Tomographic Collection of Block-Based Sparse STEM Images: Practical Implementation and Impact on the Quality of the 3D Reconstructed Volume

Abstract

The reduction of the electron dose in electron tomography of biological samples is of high significance to diminish radiation damages. Simulations have shown that sparse data collection can perform efficient electron dose reduction. Frameworks based on compressive-sensing or inpainting algorithms have been proposed to accurately reconstruct missing information in sparse data. The present work proposes a practical implementation to perform tomographic collection of block-based sparse images in scanning transmission electron microscopy. The method has been applied on sections of chemically-fixed and resin-embedded Trypanosoma brucei cells. There are 3D reconstructions obtained from various amounts of downsampling, which are compared and eventually the limits of electron dose reduction using this method are explored.

Keywords: Trypanosoma brucei; biological samples; electron tomography (ET); inpainting reconstruction; scanning transmission electron microscopy (STEM); sparse imaging.

Figure 1

Collection of a sparse scanning transmission electron microscopy (STEM) image, inpainting reconstruction and comparison with ground truth. (A) Sparse STEM image downsampled to 12.5% using 512 blocks of 16 × 4 pixels. The white background corresponds to uncollected pixels. (B) Sparse image A after inpainting reconstruction. (C) Ground truth fully-collected STEM image of the very same ROI on the grid. Scale bar is 200 nm. The three subfigures have the same scale.

Figure 2

Block-based sparse imaging of an FeO nanoparticle at very high magnification. (A) Overlapping blocks (64 × 64 pixels) partially covering the FeO nanoparticle, white areas correspond to uncollected zones. (B) Zoom-in on a particular location of the nanoparticle (corresponding to the black box in (A) where the crystalline pattern is visible on several overlapping blocks. Thick grey lines drawn along the crystalline lattice highlight the alignment of the atom columns over several blocks.

Figure 3

Strategy developed to collect sparse STEM images in a tomography workflow. The region of interest (in red) and the tracking and focusing areas (in dark and light pink respectively) are aligned with the tilt axis (in dark blue).

Figure 4

Comparison of the reference and sparse reconstructions. The reference reconstruction (upper row) has been computed using fully-collected images whereas the sparse reconstruction (lower row) has been computed using sparse images containing 6.25% of the pixels. For each reconstructed volume, three Z-slices were extracted: (i) below the sample (left column, Z-slices 50), (ii) at the level of the Au/Pd coating (center column, Z-slices 150) and (iii) above the sample at the level of the latex spheres (right column, Z-slices 250). The center area of Z-slices 150 (black boxes) has been zoomed-in to better visualize the details on the Au/Pd deposits. Reconstructions artifacts and latex spheres are indicated using white asterisks and white arrows, respectively. The scale bar is 250 nm. The six subfigures have the same scale.

Figure 5

Comparison of 3D volumes reconstructed from sparse images collected at different downsampling values. The central Z-slice of each reconstruction is displayed. The value in the top left corner of each image corresponds to the downsampling value. Inserts in the bottom right corner are zoom-ins of the cell membrane. Several cellular structures are pointed out: the cytoplasm (Cyt), microtubules of the flagellum (Mt), the cell membrane (Mb), the nucleus (N) and the nuclear envelope (Ne). The scale bar is 400 nm. The eight subfigures have the same scale.

Figure 6

Image quality descriptors (IQD) values of sparse reconstructions depending on the amount of downsampling. (A–C) Plots of the H, C_m and C_rms descriptors computed at each Z slice of three reconstructions (3.125%, 12.5% and 25%), respectively. Thick vertical lines mark the location of the resin section in the reconstructed volumes (between Z-slices 50 and 250). (D) IQD ratios (experimental values and fitted curves) plotted by increasing order of downsampling.