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5675916 
Journal Article 
Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission 
Klar, TA; Jakobs, S; Dyba, M; Egner, A; Hell, SW 
2000 
1 
Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
EISSN: 1091-6490 
97 
15 
8206-8210 
English 
10899992 
The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up to 6 times beyond the diffraction barrier. The simultaneous 2-fold improvement in the radial direction rendered a nearly spherical fluorescence spot with a diameter of 90-110 nm. The spot volume of down to 0.67 attoliters is 18 times smaller than that of confocal microscopy, thus making our results also relevant to three-dimensional photochemistry and single molecule spectroscopy. Images of live cells reveal greater details.