FIGURE 3.

Distribution and quantification of active RNAPII (phosphorylated at serine 2) molecules in a differentiated 16C A. thaliana leaf nucleus. After immunostaining with specific antibodies SIM (A) and PALM (B) were performed consecutively. (A) Compared to widefield and deconvolution microscopy SIM clearly delivers an increased resolution especially visible in the enlarged region (white rectangle) below. This indicates that RNAPII is distributed network-like within the nucleus. (B) After 3D-PALM the resolution becomes further increased when visualized as Gauss distribution (left). Centroid visualization allows the exact localization and counting of single molecules (center). The nucleus with a z extension of 1.2 μm contains in total 65761 molecules which were counted in 30 slices of 40 nm. The merged Gauss-centroid view (right) represents the localization of eight single molecules in a single slice of a RNAPII cluster (red rectangle) containing in total 37 molecules in five slices with a total z range of 0.2 μm.