header image life science

Life Science

Stimulated Emission Depletion Microscopy (STED)

Imaging beyond the optical diffraction limit

Stimulated emission depletion microscopy (STED) is a fluorescence microscopy technique that overcomes the diffraction limited resolution of confocal microscopes. The resolution enhancement is essentially based on switching off the fluorescence of dye molecules by stimulated emission using intense laser light in the outer regions of the diffraction limited excitation focus. This intense radiation causes almost all of the excited molecules to return to the ground state. Fluorescence from the remaining excited dye molecules in the center of the excitation focus is then detected and used to form the high resolution images. An even further resolution enhancement  is possible by applying time gates to the collected data (gated STED or gSTED). As STED creates an effectively smaller observation volume, it can also be applied to other methods such as FCS. In that case, collecting data at different observation volume diameters can help to disentangle complex 2D diffusion scenarios in heterogeneous samples such as biological membranes.

Scheme of a typical STED set-upThe STED method uses pairs of synchronized laser pulses. The first laser pulse generated by, e.g. a picosecond pulsed diode laser, is used to excite the fluorescence dye and produces an ordinary diffraction limited focus. The excitation pulse is immediately followed by a depletion pulse, which is red-shifted in frequency to the emission spectrum of the dye. By spatially arranging the STED pulse in a doughnut shape using specially designed phase plates, only the fluorescence from molecules at the periphery of the excitation focus is quenched via stimulated emission. In the center of the doughnut, where the STED laser intensity is zero, fluorescence remains unaffected and is detected by single-photon sensitive detectors (e.g., Single Photon Avalanche Diodes, SPADs).

Consequently the essential components of a STED microscope set-up are:

  • pulsed laser source for excitation and depletion
  • single photon sensitive detector
  • dichroic mirror (to overlay excitation and depletion lasers and to separate fluorescence signal from excitation light)
  • phase plate for transforming the STED pulse into a doughnut shape
  • objective
  • data acquisition unit, e.g., a TCSPC module
  • suited scanning system

Nobel Prize winner Stefan W. Hell talks about the origins of STED microscopy