Silver deposition on freeze-dried cells allows subcellular localization of cholesterol with imaging TOF-SIMS

Imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) was used for characterization and subcellular localization of organic ions in leucocytes adhering to glass surfaces. The cells were fixed by freeze drying in 0.15 m ammonium formate buffer at pH 7.2-7.4. The freeze-dried cells were sputter-coated with silver, and the silver surface was analysed with imaging TOF-SIMS. TOF-SIMS spectra were recorded by scanning the primary ion beam over the analysis area and acquiring positive mass spectra of the ions leaving the surface. The relative brightness of each pixel within the analysis area reflects the signal intensity of a selected ion in that pixel. Data were collected separately at high mass resolution m/delta m > 7000 and at high lateral resolution (= 0.5 micro m). The images were analysed by principal component analysis (PCA). The glass-adhering cells showed a well defined attachment area with a diameter of up to 20 micro m, and an equally well defined cell body, containing the nucleus, with a diameter of 8-10 micro m. On the raw data images, the obtained cholesterol distributions were consistent with a higher cholesterol content of the cell membrane in the attachment area than in the cell body. Using PCA analysis, silver-cationized molecular cholesterol was found localized mainly in the attachment area of the cells. Cholesterol was also seen at higher concentration in circular spots of