The spatial distribution analysis averaged over the measured cells.

<p>Between 5 and 35 nm, a single blurred peak is apparent and converges to a straight line at larger distances. Note the scaling of the y-axis: the peak is caused by several thousands of molecules. This indicates that Pgp-GFP shows clustered formations in hCMEC/D3 cells. The clustered formations themselves are distributed randomly across the cell membrane which can be seen by convergence to the random distribution at larger distances.</p

Flow cytometry of transduced hCMEC/D3 cells.

<p>Approximately 11% of transduced cells show GFP expression.</p

Simulated cell curvature effects on projected random molecule distributions.

<p><b>A:</b> Random molecule distribution on a chirp curvature in x-direction. <b>B:</b> Projection of the curved random distribution along the z-direction. <b>C:</b> Non-curved random molecule distribution (same as in A). Molecule aggregations along the x-direction are induced by the projection of the curved random distribution (highlighted by two arrows in section 1). These aggregations are not present in the non-curved random distribution (section 2). In a real SPDM measurement the projection is caused by a non-improved z-resolution.</p

Histogram of the localization accuracy of 112,000 detected single molecules.

<p>The average localization accuracy was 34 nm. Note that these 112,000 molecules survived the discarding process and were further kept for spatial distribution analysis.</p

Pgp-GFP fusion protein expression in hCMEC/D3 cells. A:

<p>Simulated conventional microscopy image of Pgp-GFP in hCMEC/D3 cells (by convolving a super-resolution image with a Gaussian function with a standard deviation of 200 nm). A conventional microscopy image was not acquired to prevent bleaching. <b>B:</b> Super-resolution localization microscopy image of the same cell. Each molecule is convolved with a Gaussian function with a standard deviation equal to its localization accuracy. Some Pgp-GFP molecules could be resoluted with a localization accuracy of approximately 42 nm (asterisk). <b>C–E:</b> Three selected images from a total of 20 equivalent images of hCMEC/D3 cells which express Pgp-GFP.</p

Intracellular accumulation of eFluxx-ID Gold in nontransduced and Pgp-GFP-hCMEC/D3 cells.

<p><b>A:</b> Quantitative analysis of the intracellular fluorescence intensity of eFluxx-ID Gold in native and in transduced Pgp-GFP-hCMEC/D3 cells in the absence or presence of 20 µM verapamil. <b>B:</b> The multidrug resistance activity of Pgp-GFP-hCMEC/D3 cells is increased by a factor of 2 compared to nontransduced cells, which was determined by calculating the multidrug resistance factor for each probe.</p

Selected ROIs in the super-resolution localization images.

<p>In total, 19,000 Pgp-GFP molecule counts were excluded (white) from the spatial distribution analysis to prevent artifacts due to highly curved cell boundaries.</p

Tight junction networks formed by Cld3-YFP and Cld5-YFP.

<p><b>A</b>: Localization microscopy image of the region marked in the conventional wide-field fluorescence image (<b>B</b>) of YFP labeled Cld3 in HEK293 cells. More than 450,000 molecules were detected with a mean localization accuracy of ∼20 nm. The mean distance to the next neighboring molecule in the image is ∼10 nm, thus the mean effective optical resolution is only limited by the localization accuracy and yielding ∼48 nm. Magnified images of the region marked in <b>A</b> are shown in <b>D</b> and <b>E</b>. Mesh-like structures identified and analyzed by the algorithm are indicated in white. <b>C</b> represents the same region but taken from the conventional wide-field fluorescence image. <b>F</b>–<b>J</b>: Analogue visualization of Cld5-YFP expressed in HEK293 cells. Here, ∼260,000 molecules were detected with a mean localization accuracy of ∼21 nm. The mean distance to the next neighboring molecule in this image is ∼7 nm; yielding a structural resolution of ∼50 nm.</p

Analysis of mesh-like structures.

<p><b>A</b>,<b>B</b>: Local point densities are visualized as grey values in the localization microscopy image. Positions of the detected single molecules are indicated by red dots. <b>C</b>: Radial intensity distribution of the mesh-like structure shown in <b>A</b>,<b>B</b>. <b>D</b>: First derivative of the radial intensity distribution. The first zero-crossing marks the position of the first maximum of the intensity distribution. A blue circle in <b>A</b>,<b>B</b> represents the approximation by the circle with a radius corresponding to the maximum of the radial intensity distribution. <b>E</b>: Histogram of the distances between the single molecule positions and the shape of the edge filter (indicated by yellow arrows in <b>A</b> for some of the positions). <b>F</b>: The first zero-crossing of the first derivative gives the distance of the majority of the points. This value is used to correct the underestimated size of the mesh obtained by the edge filter (red line in <b>A</b>). In <b>B</b> the corrected shape is illustrated by the two red lines. The area in between is used to determine the molecule density on the strand of the mesh.</p

Densities of detected proteins and shape of the mesh-like structures.

<p><b>A</b>,<b>B</b>: Localization microscopy images of Cld3-YFP (<b>A</b>) and Cld5-YFP (<b>B</b>) with overlay of single molecule positions represented by red crosses. <b>C</b>: Histogram of the density of detected proteins on the strands of the mesh-like structures (indicated in <b>A</b>,<b>B</b> by orange arrows). Both protein types show similar distributions with mean values of ∼3700 proteins/µm². The standard deviation of the distribution for Cld3 is 760 proteins/µm². The distribution for Cld5 is wider and provides a standard deviation of 900 proteins/µm². <b>D</b>: Histogram of the density of detected proteins beside the strands of the meshes (indicated in <b>A</b>,<b>B</b> by green arrows). Here, the distribution for Cld3 and Cld5 are very similar, too (mean values: ∼780 proteins/µm² with STDs of ∼640 proteins/µm²). For many meshes (especially the very small ones) no proteins could be detected on their inside. These are not considered in the histograms. <b>E</b>: Histograms of the minimum diameter divided by its maximum perpendicular diameter of the meshes. Both protein types show similar distributions with a maximum at ratio of ∼0.7. <b>F</b>: Histograms of the extension of the meshes parallel to the orientation of the whole TJ-network (principal axis) divided by its perpendicular extension showing that the mean orientation of the meshes of both protein types is parallel to the orientation of the whole TJ-network (mean value for Cld3: ∼1.21 with STD: ∼0.38; mean value for Cld5: ∼1.29 with STD: ∼0.42). All histograms are normalized to the total amount of analyzed meshes.</p