Stimulated emission depletion microscopy (STED and the related techniques of ground state depletion (GSD and saturated structured illumination (SSIM) are referred to as ensemble focused light imaging techniques, and are based on non-linear optical effects that typically require the application of multiple high-intensity pulsed lasers with specialized modulation filters to control the excitation beam geometry (a technique commonly termed point-spread function engineering). STED instruments utilize a raster-scan imaging scenario similar to a laser-scanning confocal microscope. In contrast, stochastic optical reconstruction microscopy (STORM), as performed using Nikon's N-STORM system, is a single-molecule approach that relies on activation of a limited subset of the overall molecular population to sequentially image and localize individual emitters on a temporal basis. This interactive tutorial explores the similarities and differences of STED and STORM microscopy.
The tutorial initializes with a pair of virtual cells appearing in the window showing the nucleus (dark gray) and surrounding cytoplasm (light gray). The photoswitchable fluorophores are represented in the cells as small green spheres. In order to operate the tutorial, click on the Auto button and adjust the Speed control. As the STED cell is scanned by a combination of excitation and depletion lasers (green and orange), fluorophores in the STORM cell are sequentially photoactivated, localized, and photobleached (marked with a + symbol). Note that STED is a scanning technique, whereas STORM is a stochastic technique that images subsets of single molecules.
Stanley A. Schwartz - Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York, 11747.
Sunita Martini and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.