Characterization of biological macromolecules by combined mass mapping and electron energy-loss spectroscopy.
The combination of scanning transmission electron microscopy (STEM) and parallel-detection energy-loss spectroscopy (EELS) was used to detect specific bound elements within macromolecules and macromolecular assemblies prepared by direct freezing. After cryotransferring and freeze-drying in situ, samples were re-cooled to liquid nitrogen temperature and low-dose (about 10(3) e/nm2) digital dark-field images were obtained with single-electron sensitivity using a beam energy of approximately 100 keV and a probe current of approximately 5 pA. These maps provided a means of characterizing the molecular weights of the structures at low dose. The probe current was subsequently increased to about 5 nA in order to perform elemental analysis. The 320 copper atoms in a keyhole limpet haemocyanin molecule (mol.wt = 8 MDa) were detected with a sensitivity of +/- 30 atoms in an acquisition time of 200 s. Phosphorus was detected in an approximately 10-nm length of single-stranded RNA contained in a tobacco mosaic virus particle (mol.wt = 130 kDa/nm) with a sensitivity of +/- 25 atoms. Near single-atom sensitivity was achieved for the detection of iron in one haemoglobin molecule (mol.wt = 65 kDa, containing four Fe atoms). Such detection limits are only feasible if special processing methods are employed, as is demonstrated by the use of the second-difference acquisition technique and multiple least-squares fitting of reference spectra. Moreover, an extremely high electron dose (about 10(10) e/nm2) is required resulting in mass loss that may be attributable to 'knock-on' radiation damage.
Leapman
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RD
Biomedical Engineering and Instrumentation Program, NCRR, National Institutes of Health, Bethesda, MD 20892.
Andrews
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SB
D000818:Animals; D005293:Ferritins / Q000737:chemistry; D006433:Hemocyanins / Q000737:chemistry* / Q000648:ultrastructure; D006454:Hemoglobins / Q000737:chemistry* / Q000648:ultrastructure; D007501:Iron / Q000032:analysis; D008855:Microscopy, Electron, Scanning; D008974:Mollusca; D010758:Phosphorus / Q000032:analysis*; D013057:Spectrum Analysis; D014027:Tobacco Mosaic Virus / Q000737:chemistry* / Q000648:ultrastructure
D006454:Hemoglobins; D010758:Phosphorus; D005293:Ferritins; D006433:Hemocyanins; D007501:Iron