title: Development of Correlative Cryo-soft X-ray Tomography and Stochastic Reconstruction Microscopy. A Study of Cholesterol Crystal Early Formation in Cells.
abstract: 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-imaging.
authors.forename: Neta
authors.initials: N
authors.lastname: Varsano
authors.forename: Tali
authors.initials: T
authors.lastname: Dadosh
authors.affiliation: Soft Matter and Functional Materials, Helmholtz-Zentrum Berlin , Albert-Einstein-Strasse 15, D-12489 Berlin, Germany.
authors.forename: Sergey
authors.initials: S
authors.lastname: Kapishnikov
authors.affiliation: ALBA Synchrotron Light Source, MISTRAL Beamline-Experiments Division, 08290 Cerdanyola del Valles, Barcelona, Spain.
authors.forename: Eva
authors.initials: E
authors.lastname: Pereiro
authors.forename: Eyal
authors.initials: E
authors.lastname: Shimoni
authors.affiliation: Experimental Atherosclerosis Section, National Heart, Lung, and Blood Institute, National Institutes of Health , Bethesda, Maryland 20892-1422, United States.
authors.forename: Xueting
authors.initials: X
authors.lastname: Jin
authors.affiliation: Experimental Atherosclerosis Section, National Heart, Lung, and Blood Institute, National Institutes of Health , Bethesda, Maryland 20892-1422, United States.
authors.forename: Howard S
authors.initials: HS
authors.lastname: Kruth
authors.forename: Leslie
authors.initials: L
authors.lastname: Leiserowitz
authors.forename: Lia
authors.initials: L
authors.lastname: Addadi
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002784:Cholesterol / Q000138:chemical synthesis* / Q000737:chemistry; D020285:Cryoelectron Microscopy; D003460:Crystallization; D008264:Macrophages / Q000737:chemistry* / Q000166:cytology; D051379:Mice; D008856:Microscopy, Fluorescence; D010316:Particle Size; D000067996:RAW 264.7 Cells; D013269:Stochastic Processes; D013499:Surface Properties; D014056:Tomography, X-Ray
chemical_list: D002784:Cholesterol