Dual-axis electron tomography of biological specimens: Extending the limits of specimen thickness with bright-field STEM imaging.

Experimental Electron tomography

Electron microscopy was performed in a Tecnai TF30 electron microscope (FEI Company, USA) operating with a Shottky field-emission gun and at an acceleration voltage of 300 kV. This instrument is equipped with a 2048 × 2048 pixel Ultrascan CCD camera situated after a Tridiem post-column imaging filter (Gatan, USA). In addition, this microscope is fitted with an in-column high-angle annular dark-field STEM detector (Fischione, USA) as well as an axial bright-field STEM detector (Gatan) located below the viewing screen. Samples for electron tomography consisted of plastic-embedded and post-stained sections of chemically fixed pancreatic beta cells. Particularly for STEM tomography of thick sections, introducing unnecessarily large amounts of heavy-metal stain should be avoided. This is because introducing too much stain would most probably not reveal any additional ultrastructural detail, but might lead to an increased broadening of the STEM probe and thus to a lower resolution towards the bottom sample surface. Three distinct electron tomography experiments were performed: (1) conventional ET (with zero-loss filtering) on 300-nm thick sections; (2) most-probable energy-loss tomography (MPEL) on 1-µm thick sections; and (3) axial bright-field STEM tomography also on 1-µm thick sections. For the STEM tomographic tilt series, the regions to be analyzed were first pre-irradiated and stabilized with a broad beam in transmission mode before switching to STEM. In all these experiments, we recorded dual-axis tilt series ranging from −60° to +60° in increments of 1.5° and with a total electron dose of 3.9 × 10 4 e/nm 2 . The pixel size of the images in the tilt series was 4 nm, which corresponded to specimen fields approximately 8.2 × 8.2 µm 2 in area. A STEM tomographic tilt series with pixel size of 1.4 nm and dose of 3.2 × 10 5 e/nm 2 was also recorded. MPEL tomography was carried out with a 30 eV energy window centered for each tilt angle at the maximum in the energy-loss spectrum. Axial STEM tomography was performed with a convergence semi-angle of the incident probe of 1.6 mrad and a BF STEM detector outer semi-angle of 9.3 mrad. For both MPEL and axial STEM tomography, the 1-µm-thick samples were flipped upside-down in addition to being rotated by 90° during acquisition of the second orthogonal tilt series. 3-D reconstruction of the tilt series was computed using the weighted back-projection algorithm included in the IMOD package (University of Colorado, USA) ( Kremer et al., 1996 ). All tomograms were generated with a binning factor of 2, and were post-processed with a median filter of size 3. Monte Carlo electron trajectory simulations A Monte Carlo simulation program was written to generate images of gold particles situated at the top and bottom surfaces of thick sections. In the simulations, the matrix consisted of a uniform mixture of carbon and osmium atoms to represent a stained biological specimen. The main workflow of the computer program, which is largely based on the algorithms outlined by Joy (1995) and Hovington et al. (1997) , has been described elsewhere ( Sousa et al., 2009 ). For samples such as these consisting of gold particles embedded in a matrix of uniform composition, the calculations must take into account the change in sample composition when an electron travels from matrix to particle and vice versa. We thus followed a scheme described by Gauvin et al. (1995) that enables the proper calculation of electron trajectories in this type of heterogeneous specimen. To simulate images of gold particles, we begin by performing Monte Carlo calculations along a line profile across the particles ( Fig. 1A ). Specifically, at each 1-nm pixel in the profile we compute the trajectories of 1–5 million electrons as they traverse the sample ( Fig. 1A ). We then use the net scattering angle of each electron at the exit surface of the sample to determine whether a STEM detector of given collection angle intercepts that electron trajectory. The line profile with the fraction of collected electrons ( Fig. 1B ) is then rotated in plane by 360° giving rise to an image of a gold particle ( Fig. 1C ). We finally multiply the resulting image by a given number of incident electrons and add Poisson noise ( Fig. 1D ). Fig. 1 shows a gold particle at the bottom of a thick sample with a slightly blurrier, more diffuse outline, in agreement with beam broadening of the incident beam in thick specimens. Using the above method, we simulated BF and HAADF STEM dual-axis tilt series for a specimen consisting of 10-nm gold particles located at the top and bottom surfaces of a 1-µm-thick section composed of 2 atomic % osmium in carbon. The series were calculated for tilt angles ranging from −60° to +60° in increments of 2° and for an electron dose of 10 4 incident electrons per nm 2 in each image. The outer semi-angle of the BF STEM detector was 10 mrad, and the HAADF detector had a collection angle of 40–200 mrad.

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