The existence of generic and relatively long-lived dark states, such as triplet or radical-ion states, exhibited by many synthetic fluorescent probes can be exploited to temporarily photoswitch single molecules into an off state for stochastic readout in the construction of super-resolution images. These photoinduced switching mechanisms can be controlled with the careful selection of buffers and laser power for both targeted (point-spread function engineering techniques, such as stimulated emission depletion; STED) and stochastic readout (single-molecule techniques, such as stochastic optical reconstruction; STORM) microscopy. This interactive tutorial explores the assembly of super-resolution images using molecular blinking techniques, as performed using Nikon's N-STORM system.
The tutorial initializes with a series of images resulting from irradiation of a specimen labeled with synthetic probes using high laser power in the Single-Molecule Image window. As the tutorial progresses, the corresponding super-resolution image is progressively assembled in the adjacent Super-resolution Image window. The stage of the process can be controlled using the Tutorial Progress slider, or the tutorial can be automatically run using the Auto button. Alternative image sets are available using the pull-down menu.
A large number of synthetic probes, including the Alexa Fluor, ATTO, and carbocyanine dyes, have been demonstrated to stochastically photoswitch under the appropriate conditions and are therefore useful in super-resolution microscopy techniques based on stochastic readout where the ability to control photoswitching of single emitters is critical.
Stanley A. Schwartz - Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York, 11747.
Alex B. Coker, Sunita Martini, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.