ABSTRACT
From the early 1990s onward, several methods of signi cantly improving resolution have been developed and continue to improve. One method of note is stimulated-emission depletion (STED) (Hell and Wichmann, 1994). is method can increase t he lateral resolution to b eyond t he d i raction l imit in far- eld uorescence microscopy, and works by exploiting STED in the sample (Klar and Hell, 1999).
It requires two laser beams, one for uorescence excitation and one for depleting the excitation of the surrounding area. is second so-called STED beam typically has a z ero in the beam intensity at t he center o f t he focus, a nd i s u sed to de plete t he e xcited s tates o f t he mole cules i n t he outer re gion of the focal spot. e depletion is done by stimulated emission, which has the advantage that the emitted uorescence and the STED beam have the same wavelength, and both may be eliminated by the use of a suitable optical lter. is leaves a subdi raction l imited spot at t he center of the focal spot, where uorescence c an b e gener ated b y t he e xcitation b eam. e ex citation bea m must, t herefore, be o f a di erent wavelength. If the STED beam intensity is high enough, saturation of the STED process will occur, which can further reduce the e ective dimensions of the uorescence-emitting spot at the center of the beams. is technique has been shown to be able to produce an improvement in the resolution of close to 10 times, when compared with conventional optical microscopy of uorescent samples (Hell 2003). e s aturation e ect comes i nto play i n STED m icroscopy by en hancing t he depletion of t he excitation of areas around the center of the focal spot. Saturation typically has the e ect of “squaring o ” the edges of a smooth function (which is in this case the intensity pro le of the STED beam) and e ectively increases the abruptness of the transition between the depleted region in the sample and the uorescence-emitting region at t he center of t he focal spot. For STED, it i s t he geometrical e ect of the saturation that improves the resolution above what would be possible with standard beam-shaping techniques. If the STED beam intensity pro le appears as a do nut shape, and the uorescence signal of i nterest i s gener ated f rom t he region i nside t he donut hole, t hen t he re solution c an be a rbitrarily increased by decreasing the size of the hole. e optimization of the beam-forming optics alone cannot reduce the hold at t he center of the STED beam but i f the intensity of the STED beam increases and saturates, then the shoulders of the STED beam encroach further and further on the donut hole, reducing the e ective size of the spot that generates the uorescence signal.
