Research ArticleCELL BIOLOGY

The role of electron irradiation history in liquid cell transmission electron microscopy

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Science Advances  20 Apr 2018:
Vol. 4, no. 4, eaaq1202
DOI: 10.1126/sciadv.aaq1202
  • Fig. 1 Overview of new multiwindow devices for improved LC-TEM.

    (A) Standard commercially available nanofluidic device with a single 200 × 50–μm window centered on the device (black arrow). (B) Schematic of available imaging area when devices from (A) are assembled with windows oriented perpendicular to each other. (C) Custom nanofluidic device with five windows of 50 μm width (black arrow). Gold spacer material can be seen on the devices (white arrow). (D) Schematic of available imaging area when devices from (B) are assembled with windows oriented perpendicular to each other, where a 5 × 5 grid of windows is created. (E) Nanofluidic devices from (C) that have been patterned with Au grid bars (orange arrow) crossing the windows for use as focusing aids. (F) Schematic of available imaging area when devices from (E) are assembled with windows oriented perpendicular to each other. Grid bars are shown bisecting the center of each window for focusing applications. Scale bar represents 1 mm for (A), (C), and (E), whereas (B), (D), and (F) are illustrations of window overlaps not shown to scale.

  • Fig. 2 Cumulative electron flux effects on silver nanoparticle growth.

    (A) Individual frames showing growth of silver nanoparticles by reducing a 0.1 mM AgNO3 aqueous precursor solution with the electron beam. LC-STEM frames taken from video captures 2, 6, and 12 are compared across time. The scan area was 1024 × 1024 pixels, the dwell time was 3 μs, the beam current was 5.85 pA, and the pixel size was 1.47 nm, resulting in an electron flux of 0.51 e2 per scan. (B) Diameter of particles from all videos plotted over the course of the 200 scan exposures. Only particles in focus, contained entirely within the viewing area, and nonoverlapping are used in the growth rate analysis. (C) Total number of particles nucleated in each capture video, where particles on both windows (in focus and out of focus) are counted. (D) Number of particles nucleated for a 0.1 mM AgNO3 precursor solution (blue squares) and a 0.5 mM AgNO3 precursor solution (orange circles) without flow of fresh precursor during imaging. Each experiment was performed in an adjacent window for 200 STEM scans. The electron flux was 0.51 e2 per scan and 101.4 e2 per video. (E) Number of particles nucleated for a 0.1 mM AgNO3 precursor solution (blue squares) and a 0.5 mM AgNO3 precursor solution (orange circles) while flowing fresh precursor solution at a rate of 0.5 μl/min. The electron flux is equivalent to that in (D). Scale bar, 1 μm (A).

  • Fig. 3 Low-dose image acquisition empowered by patterned in situ focusing aides.

    (A) Magnified view of five-window chip with grid bar patterning. (B) In situ image of grid bars over a window once assembled in a commercial LC-TEM holder. (C) Demonstration of low-dose capability allowed by using grid bars as focusing aids. Optimal focus is found on grid bars followed by imaging the sample area in a single shot with minimal previous electron exposure. Scale bars, 500 μm (A), 20 μm (B), 10 μm (C, Search), 500 nm (C, Focus), and 500 nm (C, Acquire).

  • Fig. 4 Cumulative electron flux effects on biological cells.

    (A) Damage series of C. metallidurans, imaged using a low-dose imaging regime at an electron flux of 1 e2 per acquisition. Time between each subsequent acquisition was 20 min, where the beam was blanked during that time. Flux labeled is total cumulative flux for the frame. Total field of view, 3.5 μm × 3.5 μm. (B) Overlay of cell boundary from initial image (A) (1 e2) highlighting decreasing size and morphological changes of cells as a function of increasing cumulative electron flux. (C) Magnification (×10) of region depicted in (A), where the edge of the cells (yellow arrows at top and bottom of each frame) can be seen moving relative to the metal cluster with increasing electron flux.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/4/eaaq1202/DC1

    fig. S1. Multiwindow chips compatible with commercial LC-TEM holders.

    fig. S2. Additional experimental data from Fig. 2.

    fig. S3. Imaging path and thickness of multiwindow devices for no flow versus flow.

    fig. S4. Low-dose imaging of biological samples.

    fig. S5. Improved sampling of nonuniform samples with multiwindow areas.

    fig. S6. Illustration of microfabrication workflow.

    Supplementary protocol for fabrication of multiwindow devices

    movie S1. Demonstration of in situ liquid cell “low-dose” imaging.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Multiwindow chips compatible with commercial LC-TEM holders.
    • fig. S2. Additional experimental data from Fig. 2.
    • fig. S3. Imaging path and thickness of multiwindow devices for no flow versus flow.
    • fig. S4. Low-dose imaging of biological samples.
    • fig. S5. Improved sampling of nonuniform samples with multiwindow areas.
    • fig. S6. Illustration of microfabrication workflow.
    • Supplementary protocol for fabrication of multiwindow devices
    • Legend for movie S1

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mp4 format). Demonstration of in situ liquid cell “low-dose” imaging.

    Files in this Data Supplement:

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