• 3D reconstruction;
  • confocal/4Pi/electron/correlative microscopy;
  • intra-Golgi transport;
  • threshold;
  • zero-crossing

Two problems have hampered the use of light microscopy for structural studies of cellular organelles for a long time: the limited resolution and the difficulty of obtaining true structural boundaries from complex intensity curves. The advent of modern high-resolution light microscopy techniques and their combination with objective image segmentation now provide us with the means to bridge the gap between light and electron microscopy in cell biology applications. In this study, we provide the first comparative correlative analysis of three-dimensional structures obtained by 4Pi microscopy and segmented by a zero-crossing procedure with those of transmission electron microscopy (TEM). The distribution within the cisternae of isolated Golgi stacks of the cargo protein procollagen 3 was mapped by both 4Pi microscopy and TEM for a detailed comparative analysis of their imaging capabilities. A high correlation was seen for the structures, indicating the particular accuracy of the 4Pi microscopy. Furthermore, for the first time, transport of a cargo molecule (vesicular stomatitis virus G protein-pEGFP) through individual Golgi stacks (labeled by galactosyl transferase-venusYFP) was visualized by 4Pi microscopy. Following the procedures validated by the correlative analysis, our transport experiments show that (i) VSVG-pEGFP rapidly enter/exit individual Golgi stacks, (ii) VSVG-pEGFP never fills the GalT-venusYFP compartments completely and (iii) the GalT-venusYFP compartment volume increases upon VSVG-pEGFP arrival. This morphological evidence supports some previous TEM-based observations of intra-Golgi transport of VSVG-pEGFP and provides new insights toward a better understanding of protein progression across Golgi stacks. Our study thus demonstrates the general applicability of super-resolution fluorescence microscopy, coupled with the zero-crossing segmentation procedure, for structural studies of suborganelle protein distributions under living cell conditions.