The scanning transmission electron microscope (STEM) has made significant contributions to structural biology by providing accurate determinations of the molecular masses of large protein assemblies that have arbitrary shapes and sizes. Nevertheless, STEM mass mapping has been implemented in very few laboratories, most of which have employed cold-field emission gun (FEG) electron sources operating at acceleration voltages of 100 kV and lower. Here we show that a 300 kV commercial transmission electron microscope (TEM) equipped with a thermally assisted Shottky FEG can provide accurate STEM mass measurements of disease-related amyloid fibrils. Morphology of aggregation intermediates and polymorphism of amyloid fibrils as well as aggregation kinetics of the Arctic mutant of the 40-amino acid Alzheimer's amyloid beta peptide (E22G), were explored using electron microscopy together with atomic force and circular dichroism measurements, which enabled the kinetics of aggregate formation to be correlated with the secondary structure of amyloid beta peptide. Scanning transmission electron microscopy (STEM) was employed to explore the fibril morphology and to measure mass-per-length of different fibril polymorphs. Characteristic dimensions of both intermediate aggregates and fibrils were measured during the aggregation process. Critical concentrations of fibrillization of both arctic mutation and wild type peptide were studied. At the end of a lag-period with almost indistinguishable changes in morphology of diffuse aggregates, the arctic mutation peptide underwent an abrupt transition to compact spherical aggregates with a highly distinct and homogeneous morphology. Aggregation then proceeded with a rapid growth of amyloid fibrils to produce a variety of morphologies, while the spherical aggregates eventually disappeared. It was found that amyloid fibrils exhibited a variety of polymorphs. At least four fibril polymorphs were identified by TEM and STEM and their mass-per-length statistics suggested supramolecular structures with two, four and six beta-sheet laminae. Real time aggregation dynamics of the fibrils revealed that different fibril morphologies not only co-exist in the same sample, but they also grow or dissociate at different rates. These results suggest an alternative pathway of fibrillogenesis for full-length Alzheimer's peptides with small and structurally ordered transient spherical aggregates as immediate precursors of amyloid fibrils. These findings could be significant in studying aggregation kinetics, morphology and interaction of peptide with inhibitors and phospholipid membranes of several other mutations of the Alzheimer's beta amyloid peptide. To elucidate how the components of an extremely simple cell are spatially organized, STEM mass mapping has been applied to analyze the molecular inventory of Spiroplasma melliferum, a wall-less free-living bacterium with an exceedingly small genome and dynamic helical geometry. Together with other biophysical measurements, STEM determination of the mass-per-length of whole Spiroplasma, the mass-per-length of the cell's cytoskelatal motor assemblies, and the mass-per-area of its membrane fractions, provide a general framework for a minimal inventory and arrangement of major cellular components that are needed for a minimal viable cell.

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National Institute of Biomedical Imaging and Bioengineering
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Trachtenberg, Shlomo; Schuck, Peter; Phillips, Terry M et al. (2014) A structural framework for a near-minimal form of life: mass and compositional analysis of the helical mollicute Spiroplasma melliferum BC3. PLoS One 9:e87921
Sousa, Alioscka A; Leapman, Richard D (2013) Mass mapping of amyloid fibrils in the electron microscope using STEM imaging. Methods Mol Biol 950:195-207
Sarin, Hemant; Kanevsky, Ariel S; Wu, Haitao et al. (2009) Physiologic upper limit of pore size in the blood-tumor barrier of malignant solid tumors. J Transl Med 7:51
Sousa, A A; Aronova, M A; Wu, H et al. (2009) Determining molecular mass distributions and compositions of functionalized dendrimer nanoparticles. Nanomedicine (Lond) 4:391-9
Sarin, Hemant; Kanevsky, Ariel S; Wu, Haitao et al. (2008) Effective transvascular delivery of nanoparticles across the blood-brain tumor barrier into malignant glioma cells. J Transl Med 6:80