During STORM, single fluorophores “blink” by a process of random activation from an off or dark state, to an on or emission state, quickly followed by a switch back to a dark state or photobleaching. This process is sequentially repeated many times until most fluorophores have been imaged. For a successful STORM imaging, individual fluorophores must be sparse enough, so that only one molecule is activated within a diffraction-limited region at any given time.

In the STORM reconstruction process, individual molecules can be precisely localized by determining their position coordinates from the photons detected for each activation event (often as a Gaussian function).

By stochastically imaging small subsets of photoswitchable fluorophores over time, STORM microscopy allows to spatially resolve the localization of individual molecules with high precision even in dense populations. STORM microscopes can produce high-quality images of cellular components resolved to under 20 nm, changing the way scientists visualize molecular structures, interactions within organelles and biological processes.

While STORM is an excellent localization-based super-resolution technique, its use for live imaging is limited, mainly due to phototoxicity issues. In this case, PALM microscopy, which uses different photoactivatable fluorophores, is better suited for live imaging and single-particle tracking. Equally, STORM requires specific antibodies for the target of choice, if such antibodies are not available, PALM can be an alternative.

There are two variants of STORM. The first one, known as direct STORM or dSTORM microscopy, is compatible with many commonly used fluorophores, which can be converted to an off state using specific excitation parameters and in combination with specialized oxygen-scavenging imaging buffers. Ideal fluorophores for STORM should be very bright, have a high rate of photoswitching and exhibit minimal photobleaching in thiol-containing buffers. Some of the most common dSTORM fluorophores are Alexa Fluor™ and CFⓇ Dyes.

The second type of STORM microscopy uses a pair of activator-reporter dyes; the “activator” induces a switching and the “reporter” emits the signal detected. While the multiple activator-report pairs available allow multicolor STORM applications, this system requires dual labelling of the same target antibody and relies on the proximity of the two fluorophores. STORM without the activator dye, dSTORM, is currently widely used with different report dyes, as the photoswitching induced is efficient enough with substantial laser power.