STED-Microscopy...
 
... overcomes the diffraction limit in a fundamental way.
 
Scientists involved:
    
Marcus Dyba
Alexander Egner
Stefan Jakobs
 
The perhaps most straightforward way to sharpen the fluorescence focal spot is to selectively inhibit the fluorescence at its outer part [1, 2]. If this is applied to an otherwise diffraction-limited spot, one would expect that the diffraction barrier can be overcome since scanning with a smaller fluorescent spot signifies increased spatial resolution. A phenomenon that stops fluorescence (=spontaneous emission) is that of stimulated emission. This is one of the key ingredients of the Stimulated Emission Depletion (STED-) microscope. However, STED by itself could not really break the diffraction barrier since the beams with which STED is accomplished are diffraction-limited as well. Therefore the real physical ingredient for breaking the diffraction barrier is the saturation of the fluorescence inhibition by stimulated emission, as we will argue below.
 
The setup: The STED-microscope relies on pairs of synchronized laser pulses [1, 2, 20]. To this end, excitation is performed by a subpicosecond laser pulse that is tuned to the absorption spectrum of the dye. The excitation pulse is focused into the sample, producing an ordinary diffraction limited spot of excited molecules. The excitation pulse is immediately followed by a depletion pulse, dubbed 'STED-pulse'. The STED pulse is red-shifted in frequency to the emission spectrum of the dye, so that its lower energy photons act ideally only on the excited dye molecules, quenching them to the ground state by stimulated emission. The net effect of the STED pulse is that the affected excited molecules cannot fluoresce because their energy is dumped and lost in the STED pulse. By spatially arranging the STED pulse in a doughnut mode, only the molecules at the periphery of the spot are ideally quenched [9, 21]. In the center of the doughnut, where the STED pulse is vanishing, fluorescence ideally remains unaffected. Click on the image for enlargement!
No resolution limit: By increasing the STED pulse intensity, the depletion becomes complete at the spot's periphery and increasingly more effective towards the middle. At the doughnut hole, however, the fluorescence is ideally not affected at all. Therefore, by increasing the intensity of the doughnut-shaped STED-pulse, the fluorescent spot can be progressively narrowed down, in theory, even to the size of a molecule. This concept signifies a fundamental breaking of the diffraction barrier. The essential ingredient is the saturated reduction of the fluorescence (= depletion) at any coordinate but the focal point.
Comparison with confocal fluorescence microscopy: This microscopy is in stark contrast to the presently known superresolution methods like the confocal, the multiphoton or related fluorescence microscopes, which can never surpass Abbe's barrier by more than a factor 2. In a way, confocal fluorescence and two-photon microscopes just cross the diffraction border, without breaking it. The resolution of these systems is still limited by diffraction, in contrast to the STED-microscope [1].
Depletion means saturation: The real physical reason for the breaking of the diffraction barrier is not the fact that fluorescence is inhibited, but the saturation (of the fluorescence reduction). Fluorescence reduction alone would not help since the focused STED-pulse is also diffraction-limited. What does saturation mean in this context? Whereas the fluorescence at the middle of the doughnut is unaffected, it is fully stopped at the closest proximity of the doughnut. Thus the fluorescent region is continuously narrowed down without limit! [2]
Fundamentally enlarged passband of the optical transfer function: It is clear that the decrease in spatial extent of the effective spot or point-spread-function in a STED-microscope is associated with a fundamental increase of the passband of the effective transfer function of the microscope. The STED-microscope is not a diffraction-limited system anymore. It is the first to provide conceptionally unlimited optical resolution, in spite of the fact that it relies on visible light and regular objective lenses [1, 2].
Experiments: To date an improvement beyond the diffraction barrier of 3 in the transverse direction and up to 6 along the optical axis has been experimentally demonstrated. The viability of the STED-concept has been exemplified in a number of simple experiments. Its practicability and the maximum spatial resolution depend very much on the level of saturation that can be obtained and on the deepness of the doughnut hole, which should be ideally zero. So far, experiments show that the level of saturation will be determined by the bleaching that is inflicted on the dye. Moreover, it will be interesting to see to which extent dyes can be switched off and if STED is applicable to all dyes, including those that are endogenous to the cell [21]. Click on the image for enlargement!
Ground-State-Depletion- (GSD) Microscopy, a cousin of STED: An alternative to quenching the excited state is to deplete the ground state of the dye. This depletion could be achieved by shelving the dye into the triplet state or another long-lived state. As in the concept of STED, the real ingredient is the saturation of the depletion. Saturation entails a non-linear relationship between the (residual) fluorescence and the applied intensity [2].
[1]
Hell, S. W. and J. Wichmann (1994). "Breaking the diffraction resolution limit by stimulated emission." Opt. Lett. 19(11): 780-782.
[2]
Hell, S. W. (1997). "Increasing the Resolution of Far-Field Fluorescence Microscopy by Point-Spread-Function Engineering." Topics In Fluorescence Spectroscopy; 5: Nonlinear and Two-Photon-Induced Fluorescence, edited by J. Lakowicz. Plenum Press, New York: 361-426.
[9]
Klar, T. A., S. Jakobs, M. Dyba, A. Egner and S. W. Hell (2000). "Fluorescence microscopy with diffraction resolution limit broken by stimulated emission." Proc. Natl. Acad. Sci. USA 97(15): 8206-8210.
[20]
Hänninen, P. E. and S. W. Hell (1994). "Femtosecond pulse broadening in the focal region of a two-photon fluorescence microscope." Bioimaging 2: 117-122.
[21]
Klar, T. A., E. Engel and S. W. Hell (2001). "Breaking Abbe's diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes." Phys. Rev. E 64: 066613, 066611-066619.
 
 List of reported STED dyes now online.