... the combination means optical nanoscopy for the first time.
3 x 5 = 15 ?
The setup: The 4Pi-STED-microscope is the result of combining the two unrelated concepts of STED- and 4Pi-microscopy. Here, the fluorescent sample is placed in the common focus of two opposing lenses, but excitation and detection are performed through a single lens. The green excitation pulse is immediately followed by a red STED-pulse, which enters the focal region through both lenses inducing stimulated emission of the excited fluorescent molecules to the ground state .
The combination: To reduce the size of the focal spot, the PSF of the STED irradiation has to vanish at the center of the focal region, but must be high elsewhere, as we have demonstrated in our STED-microscope. This can be accomplished by employing a standing wave. A planar standing wave, however, exhibits many minima in which fluorescence would still be present. To create a single minimum, we make use of the large focusing angle and symmetry of the 4Pi-microscope .
From λ/3 to λ/23: The "diffraction barrier" of about realized by Ernst Abbe in 1873 is no longer limiting the resolution in light microscopy. In our experiments, we have shown spots of excited molecules of 33 nm width with focused light at 760 nm. These sub-diffraction spots enable for the first time far-field microscopy with resolution at the tens of nanometer scale .
Biological image: The upper figure shows a standard confocal xz-image of membrane labeled bacillus megaterium. In comparison to the STED-4Pi-counterpart shown on the picture below, the figure reveals the vastly improved axial resolution, but also exhibits the side-lobe effects due to incomplete supression of the side minima. Fortunately, the effect of the lobes can be dealt with mathematical filtering which is shown in the lower image .
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.
10 Dyba, M. and S. W. Hell (2002). "Focal spots of size λ/23 open up far-field fluorescence microscopy at 33 nm axial resolution." Phys. Rev. Lett. 88: 163901.