Osmium resistance of the mEos4 probes. a) Green mEos (and eGFP; 1 μM) fluorescence preservation in OsO₄ (incubation for 10 minutes before measurement). Excitation 480 nm, emission 515 nm, bandwidths 5 nm. Left, raw fluorescence values, all at same fluorimeter settings. Right, normalized data. b) Photoconversion of Eos (1 μM) from green to red in the presence of OsO₄ (incubation/photoconversion for 30 minutes before measurement). Excitation 550 nm, emission 580 nm, bandwidths 5 nm. Left, raw fluorescence values, all at same fluorimeter settings. Right, normalized data. See Methods for details of normalization. Mean and s.e.m. shown: n = 3. c) Fluorescence preservation of mitochondrially expressed mEos variants in fixed 3T3 cells, post-fixed with 1% OsO₄ and embedded in GMA. 60 nm GMA sections, 100 ms exposure time. Scale bars, 5 μm. See Methods for details of background subtraction. d) TEM images of 3T3 cells expressing mitochondrial mEos4a. Cells were post-fixed in 1% OsO₄ and embedded in GMA. EM images are of 40 nm-thick sections. Top: montage reconstruction of whole cell from tiled images. Bottom left: zoomed TEM image, showing mitochondria (M- note cristae), rough endoplasmic reticulum (RER- note ribosomes), and nuclear envelope (N- note bilayer). Bottom right: fluorescence image of a 60 nm-thick section from same cell, cut after the 40 nm-thick section used for TEM. Scale bars, 5 μm.

Correlative PALM and TEM imaging using the consecutive-section approach. a) Schematic of consecutive-section approach using conventional chemical fixation. glut = glutaraldehyde, UA = uranyl acetate, Pb = Sato’s triple lead. b) Top right: A montage of TEM images of a 60 nm GMA section of a 3T3 cell expressing mEos4b-lamin A. Cells were postfixed with 1% OsO₄ and embedded in GMA. Middle right: PALM image of a 500 nm section cut consecutively to the 60 nm section, showing localization of mEos4b-lamin A. Bottom right: manual overlay of PALM and TEM images. Left: zoomed images of boxed areas, showing mitochondrial cristae, ribosomes and nuclear bilayer. Scale bars, 500 nm.

Correlative PALM and TEM/SEM imaging using the same-section approach. a) Schematic of same-section approach using HPF-FS. glut = glutaraldehyde, UA = uranyl acetate, Pb = Sato’s triple lead. b) Left: Overlaid PALM (red) and TEM images taken on 60 nm GMA section of 3T3 cells expressing mEos4b-Lamin A. 0.5% OsO₄. Right: Zoomed image of the marked area. Scale bars, 1 μm. Yellow arrows indicate fluorescent, electron-dense gold nanoparticles used for fiducial registration. c) CLEM images of 60 nm GMA section of 3T3 cells expressing mitochondrially targeted mEos4a. 0.5% OsO₄. Top: whole cell. Middle: PALM, merge and TEM images of boxed area. Arrow indicates a mitochondrial cristae fold. Bottom: Overlaid PALM and SEM images of the same section. Scale bars, 1 μm. N = nucleus, M = mitochondrion, RER = rough endoplasmic reticulum.