Focal Spots of Size l 23 Open Up Far-Field Florescence Microscopy at 33 nm Axial Resolution

We report spots of excited molecules of 33 nm width created with focused light of l 760 nm wavelength and conventional optics along the optic axis. This is accomplished by exciting the molecules with a femtosecond pulse and subsequent depletion of their excited state with red-shifted, picosecondpulsed, counterpropagating, coherent light fields. The l 23 ratio constitutes what is to our knowledge the sharpest spatial definition attained with freely propagating electromagnetic radiation. The sub-diffraction spots enable for the first time far-field fluorescence microscopy with resolution at the tens of nanometer scale, as demonstrated in images of membranes of bacillus megaterium.