Analysis of directly frozen macromolecules and tissues in the field-emission STEM.
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 (approximately 10(3) e/nm2) 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 micron2. 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-nm2 regions within Purkinje cell dendrites with an uncertainty of +/- 2 mmol/kg dry weight, equivalent to +/- 12 atoms.
Leapman
R D
RD
Biomedical Engineering and Instrumentation Program, NCRR, National Institutes of Health, Bethesda, MD 20892.
Andrews
S B
SB
D000818:Animals; D001921:Brain / Q000648:ultrastructure; D002525:Cerebellar Cortex / Q000648:ultrastructure; D003712:Dendrites / Q000648:ultrastructure; D004577:Electron Probe Microanalysis; D005612:Freeze Drying; D005629:Frozen Sections; D006433:Hemocyanins / Q000648:ultrastructure; D051379:Mice; D008855:Microscopy, Electron, Scanning; D008870:Microtubules / Q000648:ultrastructure; D008974:Mollusca; D015388:Organelles / Q000648:ultrastructure; D011689:Purkinje Cells / Q000648:ultrastructure; D013053:Spectrophotometry; D014027:Tobacco Mosaic Virus / Q000648:ultrastructure
D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin