Computational protocol: Correlative super resolution fluorescence and metal replica transmission electron microscopy

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Protocol publication

[…] Samples were critical-point-dried and coated with platinum and carbon as previously described. The coated samples were imaged with 10x phase contrast light microscopy to locate the regions of interest (ROIs). A pioloform and carbon coated 50-mesh, 3 mm copper grid was plasma discharged and dipped in a 1:5 dilution of goat anti-mouse 10 nm immunogold conjugate (EM.GMTA10, BBinternational, Cardiff, UK) rinsed and dried on filter paper. This resulted in sparsely scattered gold nanoparticles that were used as fiducials for tomogram alignment.The platinum-carbon replica was lifted off of the coverslip by floating the sample on 5% hydrofluoric acid. The replicas were rinsed using successive dilutions with water, lifted out of the water and placed onto the grid using a Perfect Loop (Electron Microscopy Sciences). The replica was again imaged with 10x phase contrast light microscopy to find the placement of the ROIs with respect to the grid. In some cases, there was loss of a ROI because it was placed over a grid bar.Transmission electron microscopy was performed on a JEOL 1400 running SerialEM freeware and equipped with a XR-111 CCD camera (AMT, Wobum, MA) Montages of entire unroofed cells were produced at 15000x with 10% overlap. Single axis tilt series (−60° to 60°, 1° increments) were collected at 8000x. The montages were stitched together and the tilt series were reconstructed into tomograms using IMOD software,. [...] Two-dimensional EM micrographs were analyzed in ImageJ and elliptical regions of rx and ry (radii along the X and Y axes) were drawn to best fit the shape of visible clathrin lattices. Clathrin structures were split into two categories: flat or slightly curved domes, and highly invaginated pits. Clathrin structures were omitted from this analysis only if they were at the edge of the cell, did not fit well to an ellipse, within 0.5 μm of a gold nanorod, or right next to another clathrin structure. In Matlab (Mathworks), the coordinates of PALM localizations were mapped onto the coordinates of the ellipses and assigned a fractional position from −2rx to 2rx in the X dimension and −2ry to 2ry in the Y dimension. The fractional coordinates from each ellipse were binned into a 40 × 40 two-dimensional histogram. The resulting image was normalized prior to averaging the images from all regions of a specific category ( and ). Radial scans were produced by averaging pixels based on their distance away from the center of the image. The standard error of pixel sampling takes into account the total number of pixels included in the final data point (from each separate 2D histogram). […]

Pipeline specifications

Software tools SerialEM, IMOD, ImageJ
Applications cryo-EM, Microscopic phenotype analysis