Abstract
A secondary ion mass spectrometry (SIMS)-based isotopic imaging technique of ion microscopy was used for observing calcium influx in single renal epithelial LLC-PK(1) cells. The CAMECA IMS-3f SIMS instrument, used in the study, is capable of producing isotopic images of single cells at 500 nm spatial resolution. Due to the high-vacuum requirements of the instrument the cells were prepared cryogenically with a freeze-fracture method and frozen freeze-dried cells were used for SIMS analysis. The influx of calcium was imaged directly by exposure of cells to (44) Ca stable isotope in the extracellular buffer for 10 min. The (44) Ca influx was measured at mass 44 and the distribution of endogenous calcium at mass 40 ((40) Ca) in the same cell. A direct comparison of interphase cells to cells undergoing division revealed that calcium influx is restricted in metaphase and post-metaphase stages of cell division. This restriction is lifted in late cytokinesis. The net influx of (44) Ca in 10 min was approximately half under calcium influx restriction in comparison to interphase cells. Under calcium influx restriction the (44) Ca concentration was the same between the mitotic chromosome and the cytoplasm. These observations indicate that the endoplasmic reticulum (ER) calcium uptake is compromised under calcium influx restriction in cells undergoing division.