In the recent past, a variety of methods have been developed to circumvent the diffraction barrier of light which restricts
optical resolution to about 200 nm in the image plane. Single-molecule based photoswitching microscopy such as direct
stochastic optical reconstruction microscopy (dSTORM) has been successfully implemented for subdiffraction-resolution
fluorescence imaging. The major drawback of this technique has been that the reconstruction of subdiffraction-resolution
images requires substantially more time than the actual experiment and prevented real-time imaging. Here we present a
new computational algorithm enabling subdiffraction-resolution fast imaging of cellular structures with ~20 nm optical
resolution in less than 10 seconds.
High-resolution fluorescence imaging has a vast impact on our understanding of intracellular organization. The
key elements for high-resolution microscopy are reversibly photo-switchable fluorophores that can be cycled
between a fluorescent and a non-fluorescent (dark) state and can be localized with nanometer accuracy. For
example, it has been demonstrated that conventional cyanine dyes (Cy5, Alexa647) can serve as efficient photoswitchable
fluorescent probes. We extended this principle for carbocyanines without the need of an activator
fluorophore nearby, and named our approach direct stochastic optical reconstruction microscopy (dSTORM).
Recently, we introduced a general approach for superresolution microscopy that uses commercial fluorescent
probes as molecular photoswitches by generating long lived dark states such as triplet states or radical states.
Importantly, this concept can be extended to a variety of conventional fluorophores, such as ATTO520, ATTO565,
or ATTO655. The generation of non-fluorescent dark states as the underlying principle of superresolution
microscopy is generalized under the term photoswitching microscopy, and unlocks a broad spectrum of organic
fluorophores for multicolor application. Hereby, this method supplies subdiffraction-resolution of subcellular
compartments and can serve as a tool for molecular quantification.