CORRELATIVE CRYO-SOFT X-RAY TOMOGRAPHY AND STORM STUDY OF CHOLESTEROL CRYSTAL EARLY FORMATION IN CELLS

Accumulation of cholesterol in the blood vessel walls is a prominent feature of atherosclerosis (1), a major precursor of many cardiovascular diseases. It is well accepted that precipitation of cholesterol crystals in atherosclerotic lesions is a crucial part of the pathological progression (2). Cholesterol monohydrate crystals can increase the inflammatory response and cause expansion of the lesion core leading to arterial thrombosis (2b). What initiates cholesterol crystal formation is, however, still not well-understood (3). Studies in cell culture (4) and in supported lipid bilayers (5) showed that when a bilayer is loaded with high levels of cholesterol, 2 dimensional (2D) crystalline domains of cholesterol are formed and it was suggested to be an initial step in atherosclerosis development (4). We indeed demonstrated that such 2D cholesterol domains can serve as nucleation sites for the formation of 3D cholesterol crystals in vitro (6). The question of whether this process occurs also in biological membranes was then approached.

High resolution imaging is required to directly visualize cholesterol domains and identify the initial stages of cholesterol segregation and crystal formation in cell membranes. For this, we developed a correlative method that combines cryo soft X-ray tomography (cryo-SXT) with stochastic optical reconstruction microscopy (STORM). The correlative approach provides imaging of the domain distribution in cell membranes and identification of 3D cholesterol crystals. This is done by using a specific antibody (MAB 58B1) which labels cholesterol 2D and 3D crystals (7). To the best of our knowledge, this is the first example of correlative STORM and cryo-SXT providing information on cellular systems at resolution of tens of nanometers and in relatively large volumes (tens of micrometers). Our results show that cholesterol crystals can be imaged in a very early stage at their exact locations inside the cell. The cells were observed under cryo-conditions, without dissolving the crystals or using any other dehydration methods, such that the biological system is as close to the natural condition as possible.

In the future, the method will be applied to determine the crystal nucleation location and mechanism for intracellular, interfacial as well as extra-cellular particles. If our hypothesis will be proven correct, it can provide new inspiration on how to inhibit the critical pathological step of cholesterol crystal nucleation in early stages of atherosclerosis

References:

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