My current research involves the development of techniques for electron microscopy and their application to structural and cell biology. I am developing new digital imaging methods for high-resolution, scanning transmission electron microscopy (STEM) as well as sensitive new analytical methods based on electron energy loss spectroscopy (EELS). These techniques are being used to measure various quantities including molecular weights of large protein assemblies and ion concentrations in small cellular organelles. Application of STEM to structure determination of the cytoskeleton has yielded mass maps of the cytoplasmic motor protein, kinesin, attached to intact microtubules (collaboration with the Laboratory of Neurobiology, NINDS). These results show the first direct evidence that single kinesin molecules can cross-bridge microtubules providing a mechanism for microtubule sliding. I have also recently demonstrated that EELS can detect very small numbers of specific atoms bound to macromolecular assemblies. By combining STEM and EELS it has been possible to determine the quaternary structure and phosphorylation level of native neurofilaments from the squid giant axon. In these experiments STEM provides the total mass per unit length while EELS gives the number of phosphorylation sites associated with each protein subunit. By combining STEM with x-ray spectroscopy and by employing cryo-preparation techniques, I am also investigating calcium regulation in dendrites of mouse hippocampal neurons maintained under different physiological conditions in organotypic cultures (collaboration with LN, NINDS). I am using similar methods to study a wide range of other systems, including water regulation in amoebae and secretion in cells of the endocrine system.
Andrews, S.B., Gallant, P.E., Leapman, R.D., Schnapp, B.J., and Reese, T.S. Single kinesin molecules cross bridge microtubules in vitro; Proc. Nat. Acad. Sci. (USA) 90: 6503-6507, 1993.
Leapman, R.D., Hunt, J.A., Buchanan, R.A., and Andrews, S.B. Measurement of low calcium concentrations in cryosectioned cells by parallel-EELS mapping; Ultramicroscopy 49: 225-234, 1993.
Leapman, R.D., and Newbury, D.E. Trace elemental analysis at nanometer resolution by parallel electron energy loss spectroscopy; Anal. Chem. 65: 2409-2414, 1993.
Sun, S, Shi S, Hunt, J.A., Leapman, RD. Quantitative water mapping of cryosectioned cells by electron energy loss spectroscopy. J Microsc 1995; 177: 18-30.
Leapman, RD, Hunt, JA. Compositional imaging with electron energy loss spectroscopy. J Micros Soc Am 1995; 1(3): 93-108.
Leapman, RD, Sun, S. Cryo-electron energy loss spectroscopy: observations on vitrified hydrated specimens and radiation damage. Ultramicroscopy 1995; 59: 71-79.
Document date: 11-30-95