Live cell imaging by multifocal multiphoton microscopy

Multifocal multiphoton microscopy (MMM) permits parallel multiphoton excita tion by scanning an array of high numerical aperture foci across a plane in the sample. MMM is particularly suitable for live cell investigations sinc e it combines advantages of standard multiphoton microscopy such as optical sectioning and suppression of out-of-focus phototoxicity with high recordi ng speeds. Here we describe several applications of MMM to live cell imagin g using the neuroendocrine cell line PC12 and bovine chromaffin cells. Stai nings were performed with the acidophilic dye acridine orange and the lipop hilic dyes FM1-43 and Fast DiA as well as by transfection of the cells with GFP. In both bovine chromaffin and PC12 cells structural elements of nucle ar chromatin and the 3-D distribution of acidic organelles inside the cells were visualized. In PC12 cells differentiated by nerve growth factor examp les of neurites were monitored. Stainings of membranes were used to reconst ruct the morphology of cells and neurites in three dimensions by volume-ren dering and by isosurface plots. 3-D reconstructions were composed from stac ks of about 50 images each with a diameter of 30-100 mu m that were acquire d within a few seconds. We conclude that MMM proves to be a technically sim ple and very effective method for fast 3-D live cell imaging at high resolu tion.