This research group will develop methods for mapping the composition of cell membranes and organelles at 2nm-3nm resolution, with a 200kV field emission gun ?FEG!-equipped electron microscope interfaced with a parallel electron energy loss spectrometer (parallel EELS) and an X-ray detector. Physicists and biologists will collaborate to develop the special techniques required for quantitative imaging of low-concentrations of elements (e.g., Ca, P, Gd) in biological cryosections. The major obstacle to EELS, the large background due to the thickness of cryosections, will be overcome by the use of 200kV excitation and through theoretical and experimental development of optimal methods of spectral collection and data analysis. EELS spectra collected at each scanned pixel will be multiple least squares fitted with algorithms that take into account the contributions of plural plasmon scattering and, in the higher loss region, the carbon energy loss and its fine structure, and L-edges (e.g. potassium)to the EELS background. Electron optical components will be designed and added to an existing parallel EELS system to correct the gain variations that reduce detection sensitivities. High resolution compositional mapping of Ca-bound to membranes or sequestered in organelles and of phosphorylation sites will have broad biological applications and will be explored.
