Development of Correlative Stochastic Optical Reconstruction Microscopy and Cryo-solf X-ray Tomography of Cholesterol Crystal Early Formation in Cells
2Department of Structural Biology, Weizmann Institute of Science, Rehovot
3Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot
4Soft Matter and Functional Materials, Helmholtz-Zentrum Berlin, Berlin
5ALBA Synchrotron Light Source, MISTRAL Beamline-Experiments Division, Barcelona
6Experimental Atherosclerosis Section National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
One of the most versatile imaging techniques in biology, fluorescence microscopy, allows noninvasive imaging with molecular specificity. However, the resolution of a conventional wide-field fluorescence microscope is limited by the diffraction of light (∼200-300 nm). Recent advances in far-field optical nanoscopy provide spatial resolution of 20-50 nanometers that is well below the diffraction limit. Combining super resolution fluorescence microscopy with additional high resolution imaging technique is highly attractive. Correlation of two imaging approaches allows specific cellular characterization together with a highly complete view of the cell ultrastructure. We therefore were inspired to develop a high resolution correlative method involving cryo-soft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM), which provides information in three dimensions on large cellular volumes at 70 nm resolution. Cryo-SXT morphologically identifies and localizes aggregations of carbon-rich materials. STORM identifies specific markers on the desired epitopes, enabling colocalization between the identified objects, in this case cholesterol crystals, and the cellular environment. The samples were studied under ambient and cryogenic conditions without dehydration or heavy metal staining. The early events of cholesterol crystal development were investigated in relation to atherosclerosis, using as model macrophage cell cultures enriched with LDL particles. Atherosclerotic plaques build up in arteries in a slow process involving cholesterol crystal accumulation. Cholesterol crystal deposition is a crucial stage in the pathological cascade. Our results show that cholesterol crystals can be identified and imaged at a very early stage 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.
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