Relative Quantification of Exogenous Phospholipids Regulating Exocytosis by TOF SIMS of Hydrated Single Cells

Phospholipids in single cells have been studied by time of flight secondary ion mass spectrometry (TOF SIMS) in combination with a newly developed spring-loaded freeze fracture device. The freeze fracture device sandwiches the cell suspension between two silicon shards and is fast frozen in liquid propane at liquid nitrogen temperature. This procedure provides a snapshot of the chemical distribution in a living hydrated cell. A TOF SIMS IV instrument from ION-TOF GmbH equipped with a Bi liquid metal cluster primary ion source to image cells under static conditions was used.Single frozen hydrated rat pheochromocytoma (PC12) cellshave been used. These cells have diameters between 10 and 20 µm and are commonly used to study exocytosis. In amperometric studies we have found that incubation with lipids can alter the rate of exocytosis, apparently by altering the membrane composition. By use of a Bi cluster primary ion source, sub cellular images of phospholipids in single PC12 cells have been obtained without the need of exogenous labeling. Fragment ions from phosphatidylcholine (PC) are found at m/z 184 and 224 and fragment ions from phosphoethanolamine at m/z 124 and 142.Using the freeze fracture device we relatively quantify exogenous deuterized phospholipids that are incorporated into the plasma membrane by PC12 cells during incubation. Incubation under the same conditions as for the amperometry experiments results in membrane composition changes that are a fraction of a percent, apparently indicating that a very small change in composition leads to a significant change in the rate of exocytosis.