Eponate 12

Tissues were harvested, cleaned by flushing with cold PBS, immediately placed into EM-fixative (2 % Gluteraldehyde, 2 % Paraformaldehyde, 0.1 M Sodium Cacodylate, 3 mM MgCI₂, pH to 7.2 with NaOH), and incubated overnight at 4 °C. Fixed tissue was washed with EM-wash-1 (0.1 M Cacodylate, 3 mM MgCI₂, 3% sucrose pH 7.2), 3 × 15 minutes, then permeated with 1 % osmium tetraoxide for 1 hour. Permeated tissues were washed twice for 5 minutes in EM-wash-1, and washed three times for 15 minutes in EM-wash-2 (0.1 M maleic di-sodium salt, 3 % sucrose, adjust to pH 6.2). Tissues were then incubated in 2 % uranyl acetate for 1 hour at room temperature, passed through an ascending graded Ethanol series from 30 – 100 % ethanol, and washed three times for 15 minutes in PO-solution (1:1 ethanol:propylene-oxide). Propylene-oxide equilibrated tissues were incubated overnight in Eponate-12 (Ted Pella, Redding CA) plastic solution without catalyst, then washed three times for 2 hours in Eponate-12 with DMP-30 catalyst. Tissues were cured covered in Eponate-12 for 2 days at 60 degrees, then sectioned to 70 nm using a diamond microtome. EM images were collected using a Hitachi 7600 transmission electron microscope operated at 80.0 kV. Images were acquired at 25,000 to 75,000 × direct magnification, corresponding to 0.002900 μm/pixel to 0.002416 μm/pixel, respectively.

Constructs driving mitochondrial mEos2, mEos4a or mEos4b expression (same as those used in Fig. 3) were transfected into 3T3 cells and allowed to express for 24 hours. After mEos expression, cells were primary-fixed with 4% PFA / 0.2% glutaraldehyde and secondary-fixed with 1% OsO₄, dehydrated in 100% ethanol and finally resin infiltrated with EPON (Eponate 12 ^TM, Ted Pella). 100 nm sections were then cut on an ultramicrotome and imaged on an epifluorescence microscope. Images were acquired on a Nikon Eclipse Ti Inverted microscope with a CFI Plan Apo Lambda 60x oil-immersion objective, numerical aperture 1.4. Light source was a SPECTRA X light engine (Lumencor), with excitation centered around 470 nm for the green channel and 555 nm for the red channel. Emission light was collected with 515/30 nm and 595/40 nm bandpass filters, respectively. Through the microscope objective, all six images looked completely black. To show that cells were present and in focus in the fields of view, 16-bit images were taken on a Zyla 4.2 sCMOS camera (Andor) and adjusted in ImageJ to artificially enhance contrast. Histogram adjustment to show very dim fluorescent pixels clearly reveals section edges (visible at top of each field of view), as well as 3T3 cells (the dark excluded regions). Results are shown in Supplementary Fig. 9.

P. acnes ATCC 6919 at 3x10⁸ CFU/mL were incubated untreated or with 25.7μM of Pentobra, pen peptide or tobramycin for 3 hours, washed twice with PBS, and resuspended in PBS with 2% glutaraldehyde. The bacteria were fixed for 5 minutes with 0.05% OsO₄, dehydrated in graded ethanol, and then embedded in Eponate 12 (Ted Pella). 60–70nm slices were cut with a Reichert-Jung Ultracut E ultramicrotome, which were picked up on formvar coated copper grids. Samples were stained with uranyl acetate and Reynolds lead citrate and visualized at 80 kV on a JEOL 100CX electron microscope.