The fundamental principle behind stochastic optical reconstruction microscopy (STORM) and related methodology is that the activated state of a photoswitchable molecule must lead to the consecutive emission of sufficient photons to enable precise localization before it enters a dark state or becomes deactivated by photobleaching. Additionally, the sparsely activated fluorescent molecules must be separated by a distance that exceeds the Abbe diffraction limit (in effect, greater than approximately 250 nanometers) to enable the parallel recording of many individual emitters, each having a distinct set of coordinates in the lateral image plane. This interactive tutorial explores the various steps associated with acquiring a typical STORM image.
The tutorial initializes with a widefield image of the specimen appearing in the left-hand window entitled Specimen adjacent to a blank window named Resolved Image. In order to operate the tutorial, use the mouse cursor to translate the STORM Process slider to progress through the various stages involved in acquiring a STORM image. Each step is briefly described in the text box beneath the image windows as the tutorial transitions through the process. The superresolution image is slowly assembled and displayed in the Resolved Image window as the slider moves from left to right. In order to view a different specimen, choose an alternative candidate from the pull-down menu.
The basic steps involved in creating a STORM superresolution image are presented using a series of cartoon drawings in Figure 1. The target structure illustrated in Figure 1(a) shows a hypothetical densely labeled filamentous network of intracellular structures that can represent cytoskeletal biopolymers constructed with monomeric units of tubulin or actin. In Figure 1(b), a sparse set of the fluorescent probes are activated to produce single-molecule images (represented by orange circles) that do not overlap.
After capturing the images with a digital camera, the point-spread functions of the individual molecules are localized with high precision based on the photon output before the probes spontaneously photobleach or switch to a dark state. The positions of localized molecular centers are indicated with black crosses. The process is repeated in Figures 1(c) through 1(e) until all of the fluorescent probes are exhausted due to photobleaching or because the background fluorescence becomes too high. The final superresolution image (Figure 1(f)) is constructed by plotting the measured positions of the fluorescent probes.
Joel S. Silfies and Stanley A. Schwartz - Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York, 11747.
Stephen P. Price and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.