Journal of Microscopy, Vol. 230, Pt 3 2008, pp. 388–395 Received 27 June 2007; accepted 14 December 2007 Immuno-EM using colloidal metal nanoparticles and electron spectroscopic imaging for co-localization at high spatial resolution R. BLEHER ∗ , I. KANDELA †, D.A. MEYER ∗ & R.M. ALBRECHT ∗ †‡ ∗ Department of Animal Science, University of Wisconsin, 1675 Observatory Drive, Madison, WI 53706, U.S.A. †Department of Pharmaceutical Sciences, University of Wisconsin, 777 Highland Avenue, Madison, WI 53706, U.S.A. ‡Department of Pediatrics, University of Wisconsin, 600 Highland Avenue, Madison, WI 53706, U.S.A. Key words. Colloidal nanoparticles, core-shell nanoparticles, electron spectroscopic imaging, EM immunolabelling, energy filtering transmission electron microscopy, gold, multiple labelling, palladium, platinum. Summary Multiple-labelling immuno-EM is a powerful tool for localizing and co-localizing different antigens simultaneously in cells and tissues at high spatial resolution. Commonly used labels for this purpose are differently sized gold spheres. A comparison of results obtained with differently sized markers is often difficult, because the diameters of markers influence labelling efficiency. In the current study, we investigate a method for high- resolution multiple-labelling immuno-EM, using equally sized colloidal markers made of different metals. Energy filtering transmission electron microscopy is used to differentiate particles based on elemental composition. The labels consist of colloidal gold, palladium and platinum-core gold-shell particles of approximately 6 nm in diameter, which are conjugated to different primary antibodies. Applicability of the electron spectroscopic imaging, methodology is demonstrated by labelling of actin, α-actinin and myosin on ultra-thin cryosections of skeletal muscle tissue. Introduction Multiple-labelling studies for simultaneous co-localization of two or more species require different markers that can be distinguished unambiguously. In light microscopic studies, a variety of fluorescent dyes having differing excitation and emission wavelengths are used for this purpose. For EM studies, in which simultaneous Correspondence to: Ralph Albrecht. Tel: 608-263-3952; fax: 608-262-5157; e-mail: albrecht@ansci.wisc.edu localization and co-localization of specific molecular species is desirable, systems similar to those used for LM are limited. Markers for high-resolution studies in immuno-EM need to be electron dense to allow detection. Currently, one of the more effective detection systems in immuno-EM is the application of colloidal gold conjugated to antibodies or other probes (Albrecht et al., 1993; Albrecht & Meyer, 2007). The most commonly employed methodology for multiple-labelling studies involves the use of antibody-conjugated gold spheres of different sizes to localize different antigens. This method has disadvantages, in that antibodies bound to larger markers can bind more antigenic sites per unit than antibodies conjugated to smaller markers; larger spheres can also mask or block more binding sites than smaller labels (Park et al., 1989). These differences influence labelling efficiencies and spatial resolution and thus impair direct or quantitative comparison of label densities. Also, due to the variability of particle diameters in any nominal size range, the diameters of the markers have to differ considerably in order to avoid size overlap. This limits the number of different labels that can be applied simultaneously. This can be problematic when a particular spatial resolution is required and when the location of different individual molecular species relative to each other needs to be defined. In the multiple-labelling technique presented here, equally sized colloidal gold, platinum and palladium markers conjugated to primary antibodies against actin, α-actinin and myosin were used for multiple-labelling of ultra-thin cryosections of skeletal muscle tissue, and the elemental composition of the markers was identified by EFTEM. For EFTEM, inelastically scattered electrons are used to generate elemental distribution maps of specimens (Egerton, C  2008 The Authors Journal compilation C  2008 The Royal Microscopical Society