Handbook Of Biological Confocal Microscopy

pp 561-570

4Pi Microscopy

  • Jörg BewersdorfAffiliated withMax Planck Institute for Biophysical Chemistry
  • , Alexander EgnerAffiliated withMax Planck Institute for Biophysical Chemistry
  • , Stefan W. HellAffiliated withMax Planck Institute for Biophysical Chemistry

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The axial (z-) resolution of any fluorescence microscope using a single lens is limited by diffraction to >500nm. While a modest improvement by up to a factor of 2 may be achieved by mathematical deconvolution, a substantial improvement of the axial resolution requires a radical change of the physics of imaging Since the 1990s, two families of methods have evolved that accomplished substantially improved axial resolution in threedimensional (3D) imaging. The first family, comprising 4Pi microscopy and I5M, coherently combines the aperture of two opposing lenses (Hell and Stelzer, 1992a, 1992b; Gustafsson et al., 1995, 1999; Eģner and Hell, 2005). The second family, of which stimulated emission depletion (STED) microscopy (Hell and Wichmann, 1994; see also Chapter 31, this volume) is the most established member, exploits photophysical or photochemical properties of the dye to break the diffraction barrier.