Localisation of organic ions with imaging TOF-SIMS

Abstract

A new method for localisation and determination of organic ions in cells has been developed. Based on rapid freezing of polymorphonuclear leukocytes (PMNLs) and short time freeze-drying in a volatile buffer, it was possible to produce cell samples with highly preserved morphology. The cell samples were imprinted on silver surfaces and the silver surfaces were analysed by time of flight secondary ion mass spectrometry (TOF-SIMS). The imprinting technique was characterised by immunofluorescence and scanning electron microscopy (SEM), which showed that both cell membranes and cell nuclei were transferred to the silver surfaces.With the use of TOF-SIMS it was possible to detect organic ions from several lipid species as well as unfragmented, Ag-cationised molecules, at a spatial resolution of < 1 µm. High resolution TOF-SIMS images (lateral resolution < 1 mm, m/Dm = 800 - 1000) and high-resolution spectra (lateral resolution 3-4 mm, m/Dm = 7000 - 8000) were recorded separately. The method was used to study the spatial distribution of phosphatidylcholine and cholesterol in cells. The complementary nature between phosphocholine and cholesterol could be seen in the TOF-SIMS ion images. Cholesterol could be seen preferentially located in the plasma membrane, while the phosphocholine showed highest concentration in the nuclear membranes. By stimulating the PMNLs with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (f-MLP), a reorganisation of cholesterol in the TOF-SIMS ion images could be seen between unstimulated and f-MLP stimulated cells. The reorganisation could also be seen with flotillin-1 antibody immunofluorescence. This suggests the involvement of cholesterol-rich microdomains, rafts, during PMNL peptide stimulation. TOF-SIMS imaging proved to be a suitable method for localisation of lipid membrane species on silver imprints of freeze-dried human polymorphonuclear cells. The technique makes it possible to directly visualise cholesterol and phosphocholine organisation in cell membranes.