4Pi-confocal microscopy of live cells

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion 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 confocal 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 range are presented here, thus establishing a new resolution benchmark in live cell microscopy.

[1]  J. Pawley,et al.  Handbook of Biological Confocal Microscopy , 1990, Springer US.

[2]  S. Paddock,et al.  Confocal laser scanning microscopy. , 1999, BioTechniques.

[3]  J. E. Celis,et al.  Cell Biology: A Laboratory Handbook , 1997 .

[4]  H. M. Voort,et al.  Restoration of confocal images for quantitative image analysis , 1995 .

[5]  G. M. P. VAN KEMPEN,et al.  A quantitative comparison of image restoration methods for confocal microscopy , 1997 .

[6]  S. Hell,et al.  Properties of a 4Pi confocal fluorescence microscope , 1992 .

[7]  S W Hell,et al.  Far‐field fluorescence microscopy with three‐dimensional resolution in the 100‐nm range , 1997, Journal of microscopy.

[8]  Martin Schrader,et al.  Three-dimensional super-resolution with a 4Pi-confocal microscope using image restoration , 1998 .

[9]  Pekka Hänninen,et al.  Two-photon excitation 4Pi confocal microscope: enhanced axial resolution microscope for biological research , 1995 .

[10]  E. H. Linfoot.  Principles of Optics , 1961 .

[11]  K Bahlmann,et al.  Polarization effects in 4Pi confocal microscopy studied with water-immersion lenses. , 2000, Applied optics.

[12]  S W Hell,et al.  4Pi-confocal microscopy provides three-dimensional images of the microtubule network with 100- to 150-nm resolution. , 1998, Journal of structural biology.

[13]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[14]  S. Hell,et al.  Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index , 1993 .

[15]  Andrew G. Glen,et al.  APPL , 2001 .

[16]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.