Jem 1400

EMVs were visualized by TEM as previously reported (Yokoyama et al., 2017 (link)). Two microliters of the EMV samples were adsorbed onto hydrophilized carbon-coated copper grids and then treated twice with 2% uranyl acetate to negatively stain the samples. The TEM images were obtained with a JEM-1400 transmission electron microscope (JEOL, Ltd., Tokyo, Japan) at an accelerating voltage of 120 kV. Images were acquired using a charge-coupled device (CCD) camera (a built-in camera in the JEM-1400).
The intact structure of EMVs in buffer was visualized by cryo-electron microscopy (cryo-EM) at −175°C with a side-entry type cryo-specimen holder (Model 626.DH holder, Gatan Inc., Pleasanton, CA, United States). The samples were loaded onto microgrids (lacey carbon film, Ted Pella Inc., Redding, CA, United States) and treated using the same procedures as described previously (Yokoyama et al., 2017 (link)).

TEM imaging was performed on purified encapsulin, EncFtn, and EncFtn-Enc and apoferritin. Purified protein at 0.1 mg/ml concentration was spotted on glow-discharged 300 mesh carbon-coated copper grids and excess liquid wicked off with filter paper (Whatman, UK). The grids were washed with distilled water and blotted with filter paper three times before staining with 0.2% uranyl acetate, blotting and air-drying. Grids were imaged using a JEM1400 transmission electron microscope and images were collected with a Gatan CCD camera. Images were analyzed using ImageJ (NIH, Bethesda, MD) and size-distribution histograms were plotted using Prism 6 (GraphPad software). To observe iron mineral formation by TEM, protein samples at 8.5 µM concentration including EncFtn_sH, encapsulin, EncFtn-Enc and apoferritin were supplemented with acidic Fe(NH₄)₂(SO₄)₂ at their maximum iron loading ratio in room temperature for 1 hr. The mixtures were subjected to TEM analysis with or without uranyl acetate staining. TEM experiments without Fe loading were repeated three times, a representative set of images are presented here. Proteins loaded with Fe and imaged by TEM were from single preparation.

The samples for transmission electron microscopy (TEM) were prepared by incubating the confluent culture of melanoma cancer cells with the concentration of about 5 x 10⁶ in the presence of our tag structures with the cell to tag ratio of 1:5 in an 8-well Lab-Tek chambered cover-glass dish. After 18 h incubation period under standard conditions (37°C, 5% CO2, humidified), the samples were prepared for TEM sectioning. The preparation procedure [17 (link)] started with fixing the samples with fixative solution at room temperature for about 40 minutes. The solution was made of 0.1 M Sodium Cacodylate (pH 7.4) with the same percentages of 2% glutaraldehyde and 4% p-formaldehyde. The samples were then fixed again in 1% osmium tetroxide solution. After rinsing the samples with distilled water, the samples were stained with uranyl acetate. The samples then were dehydrated in ethanol solution. Finally the samples filtered and embedded in Epon for manual sectioning with a diamond knife. The sections were then imaged with JEOL JEM1400 images using a Gatan Orius model 832.