Visualization and Quantitative Analysis of Reconstituted Tight Junctions Using Localization Microscopy

Tight Junctions (TJ) regulate paracellular permeability of tissue barriers. Claudins (Cld) form the backbone of TJ-strands. Pore-forming claudins determine the permeability for ions, whereas that for solutes and macromolecules is assumed to be crucially restricted by the strand morphology (i.e., density, branching and continuity). To investigate determinants of the morphology of TJ-strands we established a novel approach using localization microscopy

Beschleunigung biomedizinischer Bildverarbeitung und -rekonstruktion mit FPGAs

Acceleration of Biomedical Image Processing and Reconstruction with FPGAs Increasing chip sizes and better programming tools have made it possible to increase the boundaries of application acceleration with reconfigurable computer chips. In this thesis the potential of acceleration with Field Programmable Gate Arrays (FPGAs) is examined for applications that perform biomedical image processing and reconstruction. The dataflow paradigm was used to port the analysis of image data for localization microscopy and for 3D electron tomography from an imperative description towards the FPGA for the first time. After the primitives of image processing on FPGAs are presented, a general workflow is given for analyzing imperative source code and converting it to a hardware pipeline where every node processes image data in parallel. The theoretical foundation is then used to accelerate both example applications. For localization microscopy, an acceleration of 185 compared to an Intel i5 450 CPU was achieved, and electron tomography could be sped up by a factor of 5 over an Nvidia Tesla C1060 graphics card while maintaining full accuracy in both cases.Weiterentwicklungen in der Mikrochipherstellung und verbesserte Programmierwerkzeuge ermöglichen es, rekonfigurierbare Hardware auch zunehmend für die Beschleunigung umfangreicherer Anwendungen einzusetzen. In dieser Dissertation wurde die Eignung von Field Programmable Gate Arrays (FPGAs) für die Ausführungsbeschleunigung von Anwendungen untersucht, die zur Bildverarbeitung und -rekonstruktion in der Biomedizin eingesetzt werden. Anhand der Datenflussbeschreibung wurde die Bildanalyse für die Lokalisationsmikroskopie und die Bildrekonstruktion in der dreidimensionalen Elektronentomografie erstmalig von einer imperativen Beschreibung auf den FPGA zu portieren. Nachdem die Grundelemente der Bildverarbeitung auf dem FPGA beschrieben wurden, untersucht die Arbeit, wie imperativer Quellcode analysiert und zu einem Pipeline-System in Hardware umgeschreiben werden kann, so dass jeder Knoten Bilddaten parallel verarbeitet. Diese Grundlage wird dann verwendet, um beide Anwendungen zu beschleunigen. Für die Lokalisationsmikroskopie konnte ein Beschleunigungsfaktor von 185 gegenüber einer CPU (Intel i5 450) erreicht werden, und die Elektronentomografie wurde um einen Faktor fünf gegenüber einer Grafikkarte (Nvidia Tesla C1060) beschleunigt. In beiden Fällen konnte die Genauigkeit der Ergebnisse erhalten werden

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

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

Visualization of local densities of detected proteins in and around TJ-networks formed by Cld3 and Cld5.

<p><b>A</b>: Localization microscopy image of Cld3-YFP labeled HEK293 cells. The local density was determined within a radius of 50 nm for every detected molecule and color coded in the image. The dynamic range was chosen between 0 and 3700 molecules/µm² - the mean density of detected molecules on the strands of the analyzed meshes. <b>B</b>: Magnified image of the region marked in <b>A</b>. <b>C</b>,<b>D</b>: Analogue visualization of Cld5-YFP. Gaps in the meshes are indicated by white arrowheads in <b>D</b>.</p

Reconstitution of TJ-strands formed by claudin proteins.

<p><b>A</b>: Schema of reconstitution of TJ-strands. HEK293 cells without endogenous TJ are transfected with claudins. TJ-strands are formed at contacts between claudin-expressing cells in the lateral membrane and at apical cellular protrusions. Confocal images of living (<b>B</b>) or fixed (<b>C</b>–<b>H</b>) cells. <b>B</b>: Cld3-YFP (green) is enriched at contacts between two claudin-expressing cells (white arrow). Plasma membrane (red) of living cells was labeled with CellMask™ Deep Red (Invitrogen, 1.25 µg/ml CellMask™ Deep Red in Hank's Buffered Salt Solution for 20 min). <b>C</b>: Cld3-YFP strands in the lateral membrane running in z-direction along the optical axis (white arrow). Cld3-YFP strands running in the apical membrane in xy-direction in the object plane (green arrow). <b>D</b>: Higher magnification of Cld3-YFP strands running in xy-direction (green arrow). <b>E</b>–<b>H</b>: Sequential images of a 3D projection of Cld3-YFP at cell-cell contact (angle: 0°, 50°, 140°, 200°).</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

Diameter of the mesh-like structures.

<p><b>A</b>: Size measurements of Cld3 tight junction networks. The histogram of diameters shows one distribution for very small sizes (diameter of ∼100 nm) overlaying a second distribution for a diameter of ∼360 nm. Results of the measurements of Cld5 are depicted in <b>B</b>. The histogram also shows two distributions (∼60 nm and ∼480 nm). Both data sets were fitted with the sum of two Gaussians. The results of the fits are plotted in orange; fitted parameters are given including standard errors.</p

Networks of TJ-strands and meshes visualized by freeze fracture EM and localization microscopy.

<p><b>A</b>: Freeze-fracture EM of Cld5-YFP/Cld3-cotransfected HEK293 cells. <b>B</b>: Localization microscopy of Cld5-YFP transfected HEK293 cells. In freeze fracture EM the Cld5/Cld3-strands appear as chains of 10 nm intramembranous particles (arrow) on the exoplasmic face (E) and protoplasmic face face (P) of the plasma membrane. Due to lower resolution of localization microscopy the strands appear thicker (arrow).</p