In STED microscopy, the specimen is illuminated by two synchronized ultrafast co-linear sources consisting of an excitation laser pulse followed by a red-shifted depletion laser pulse that is referred to as the STED beam. Generally, the excitation laser pulse width is of shorter duration than that of the STED pulse (although both are usually in the 10 to 300 picosecond range). Pulsed lasers take advantage of the time scales for molecular relaxation and interference of coherent light to produce radially symmetric depletion zones. Fluorophores positioned within the zero node region of the STED beam are allowed to fluoresce upon exposure to the excitation beam, whereas those fluorophores exposed to the STED beam are transferred back to their ground (non-fluorescent) state by means of stimulated emission. The non-linear depletion (following an exponential curve) of the excited fluorescent state by the STED beam constitutes the basis for imaging at resolutions that are below the diffraction barrier.
The tutorial initializes with the image of a STED wavefield superimposed on the excitation beam profile (labeled Excitation Laser Point-Spread Function). In order to operate the tutorial use the STED Laser Power slider to adjust the depletion laser power, which is indicated in a box adjacent to the slider. The Resolution produced at the focal point of the excitation laser is indicated in a box on the opposite side of the slider. Note how the resolution increases with increasing depletion laser power. The Wireframe radio button can be activated to view the STED beam in a wireframe model.
The doughnut-shaped depletion laser wavefield in STED effectively narrows the point-spread function of the excitation laser to increase resolution beyond the diffraction limit, which in the best cases can approach 20 nanometers in the lateral dimension. Sharpening of the focal spot through point-spread function engineering is thus equivalent to expanding the microscope spatial frequency passband. In order to obtain a complete image, the central zero produced by the STED lasers is raster scanned across the specimen, similar to the action of a confocal microscope. Among the benefits of STED microscopy are that the effective resolution increase is completely dictated by the experimental configuration and the laser powers applied to the specimen. Furthermore, the image is recorded as the beam scans along the specimen and requires no additional processing, and image acquisition times can approach the speed of any laser scanning confocal microscope. The effective resolution increase with STED is proportional to the power of the depletion laser, but can become problematic at extremely high laser powers that are likely to result in rapid photobleaching and destruction of the probe. Regardless, a wide range of fluorophores have been successfully used with STED, including fluorescent proteins, ATTO dyes, Alexa Fluors, DyLights, and several other synthetics.
Contributing Authors
Kevin A. John and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.