Superresolution microscopy using stimulated emission depletion (STED) creates sub-diffraction limit features by altering the effective point spread function of the excitation beam using a second laser that suppresses fluorescence emission from fluorophores located away from the center of excitation. The suppression of fluorescence is achieved through stimulated emission that occurs when an excited-state fluorophore encounters a photon that matches the energy difference between the ground and excited state. Upon interaction of the photon and the excited fluorophore, the molecule is returned to the ground state through stimulated emission before spontaneous fluorescence emission can occur. Thus, the process effectively depletes selected regions near the focal point of excited fluorophores that are capable of emitting fluorescence.
The tutorial initializes with a grayscale image appearing in the Widefield Image window and the corresponding STED image in the adjacent window. The tutorial runs automatically with the STED beam raster-scanning the specimen starting at the upper left-hand portion of the window and proceeding down. The STED focal spot is simulated by a small green sphere (the illumination point spread function) surrounded by the STED beam (orange). As the specimen is scanned, a superresolution STED image is produced in the right-hand window. The pull-down menu can be used to select alternative specimens, and the checkbox can be enabled to allow the tutorial to loop.
STED microscopy operates by using two laser beams to illuminate the specimen. An excitation laser pulse (generally created by a multiphoton laser) is closely followed by a doughnut-shaped red-shifted pulse that is termed the STED beam. Excited fluorophores exposed to the STED beam are instantaneously returned to the ground state by means of stimulated emission. The non-linear depletion of the fluorescent state by the STED beam is the basis for superresolution. Even though both laser pulses are diffraction-limited, the STED pulse is modified to feature a zero-intensity point at the center of focus with strong intensity at the periphery. When the two laser pulses are superimposed, only molecules that reside in the center of the STED beam are able to emit fluorescence, thus significantly restricting emission. This action effectively narrows the point spread function and ultimately increases resolution beyond the diffraction limit. To generate a complete image, the central zero is raster-scanned across the specimen in a manner similar to single-photon confocal microscopy, as illustrated in the tutorial. STED microscopy is capable of 20 nanometer (or better) lateral resolution and 40 to 50 nanometer axial resolution.
Contributing Authors
Tony B. Gines and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.