Jem 2100f field emission electron microscope

To visualize the morphology of P-EV, D-EV, and BD-EV, we fixed samples with 0.5% glutaraldehyde solution overnight for TEM. Subsequently, we centrifuged the samples at 13,000g for 3 min and dehydrated the pellets in absolute ethanol for 10 min and then dropped them onto formvar-carbon–coated copper grids (Ted Pella Inc., Redding, CA, USA). We stained the samples with 1% phosphotungstic acid for 1 min and washed them with absolute ethanol. We stored the grids in a desiccator before analysis and then observed the samples on a JEM-2100F field emission electron microscope (JEOL Ltd., Japan). For cryo-TEM analysis, P-EV, D-EV, and BD-EV were collected on lacey carbon grid (Electron Microscopy Sciences, Hatfield, PA, USA). The grids were stored in liquid nitrogen and then transferred to a cryo-specimen holder and maintained at −180°C. Images were collected on the Tecnai Twin transmission electron microscope operating at 200 kV.

Transmission electron microscopy (TEM) was carried out on a JEM-2100 electron microscope, operating at an acceleration voltage of 100 kV. Scanning transmission electron microscopy (STEM) was carried out on a JEM-2100F field-emission electron microscope, operating at an acceleration voltage of 200 kV. SEM images are captured with a JEOL Field Emission SEM. The concentration of the precursor PQDs solution was calibrated with reference to the mass of lead measured by an inductive coupled plasma optical emission spectrometer (Agilent 710). X-ray diffraction (XRD) patterns were acquired with a Bruker AXS D2 phaser X-ray diffractometer equipped with a Cu Kα radiation source (λ = 1.54 Å) at 40 kV and 30 mA. Infrared absorption spectroscopy was carried out on a BRUKER Vertex Fourier Transform Infrared spectrometer. UPS and XPS measurements were carried out on a VG ESCALAB 220i-XL surface analysis system equipped with a He discharge lamp (hν = 21.2 eV) and a monochromatic Al–Kα X-ray gun (hν = 1486.6 eV).

An experimental sample of a multivesicular body (MVB) (Figure 2) was prepared by high-pressure freezing and freeze substitution as in [24] (link). The sample was used to study breakages in the membranes of virus-induced MVB and intraluminal vesicles suggesting genome release to the cytoplasm. Also, two specific targets, echovirus 1 and α2β1-integrin receptor, were labeled with gold particles of 14nm and 6 nm of size, respectively, to study the localization and distribution of these targets in the virus-induced MVB. A series of single axis TEM images were obtained with the JEM-2100F Field Emission Electron Microscope (JEOL Ltd., Tokyo, Japan) at a voltage of 200 kV. It comprises 124 images of the MVB taken at an interval of approximately 1° in the angular range [−65°, +58°], with the magnification of 10k. Before reconstruction, the projection images were aligned by cross-correlation using the 14nm gold particles as markers. The dimensions of the original projection images were 4096×4096 which were cropped to size 958×712 pixels used for reconstruction. The pixel size was 1nm.
The projection data and reconstructions of all datasets are available at our supplementary data site: http://pioms.ucdavis.edu/pone/smap-em.