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.