Electron probe x-ray analysis of single ferritin molecules.

Abstract

Single molecules and groups of two or three ferritin molecules were subjected to electron probe x-ray microanalysis in a transmission electron microscope equipped with a liquid nitrogen cooled stage. Significant Fe Ka peaks were generated during 100-sec counts when single ferritin molecules were excited with a probe current of 0.35 nA/60 nm spot, less than the maximal current available in a thermionic gun. There was a linear relationship between the number of ferritin molecules analyzed and count rates. The experimental results are compared to the theoretically calculated Fe KI yields and to the results of Isaacson and Johnson [(1975) To realize the optimal minimal detectable mass attainable with this system it is essential to reduce to a minimum the background radiation (x-ray continuum) generated by extraneous sources. Contamination, the major source of extraneous continuum with small diameter (50 nm or less) probes in our system, can be eliminated or at least substantially reduced by operating at low temperatures (10). Therefore, the suspensions of ferritin molecules placed on thin (about 4 nm) carbon films were analyzed at -110°, using a Philips liquid nitrogen cooled specimen stage modified to permit the exit of generated x-rays towards the detector. Analysis at even lower temperatures was not feasible due to icing of the specimen. The stage drift of 1.5 nm/sec (specified by the manufacturer) is evident in the electron micrographs obtained and required continuous vigilance by the operator for maintaining the ferritin target in the center of the probe. RESULTS Electron micrographs of a single ferritin molecule before and after electron probe x-ray microanalysis with 60 nm diameter probe for 100 sec and of a group of three molecules are shown in Fig. 1. The hole formed in the carbon support is evidence of the continuous mass loss under the electron beam even at this low temperature. It may be possible to reduce or eliminate mass loss, at comparable current densities, by operating at liquid He temperatures (11) or by using lower current densities (ref. 12; Shuman and Somlyo, unpublished observations). The x-ray spectra obtained from a single ferritin molecule and from a group of three are shown in Fig. 2, and the results of analyses of 1, 2, and 3 ferritin molecules are shown in Table 1. The latter shows the linear increase in Fe Kaf count rate with the number of ferritin molecules analyzed.The theoretical count rate can be estimated from the x-ray production cross section, absolute detector efficiency, and measured probe current. The data in Table 1 were obtained with a measured probe current of 0.35 nA in a 60 nm diameter probe. The average current density is J = 6.2 A/cm2 and, assuming a gaussian probe, the current density at the center of the probe is Jo = 12.5 A/cm2. The x-ray production cross section is composed of two factors, the ionization cross section q and the x-ray yield w. For Fe K x-rays, w = 0.347 and q = 3.95 X 10-22 cm2 from Durup and Platzman's expression (7, 13) for ionization cros...

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The Electron Microscopy of Catalysts

“…The resolution of the area to be analysed will be determined by the spot size, and is increased by the electron spread within the specimen to about twice the probe area for a 100 nm thick foil [70] or a thin specimen [71]. For a conventional STEM with a spatial resolution of about 20 nm, Schuman and Somlyo [72] estimate the sensitivity (minimum detectable volume) for Fe to be about 9 x 10-20 g (10 3 atoms) by detecting X-rays. In a high resolution dedicated system, X-ray analysis of a Pt-Pd catalyst dispersed on AI 2 0 3 was found to have a sensitivity of about 100 atoms at a resolution of 0.3 nm, and an expected lower limit of detection of about 30 atoms [73].…”

Energy-filtered transmission electron microscopy of ferritin.

“…Relatively high (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) nm) resolution images showing the distribution of some constituent elements have been obtained by imaging with characteristic x-rays produced by a scanning electron beam generated with a highbrightness field emission source (1-3). However, x-ray mapping is ultimately limited by the low-fluorescence yield ofx-ray production and low-collection efficiency of x-ray detectors.…”