Daryl Meyer

Immuno‐EM using colloidal metal nanoparticles and electron spectroscopic imaging for co‐localization at high spatial resolution

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.

Colloidal Palladium Particles of Different Shapes for Electron Microscopy Labeling

The immunogold technique is a valuable method for labeling cellular macromolecules. However, multiple labeling using colloidal gold (cAu) nanoparticles of different sizes presents certain drawbacks; namely, as particle size increases, there is a decreased labeling efficiency and diminished spatial resolution with respect to the locations of labeled epitopes. Both concerns also limit the utility of heavy metal particles for comparative analysis of labeling densities. To minimize the variables due to differential labeling efficiencies, the best solution would be to conduct multiple labeling with particles of similar size. Consequently, some parameter other than size is necessary to distinguish each label type. In this study, we report the synthesis of colloidal palladium (cPd) nanoparticles of similar size but having two distinct shapes, umbonate and faceted, which are readily distinguishable from spherical colloidal gold particles. Their utility and fidelity as labels using a human platelet whole-mount model is also demonstrated.