Interactive Tutorials - Spinning Disk Fundamentals

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Superresolution Microscopy

Superresolution Microscopy with STED

The first technique successfully applied to superresolution biological imaging of fixed cells was the RESOLFT method named stimulated emission depletion (STED). This approach employs spatially modulated and saturable transitions between two molecular states to engineer improvements to the point-spread function. 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.

STED takes advantage of the RESOLFT concept by significantly modifying the shape of the excitation point-spread function by manipulating the phase, pulse width, and intensity of the excitation and depletion lasers. Although both lasers remain diffraction-limited as their beams pass through the microscope optical train, the STED pulse is modified by a phase modulator to feature a zero-intensity node at the center of focus with exponentially growing intensity toward the periphery. This configuration gives rise to a doughnut-shaped beam that surrounds the central focal point (and point-spread function) of the excitation laser. Only at the exact center of focus (the node) is the intensity of the STED beam equal to zero. The wavelength and duration of the STED beam pulse are chosen to coincide with the emission maximum and saturation intensity, respectively, of the fluorophore under investigation. Deactivation of the fluorophores occurs throughout the focal volume except at the center of focus. At the high depletion laser powers used for STED (often exceeding 250 megawatts per square centimeter), the fluorophores are almost instantaneously driven to the ground state by stimulated emission. Substantially reducing the laser power (as discussed below) enables formation of a non-fluorescent state via a number of other mechanisms, including driving the fluorophores into a metastable triplet state, formation of charge-transfer states, or photoswitching.

Contributing Authors

Stephen P. Price and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.