Development of Correlative Cryo-soft X-ray Tomography (Cryo-SXT) and Stochastic Reconstruction Microscopy (STORM). A Study of Cholesterol Crystal Early Formation in Cells
2Department of Chemical Research, Weizmann Institute of Science, Rehovot
3Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot
4Department of Soft Matter and Functional Materials, Helmholtz-Zentrum Berlin, Berlin
5MISTRAL Beamline−Experiments Division, ALBA Synchrotron Light Source, Barcelona
6Experimental Atherosclerosis Section, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland
Atherosclerosis, the major precursor of cardiovascular disease, is characterized by the deposition of excessive cholesterol in the arterial intima. 1 Atherosclerotic plaques build up in arteries in a slow process that initiates with uptake of LDL particles by macrophage cells leading to deposition of cholesterol monohydrate crystals and cell death.1 Precipitation of cholesterol crystals is a crucial part of the pathological progression. 2
We suggested that the initial step in atherosclerosis development may be from cholesterol domains segregating in cell membranes and serving as nucleation sites for the formation of 3-dimensional (3D) cholesterol crystals 3,4. To verify whether this process can be relevant to in vivo processes, we have developed a high resolution correlative method combining cryo-soft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM). 5 The approach provides 3D information on large cellular volumes at 70 nm resolution. 5 Cryo-SXT morphologically identifies and localizes aggregations of carbon-rich materials, while STORM identifies specific markers on the desired epitopes, enabling colocalization between the identified objects and the cellular environment. Using a specific antibody (MAB 58B1) which labels cholesterol crystals 6, we identify and image crystals at a very early stage (200-400 nm) on the cell plasma membrane and in intracellular locations. This technique can in principle be applied to other biological samples where specific molecular identification is required in conjunction with high resolution 3D-imaging
.jpg)
[1]Kruth, H. S. Curr. Mol. Med. 2001, 1, 633.
[2] Tangirala, Rajendra K., W. Gray Jerome, N. L. Jones, Donald M. Small, W. J. Johnson, J. M. Glick, F. H. Mahlberg, and G. H. Rothblat. J. Lipid Res., 1994 35, 93.
[3] Ong, D. S.; Anzinger, J. J.; Leyva, F. J.; Rubin, N.; Addadi, L.; Kruth, H. S. J. Lipid Res. 2010, 51, 2303.
[4] Ziblat, R.; Fargion, I.; Leiserowitz, L.; Addadi, L. Biophys. J. 2012, 103, 255
[5]Varsano, N., Dadosh T., Kapishnikov S. Pereiro E. Shimoni E. Jin X., Kruth, H. S. Leiserowitz L, and Addadi L. J. Am. Chem. Soc. 2016, DOI: 10.1021/jacs.6b07584
[6] Addadi, L., Rubin, N., Scheffer, L. and Ziblat, R., 2008.. Acc. Chem. Res., 2008, 41,254.
Powered by Eventact EMS