Determination of the inelastic mean free path of electrons in vitrified ice layers for on‐line thickness measurements by zero‐loss imaging

Abstract

SummaryThe inelastic mean free path of 120 keV electrons in vitrified ice layers has been determined in an energy-filtering TEM. From the ratio of the unfiltered and zero-loss-filtered image intensities recorded with a slow-scan CCD camera, the relative sample thickness t/L can be calculated. For calibration, the geometric ice thickness was measured by imaging a tilted view of a cylindrical hole which had been burnt into the ice layer. The total inelastic mean free path was found to be 161 nm, and the partial inelastic mean free path for an acceptance angle of 4·2 mrad was 232 nm. These results were built into a standard protocol for use in cryo-electron microscopy allowing on-line measurements of local ice-layer thicknesses by zero-loss-filtered/unfiltered imaging.

Electron energy filtering significantly improves amplitude contrast of frozen-hydrated protein at 300 kV

“…First, the mean free path, K for inelastically scattered electrons at 300 kV was measured to be $4000 Å from a tilted view of a hole in thick vitreous ice imaged with and without energy filtering (Angert et al, 1996). Grimm et al (1996) and Feja and Aebi (1999) measured K = 2000-2500 Å at 120 kV, which can be converted into 3500-4400 Å at 300 kV by multiplying it by the factor, (b 300 /b 120 ) 2 = (0.7765/0.5867) 2 , where b 120 and b 300 are the ratios of the electron velocity at 120 and 300 kV to that of light, respectively. Our estimate seems to be in good agreement with their values (Grimm et al, 1996;Feja and Aebi, 1999).…”

Observations on the behavior of vitreous ice at ∼82 and∼12 K

“…With this geometry, the depth of the ice is directly measurable as the projected length of the tunnel (Fig. 5) (Feja and Aebi, 1999). An important advantage of this method is that the measurement of ice depth is independent of tunnel diameter.…”

“…Low salt solutions of Escherichia coli pyruvate dehydrogenase multienzyme complex, Methanosarcina thermophila 20S proteasome (Calbiochem), and growing cultures of Mesoplasma Xorum behaved similarly. Tunnels were drilled and imaged similarly to the protocols of Feja and Aebi (1999). BrieXy, at 82 K, grids were scanned at low dose and magniWcation to locate areas of ice representative of those used for tomography data collection.…”

Changes in Nucleoporin Domain Topology in Response to Chemical Effectors

“…The microscope and the camera were controlled by a VIPS-1000 (Tietz Video and Image Processing Systems, Gauting, Germany). The sample thickness was determined on-line from a zeroloss filtered/unfiltered image pair by a macro routine applying the log/ratio method, 46 combined with the experimentally determined partial inelastic mean free electron path 47 . All images were recorded at 120 kV acceleration voltage.…”