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Analysis of directly frozen macromolecules and tissues in the field‐emission STEM
Author(s) -
Leapman Richard D.,
Andrews S. Brian
Publication year - 1991
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1991.tb03070.x
Subject(s) - microanalysis , scanning transmission electron microscopy , macromolecule , chemistry , electron microscope , transmission electron microscopy , analytical chemistry (journal) , resolution (logic) , elemental analysis , spectroscopy , biological specimen , materials science , nanotechnology , optics , biochemistry , chromatography , physics , organic chemistry , quantum mechanics , artificial intelligence , computer science
SUMMARY A VG Microscopes HB501 field‐emission high‐resolution scanning transmission electron microscope (STEM) was used to image and analyse rapidly frozen, isolated macromolecules and small organelles in tissue cryosections. Dark‐field images were obtained from frozen‐hydrated microtubules demonstrating that sufficient contrast is available to reveal structural information. The samples were subsequently freeze‐dried in the STEM and low‐dose (≅ 10 3 e/nm 2 ) dark‐field mass maps were recorded with single electron sensitivity. Elemental analysis of individual macromolecules was achievable at high dose using parallel‐detection electron energy‐loss spectroscopy, albeit with some structural degradation. Detection of copper (320 atoms) in di‐decameric haemocyanin molecules was easily within the limits of sensitivity. Elemental analysis of hydrated cryosections is limited by radiation damage to a resolution of approximately 1 μm 2 . For freeze‐dried sections, however, the high probe current and stable cold stage of the HB501 STEM allow energy‐dispersive X‐ray (EDX) microanalysis of low elemental concentrations in highly localized subcellular volumes. EDX spectra from cryosections of cerebellar cortex show that a 100‐s analysis time is sufficient to quantify the calcium content of 400‐nm 2 regions within Purkinje cell dendrites with an uncertainity of ± 2 mmol/kg dry weight, equivalent to ± 12 atoms.