Figure 5.

Re-localization after flat embedding. Light micrographs of HeLa cells grown on a laser-marked slide to the left to the letter X (a1). After 4′,6-Diamidin-2-phenylindol (DAPI) staining, metaphase cells were selected (a2, a3) and re-localized in SEM, already visible in the overview (a4, a5). DAPI and secondary electron (SE) image were superimposed to define a precise milling frame (a6). Scanning electron micrographs of critical point dried HeLa cells, compared with cells, embedded with different thick layers of resin. The topographic details of critical point dried cells (c) were preserved after ultra-thin embedding (c). Structural details like filopodia were clearly visible, whereas, after medium-thin embedding, they were obscured (d). Thin embedded cells, covered by a few microns of resin, could be still re-located and identified as small humps (e). Comparison of cross sections of HeLa cells embedded ultra-thin (f), medium-thin (g) and thin (h) in epoxy resin. The thicker the covering resin layer, the less topographic details of the cell surface were visible, however, the risk of curtaining is reduced by the smooth surface (h). The glass slide served as an absolute reference plane (xz) for alignment (g; dashed line). Lines, milled parallel to the regions of interest (ROI) into the slide in xz direction (h; arrow), served as a third reference plane for precise alignment of the image stack in three dimension. The X-ray signals were used for verification of resin thickness. The carbon signal of ultra-thin embedded cells, clearly visible at 25 kV (i), became blurred after thin embedding, due to the carbon portion of the covering resin (j). P, U and Os mapping of thin embedded cells revealed only a section of the cells appearing “in focus” as the higher energetic K-line of phosphorus, the M-line of uranium and the L-line of Os osmium were excited only from higher energetic electrons near the surface (Fig. 5j).