Unconventional immuno double labelling by energy filtered transmission electron microscopy

A new method of immuno double labelling of biological specimens with a very high spatial resolution is presented. The advantage over conventional techniques is the possibility of using two very small labels leading to higher labelling efficiency, better penetration into the specimen and reduced steric hindrance between labels at closely spaced sites. The two labels are distinguished by their electron energy loss spectra using principal component analysis and then identified by comparison with an external standard using discriminant function analysis. The method is tested on samples of insect flight muscle labelled with 8 nm colloidal gold and silver and the statistical reliability of the classification is assessed. Extensions of the method are suggested and its potential for biological research is discussed.