Multiple labeling for electron microscopy (EM) is typically accomplished by using colloidal gold (cAu) particles of different sizes. The size distribution of each cAu preparation, which may vary by up to 15%, is the principal factor that limits the number of labels which can be used simultaneously. Furthermore, there is no effective way to use multiple labeling for quantitative, high resolution EM studies. Such analysis requires the use of a single Fab antibody fragment conjugated to a single cAu particle in order to ensure that each particle corresponds to only one antigenic site. Whole antibody molecules cannot be used for quantitative analysis because they are at least divalent, and some, such as dimeric IgA or pentameric IgM, have even more antigen-binding sites. Consequently, it is impossible to deduce whether the presence of one whole antibody molecule corresponds to the presence of one, two, or more targets. Particle diameters ranging from 3 to 5nm are optimal for quantitation because one Fab fragment adsorbs to one particle, more than one Fab fragment may adsorb to a single particle larger than 5nm, and, when smaller than 3nm, several particles may bind to a single Fab fragment.

We are evaluating parameters apart from particle size variation to accomplish multiple labeling for both qualitative and quantitative analyses. One method relies on electron energy loss spectroscopy (EELS) to distinguish particles of several metallic compositions, including Au, Ag, Pt, Pd, Rh, and Ru. EELS is performed using a LEO 912 energy filtering transmission electron microscope (EFTEM) with an in-column Omega spectrometer.