Table 1.
The dyes used for labeling the liposomes in the STED and RICS experiments.
Fig 1.
Cartoon depicting the sample preparation.
Samples were labeled non-occlusively. Intact skin samples for imaging were mounted SC side down on a microscope cover glass for imaging. Samples for cryo sectioning were sectioned and mounted as depicted.
Fig 2.
Cryo frozen and sectioned skin labelled with the free dye, Atto-488-DPPE, in PBS buffer.
A) A STED image of the sample in which the SC is clearly visible, as the bright u-shaped layer in the center of the image; the width of the lipid layers in the SC are measured to approximately 100–120 nm. A line scan across the marked line is shown in B, and the individual lipid layers are recognizable. C) A confocal cross section (XZ plane) through a skin section. The slice is orientated with the SC to the left. Only the upper surface of the SC, away from the cover glass, is labeled. This is where the SC was directly exposed to the label. In contrast, the SC facing the glass (lower part in the z direction) is unlabeled. This is likely due to barrier properties of the SC towards the dye. In contrast, the SG and SS/SB are labeled all the way through in the z direction. Scale bars are 5 μm.
Fig 3.
Image of the surface of intact skin.
The skin samples were labeled using LUVs with Atto-488-DPPE. After labeling for 6–8 hours the samples were rinsed and patted dry, removing most of the LUVs from the surface. An intense labeling of the lipids around the corneocytes is observed. A) and B) shows a STED and confocal image, respectively, of a large area where several corneocytes can be distinguished. C) and D) are enlargements of the mark regions in A and B. The resolution difference between confocal and STED can clearly be seen. E) shows an intensity line profile across the line marked in C (blue) and D (green), again it is evident that STED reveals details not resolved in the confocal image. Both the STED and confocal image have been deconvolved. Scale bars are 5 μm for A and B, and 1 μm for C and D.
Fig 4.
STED images of FLUVs on intact skin.
Several vesicles are visible on the surface (A) and at different depths of the skin (B-D). FLUVs are not observed deeper in the skin. In the lower layers, C and D, the FLUVs are generally located at the edge of the corneocytes. Scale bars are 5 μm.
Fig 5.
Cryoembedded and -sectioned skin exposed to POPC LUVs on the skin surface prior to processing for microscopy.
Several LUVs can be seen on the surface of the SC, e.g. magnified region of interest in A. Some penetration of the dye into the superficial SC layers can be seen in B and C, where the underlying layers of the SC have become labeled by the dye included in the LUVs. LUVs are rarely found inside the SC. Scale bars are 2 μm.
Fig 6.
Cryoembedded and -sectioned skin exposed to FLUVs on the skin surface prior to processing for imaging.
Intact FLUVs were rarely found on the surface, and the SC was generally more intensely labeled by the dye from the FLUVs than from the LUVs, indicating a better penetration of the dye from the FLUVs. Sometimes the dye was observed deeply into the SC panel B) whereas in other cases the staining was more confined to the upper most layers of SC (panel A). Scale bars are 5 μm.
Fig 7.
The panels show the CC-RICS data (upper graph) and the fit (lower graph), for RhB-PE and ATTO-647N-PE labeled LUVs (panels A-C) and FLUVs (panels D-F) together with an intensity image of the ATTO-647N-PE channel. A) CC-RICS and fit for LUVs at the SC surface. A clear cross correlation is seen. B and C are CC-RICS and fit for LUVs at 4μm below the SC surface. In 15% of the measurements a cross correlation was found (panel B) while most of the measurements showed no cross correlation (panel C). D) CC-RICS and fit for FLUVs at the SC surface. A clear cross correlation is seen. E and F are CC-RICS and fit for FLUVs at 4μm below the SC surface. In 9% of the measurements a cross correlation was found (panel E) while most of the measurements showed no cross correlation (panel F). The image sizes are 22 x 22μm2.