Figure 3

3D large volume imaging of laminA-His₁₀-eGFP. (a) Schematic depiction of imaging an entire cell (stippled line), here mostly represented by the nucleus (continuous line), with SERI. The focus is shifted after a defined number of frames during a single imaging cycle to cover the entire volume. Thereafter the cells are treated with elution buffer, re-stained and imaged again. The stippled line denotes the assumed contours of the cell. (b–e) Four different single imaging rounds of a cell transfected with His10-mEGFP-LaminA. Note that measurements 1 (b) and 3 (d) were done in top-down direction while measurements 2 (c) and 4 (e) were done in reverse direction, thereby revealing the bleaching-induced decrease of the number of localizations in the direction of image acquisition (indicated by color code for z position). (f) Overlay of all four single imaging rounds. The scale bars in (b,f) correspond to 5 µm of the bottom side of the cell. The scale bar in (b) applies to panels (b–e). (g) xz-projection of a 500 nm thick section. The color bar ranges from 0 µm (red) to 4 µm (blue). (h) Intensity cross section of the lamin border. (i) Distribution of localizations over the z-positions of the nucleus of each measurement and of the merged measurements. The merged measurement demonstrates an enhanced labeling density over the z-range in comparison to single measurements. Similar results were obtained in a total of N = 4 cells.