Fig. 5: Ultrastructural in situ characterization of p62-encapsulated lipid droplets.

a Representative scanning electron microscopy image of a lamella generated from an EM grid grown with RPE1 cells. b Correlated fluorescence image of the corresponding lamella recorded with a reflected light channel and fluorescent channels of 595 nm for mCherry-p62 and 515 nm for autofluorescence. c Super-resolution cryo-confocal fluorescence image recorded at 561 nm for mCherry-p62 showed three p62-positive structures resolving a circular fluorescence signal as displayed in three corresponding intensity profiles below. The color is a heatmap, denoting signal intensity (in arb. units) (d) Left. Representative tomographic gray-scale z-slice through the homogeneous density sphere surrounded by a dark high-contrast layer with an enwrapped single membrane. Scale bar 100 nm. Right. Segmented and labeled structure superimposed on gray-scale z-slice, light red: lipid droplet with red: droplet surface, yellow: dark and high-contrast surrounding p62 layer and blue: single lipid membrane (right). e Box plot indicates a well-defined lipid droplet diameter of about 520 nm. Observed measurements are shown as diamonds, the central bar represents the median value, while the gray box corresponds to the standard deviation range. The whiskers limit the 1–99% observed data value range. The n value indicates the number of individual tomograms that contributed to the measurement. f Box plot of the measured distances of observed dark layer thickness and spacing of droplet surface to enwrapped single lipid bilayer. Observed measurements are shown as diamonds, the central bar represents the median value, while the gray box corresponds to the standard deviation range. The whiskers limit the 1–99% observed data value range. The n value indicates the number of individual tomograms that contributed to the measurement. g Tomographic z-slice of half a lipid droplet in raw grayscale (left), denoised grayscale (center) and superimposed labeled structures of droplet surface (red), dark layer (yellow), weaker protein density (black) and enwrapping single lipid bilayer (blue). Scale bar 50 nm. h Tomographic z-slice of another droplet showing the spacing of 13.3 nm between the droplet surface and a single lipid bilayer. Scale bar 50 nm. All experiments were performed on at least 7 independently prepared biological samples.