Localization of lipids in the aortic wall with imaging TOF-SIMS.

Time-of-flight secondary-ion-mass-spectrometry (TOF-SIMS) was utilized to address the issue of localization of lipids and inorganic ions in healthy rat aorta and human atherosclerotic plaque. Pieces of rat aorta were high pressure frozen, freeze-fractured and freeze dried. The samples were analyzed by imaging TOF-SIMS equipped with a Bi(1-7)(+)-source. Reference lipid samples were analyzed and compared to data obtained by analysis of the rat aorta samples. Fatty acids, cholesterol, oxysterol and diacylglycerols were detected and localized. A heterogeneous lipid distribution could be shown in the aorta, where the lamellae of the aorta, distinguished by imaging of CN(-), appeared enriched in cholesterol, oxysterol and diacylglycerols, while the smooth muscle tissue, identified by imaging of PO(3), appeared enriched in phosphocholine. Palmitic/palmitoleic acid and stearic/oleic acid appeared to be heterogeneously distributed over the aorta with high concentration areas located especially in the tunica media region of the aorta. Human atherosclerotic plaque showed an irregular cholesterol distribution mainly located in spots in the intima region with elongated diacylglycerol regions located mainly in the media region.

Distribution of cholesterol and galactosylceramide in rat cerebellar white matter.

White matter and the inner granular layer of rat cerebellum was analysed by imaging time-of-flight secondary-ion mass spectrometry (TOF-SIMS) equipped with a Bi+ ion cluster gun. Samples were prepared by high pressure freezing, freeze-fracturing and freeze drying or by plunge freezing and cryostat sectioning. The identified and localized chemical species were: sodium, potassium, phosphocholine, cholesterol and galactosylceramide (GalC) with carbon chain lengths C18:0 (N-stearoyl-galactosylceramide) and C24:0 (N-lignoceroylgalactosylceramide) with CH24:0 (hydroxy-lignoceroylgalactosylceramide). We report new findings regarding the organization of myelin in white matter. One is cholesterol-rich, ribbon-shaped 10-20 microm areas excluding Na+ and K+. The second finding is the different distribution of GalC C18 and GalC C24 in relation to these areas, where GalC C18 was localized in cholesterol-rich areas and GalC C24 was localized in Na/K-enriched areas. The distribution of GalC was in small spots, homogeneous in size, of 0.8-1.5 microm. Sample preparation with high pressure freezing allowed separate localization of sodium and potassium in tissue samples.

Localization of cholesterol, phosphocholine and galactosylceramide in rat cerebellar cortex with imaging TOF-SIMS equipped with a bismuth cluster ion source.

Time-of-flight secondary-ion-mass-spectrometry (TOF-SIMS) was utilized to address the issue of co-localization of cholesterol, phosphocholine and galactosylceramide in rat cerebellar cortex. Rat cerebellum was fixed, freeze-protected by sucrose, frozen and sectioned by cryoultramicrotomy and dried at room temperature. The samples were analyzed in an imaging TOF-SIMS instrument equipped with a Bi(1-7)+-source. The cholesterol signal (m/z 369 and 385) was localized in Purkinje cells and in nuclei of granular layer cells. The phosphocholine headgroup of phosphatidylcholine and sphingomyelin was localized by imaging a specific fragment (m/z 86). This signal was localized in the molecular layer of cerebellar cortex, in Purkinje cells and in parts of the granular layer probably representing the synapse-rich glomeruli. The galactosylceramide was localized by imaging the quasi-molecular ions at m/z 835 and 851, showed a clear colocalization with cholesterol, but also a specific localization in dots (diameter

Imaging TOF-SIMS of rat kidney prepared by high-pressure freezing.

Phosphocholine, potassium ions, and sodium ions were localized in rat kidney with imaging TOF-SIMS. Tissue preparation was performed with high-pressure freezing, freeze-fracturing and freeze-drying. The distribution of sodium ions was visualized by imaging the signal at m/z 23 of positively charged secondary ions, and the distribution of potassium ions was visualized by imaging the signal at m/z 39. Potassium was found localized within cells of the proximal tubulus epithelium and within cells of the glomeruli. High signals of sodium ions were seen in the interstitial tissue and also in epithelial cells of the collecting ducts and in glomeruli. The overlay image showed that the distribution of sodium ions and potassium ions were largely complementary with color mixing in glomeruli and in the interstitium surrounding proximal tubules. The ion distribution was further analyzed by correlation analysis. Phosphocholine-containing phospholipids were visualized by imaging the phosphocholine head group at m/z 184 of positively charged ions. The m/z 184 signal shows a ubiquitous distribution with a high intensity of phosphocholine in epithelial cells. Overlay image of m/z 184, m/z 39, and m/z 23 and multivariate analysis showed that the localization of high levels of phosphocholine colocalizes with high levels of potassium ions, as expected for an ion with intracellular localization.