Skip to main content Accessibility help

Immunolabeling for Correlative Light and Electron Microscopy on Ultrathin Cryosections

  • Irawati K. Kandela (a1), Reiner Bleher (a2) and Ralph M. Albrecht (a1) (a2) (a3)

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.

Corresponding author
Corresponding author. E-mail:
Hide All


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. 173208. 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. 151176. Oxford, UK: Oxford University Press.
Alivisatos, A.P., Gu, W. & Larabell, C. (2005). Quantum dots as cellular probes. Ann Rev Biomed Eng 7, 5576.
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, 109113.
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.
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), 914915.
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 14.
Faulk, W. & Taylor, G. (1971). An immunocolloid method for electron microscopy. Immunochemistry 8, 10811083.
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, 727734.
Horisberger, M. (1981). Colloidal gold: A cytochemical marker for light and fluorescent microscopy and for transmission and scanning electron microscopy. Scan Electron Microsc 2, 931.
Horisberger, M. & Rosset, J. (1977). Colloidal gold, a useful marker for transmission and scanning electron microscopy. J Histochem Cytochem 25, 295305.
Kandela, I.K. & Albrecht, R.M. (2007). Fluorescence quenching by colloidal heavy metal nanoparticles: Implications for correlative fluorescence and electron microscopy studies. Scanning 29, 152161.
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, 983990.
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), 11941195.
Meyer, D.A. & Albrecht, R.M. (1999). Multiple labeling for EM using particles of different shapes and metal composition. Microsc Microanal 5(Suppl. 2), 488489.
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), 3233.
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, 1318.
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, 212.
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, 803808.
Takizawa, T. & Robinson, J.M. (2003). Ultrathin cryosections: An important tool for immunofluorescence and correlative microscopy. J Histochem Cytochem 51, 707714.
Takizawa, T., Suzuki, K. & Robinson, J.M. (1998). Correlative microscopy using fluoronanogold or ultrathin cryosection: Proof of principle. J Histochem Cytochem 46, 10971102.
Tokuyasu, K.T. & Singer, S.J. (1976). Improved procedures for immunoferritin labeling of ultrathin frozen sections. J Cell Biol 71, 894906.
Warchol, J.B., Brelinska, R. & Herbert, D.C. (1982). Analysis of colloidal gold methods for labelling proteins. Histochemistry 76, 567575.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

Microscopy and Microanalysis
  • ISSN: 1431-9276
  • EISSN: 1435-8115
  • URL: /core/journals/microscopy-and-microanalysis
Please enter your name
Please enter a valid email address
Who would you like to send this to? *



Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed