![]() SXT was the ideal approach to study hemozoin crystal growth in Plasmodium: a), fully hydrated cells can be observed intact, without extraction, embedding, or sectioning, and b) the question of a lipid sphere surrounding the crystals could be answered definitively because the carbon cannot be hidden. For that purpose we developed a protocol for high pressure freezing of SXT specimens. One of the important challenges is to vitrify such thick specimens. Resolution is intermediate between traditional light and electron microscopy, about 30-50 nm depending on the zone plate design. The characteristic absorption length for water is about 10 microns, rendering the technique suitable for intact cells in many cases. Fresnel zone plates are used as objectives to produce a transmission image. Image contrast originates from the greater absorption by carbon than by oxygen, when the illuminating X-ray energy lies between the K shell edges of the two elements at 280 and 530 eV respectively. Of all available techniques, soft X-ray tomography in the “water window” provides the most direct detection of carbon density, the core of organic materials. (2014) Shigella subverts the host recycling compartment to rupture its vacuole. Mellouk N, Aulner N, Weiner A, Schmitt C, Elbaum M, Shorte S, Danckaert A, Enninga J.Weiner A†, Dahan-Pasternak N†, Shimoni E, Shinder V, von Huth P, Elbaum M, Dzikowski R (2011) 3D nuclear architecture reveals coupled cell cycle dynamics of chromatin and nuclear pores in the malaria parasite Plasmodium falciparum.With the lab of Thierry Rose at Pasteur we look at microtubule assemblies in activated T-cells. ![]() ![]() In collaboration with the lab of Jost Enninga at the Pasteur Institute, we took a correlative fluorescence-EM approach to follow host cell invasion by Shigella. We have used the FIB-SEM to follow dynamics of nuclear pore and chromatin distributions in Plasmodium. This can be done with a microtome mounted inside the scanning electron microscope (SEM), or with a dual instrument encompassing both focused ion beam milling and SEM. FIB-SEMĪ very direct approach to 3D reconstruction is to progressively cut away or polish a block while imaging the freshly exposed surfaces. These include serial surface imaging by FIB-SEM and its correlation with light microscopy, soft X-ray cryo-tomography (SXT), and cryo-scanning transmission electron tomography (CSTET). The lab is actively developing novel 3D modalities for ultrastructural imaging in cells. ![]()
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