Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission

Abstract

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.

Figure 1

Microscope. (a) Excitation pulses are followed by stimulated emission depletion pulses for fluorescence inhibition. After passing dichroic mirrors and emission filters, fluorescence is detected through a confocal pinhole by a counting photodiode. (b) Measured excitation PSF. (c) Measured STED-beam-PSF featuring local minimum at the center and intense maxima above and below the focal plane. Z denotes optic axis. The measurements of b and c are carried out with the pinhole removed.

Figure 2

(a) Fluorescence is a nonlinear function of stimulating intensity; 10% remaining fluorescence is obtained for I_STED^T corresponding to P_STED of 2.2 mW in the focus. (b) Surface plot of XZ-section (Inset) of confocal fluorescence spot for 1.4 oil immersion lens. (d) Same as b but with STED-beam PSF switched on. (c) Corresponding axial intensity profiles demonstrate 5.1-fold reduction of the axial width (FWHM) from 490 nm down to 97 nm.

Figure 3

XZ-images of 100-nm-diameter fluorescent beads (a and b) and of 100-nm-diameter negatively stained glass beads agglomerations (c and d) as observed in the confocal (a and c) and the STED-fluorescence (b and d) microscope. Note the artifacts indicated by arrows induced by the elongated spot in the confocal image and their reduction in the STED counterpart.

Figure 4

Resolution improvement in live cells. XZ-images of a S. cerevisiae yeast cell with labeled vacuolar membranes with standard confocal resolution (a) and with axial resolution improved by STED (b). Whereas the confocal mode fails in resolving the membrane of small vacuoles, the STED microscopy better reveals their spherical structure. XZ-images of membrane-labeled E. coli show a 3-fold improvement of axial resolution by STED in d as compared with their simultaneously recorded confocal counterparts in c.