. (2002). Fluorescence quenching of dye molecules near gold nanoparticles: Radiative and nonradiative effects. Phys Rev Lett 89, 203002 1–4.Google Scholar
Faulk, W. & Taylor, G. (1971). An immunocolloid method for electron microscopy. Immunochemistry 8, 1081–1083.Google Scholar
Goodman, S.L. & Albrecht, R.M. (1987). Correlative light and electron of platelet adhesion and fibrinogen receptor expression using colloidal gold labeling. Scan Microsc 1, 727–734.Google Scholar
Horisberger, M. (1981). Colloidal gold: A cytochemical marker for light and fluorescent microscopy and for transmission and scanning electron microscopy. Scan Electron Microsc 2, 9–31.Google Scholar
Horisberger, M. & Rosset, J. (1977). Colloidal gold, a useful marker for transmission and scanning electron microscopy. J Histochem Cytochem 25, 295–305.Google Scholar
Kandela, I.K. & Albrecht, R.M. (2007). Fluorescence quenching by colloidal heavy metal nanoparticles: Implications for correlative fluorescence and electron microscopy studies. Scanning 29, 152–161.Google Scholar
Kandela, I.K., Bleher, R. & Albrecht, R.M. (2007). Multiple correlative immunolabeling in light and electron microscopy using fluorophores and colloidal metal particles. J Histochem Cytochem 55, 983–990.Google Scholar
Kandela, I.K., Meyer, D.A., Oshel, P.E., Rosa-Molinar, E. & Albrecht, R.M. (2003). Fluorescence quenching by colloidal heavy metals: Implications for correlative fluorescence and electron microscopy studies. Microsc Microanal 9(Suppl. 2), 1194–1195.Google Scholar
Meyer, D.A. & Albrecht, R.M. (1999). Multiple labeling for EM using particles of different shapes and metal composition. Microsc Microanal 5(Suppl. 2), 488–489.Google Scholar
Meyer, D.A., Bleher, R., Kandela, I.K., Oliver, J.A. & Albrecht, R.M. (2006). The development of alternative markers for transmission electron microscopy and correlative transmission electron and light microscopies. Microsc Microanal 12(Suppl. 2), 32–33.Google Scholar
Nisman, R., Dellaire, G., Ren, Y., Ren, L. & Bazett-Jones, D. (2004). Application of quantum dots as probes for correlative fluorescence conventional, and energy-filtered transmission electron microscopy. J Histochem Cytochem 52, 13–18.Google Scholar
Powell, R.D., Halsey, C.M. & Hainfeld, J.F. (1998). Combined fluorescent and gold immunoprobes: Reagents and methods for correlative light and electron microscopy. Microsc Res Tech 42, 2–12.Google Scholar
Robinson, J.M., Takizawa, T., Pombo, A. & Cook, P.R. (2001). Correlative fluorescence and electron microscopy on ultrathin cryosections: Bridging the resolution gap. J Histochem Cytochem 49, 803–808.Google Scholar
Takizawa, T. & Robinson, J.M. (2003). Ultrathin cryosections: An important tool for immunofluorescence and correlative microscopy. J Histochem Cytochem 51, 707–714.Google Scholar
Takizawa, T., Suzuki, K. & Robinson, J.M. (1998). Correlative microscopy using fluoronanogold or ultrathin cryosection: Proof of principle. J Histochem Cytochem 46, 1097–1102.Google Scholar
Tokuyasu, K.T. & Singer, S.J. (1976). Improved procedures for immunoferritin labeling of ultrathin frozen sections. J Cell Biol 71, 894–906.Google Scholar
Warchol, J.B., Brelinska, R. & Herbert, D.C. (1982). Analysis of colloidal gold methods for labelling proteins. Histochemistry 76, 567–575.Google Scholar