Development of Correlative Cryo soft X ray Tomography and Stochastic Reconstruction Microscopy. A Study of Cholesterol Crystal Early Formation in Cells

We have developed 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 identified and localized aggregations of carbon rich materials. STORM identified 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 imagin