4Pi-confocal microscopy of live cells

By coherently adding the spherical wavefronts of two opposing lenses, two-p hoton excitation 4Pi-confocal fluorescence microscopy has achieved three-di mensional imaging with an axial resolution 3-7 times better than confocal m icroscopy. So far this improvement was possible only in glycerol-mounted, f ixed cells. Here we report 4Pi-confocal microscopy of watery objects and it s application to the imaging of live cells. Water immersion of 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4. 3-fold better axial resolution as compared to the best water immersion conf ocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm ran ge are presented here, thus establishing a new resolution benchmark in live -cell microscopy. (C) 2001 Elsevier Science B.V. All rights reserved.