A new class of electron microscopy (E.M.) labels will be developed that will have 5-10 times better resolution than those currently available. This class promises to be the next generation of high resolution electron microscopic labels and should have an extensive impact on molecular biology, since it will make possible direct domainal mapping of single biomolecules and macromolecular assemblies. Preliminary results have already demonstrated labeling of the biotin binding site on avidin with an undecagold cluster only about 10 Angstrom from the binding site. Several other proteins have also been specifically labeled. Another 11-tungsten atom complex has been made with a long alkyl chain which has been found to insert into biomembranes and can be seen in synthetic lipid vesicles. Stable complexes of 11-12 tungsten or gold atoms with an organic outer region have been described. Both have a dense 8 Angstrom core. We have studied the stability and visibility of these in the Scanning Transmission Electron Microscope (STEM) and have found them suitable for use as labels. They are easily visualized, do not move about as do single heavy atom reagents, and do not decompose at moderate doses. The organic region can be chemically modified to produce specific functionalities so as to link these complexes covalently to sulfhydryl and amino groups in proteins or diol groups in carbohydrates. Other molecules such as nucleotides, toxins and biotin may be similarly labeled. We will develop these labels further and use them to provide unique answers to the following six biological questions: (1) What is the structure of haptoglobin? (2) What is the domainal structure of fibronectin? Where is the glycosolyated region and where are specific sulfhydryls? (3) What is the morphology of the acetylcholine receptor? (4) How do lipids move in living cells? (5) Can a high resolution immunospecific label be derived by coupling these labels to Fab fragments? (6) What is the high resolution (5-10 Angstrom) structure of glutamine synthetase? Pertinent collaborations to provide broad expertise for each of these problems have been established, and this together with the success of preliminary experiments indicate that rapid development of this new technology is feasible.
