Validation of super-resolution 4Pi-microscopy, and its application to the study of intra-Golgi transport

Perinetti, Giuseppe (2009). Validation of super-resolution 4Pi-microscopy, and its application to the study of intra-Golgi transport. PhD thesis The Open University.



Two problems have hampered the use of light microscopy for structural studies of cellular organelles for a long time: the limited spatial resolution, and the difficulty of obtaining true structural boundaries from complex intensity curves. The advent of modern super-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 biological applications. This study provides the first comparative correlative analysis of three-dimensional (3D) structures obtained by 4Pi-microscopy with those of transmission microscopy (TEM). The distribution within the cisternae of isolated Golgi stacks of the cargo protein PC3-pEGFP is here mapped in 3D by both 4Pi-microscopy and TEM for a detailed comparative analysis of their imaging capabilities. A high correlation was seen for these structures, demonstrating the particular accuracy of 4Pi-microscopy. Furthermore, for the first time, the transport of a cargo molecule (VSVG-pEGFP) through individual Golgi stacks (labelled by GalT-venusYFP) is visualized at the 4Pi-microscope super-resolution level. Following the procedures validated by the correlative analysis, the transport experiments show that: 1) VSVG-pEGFP rapidly enter/exit individual Golgi stack; 2) VSVG-pEGFP never fills the GalT-venusYFP compartment completely; and 3) the GalT-venusYFP compartment volume increases upon VSVG-pEGFP arrival. This morphological evidence supports some previous TEM-based observations of the intra-Golgi transport of VSVG-pEGFP and provides new insights towards a better understanding of protein transport across the Golgi stack. This study thus demonstrates the general applicability of super-resolution 4Pi-microscopy coupled with the zero-crossing-based segmentation technique for structural studies of sub-organelle protein distributions in living cells.

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