dSTORM image of the tubulin cytoskeleton of U2OS cells where the depth of the tubulin fibers along the vertical axis was resolved by using astigmatism in the detection optics introduced by the addition of a cylindrical lens.




General description of single molecule localization microscopy (SMLM) techniques

The potential of utilizing single molecule fluorescence for achieving optical superresolution was initially postulated in a short and somewhat cryptic paper by Betzig, published in 1995. It took until the year 2006, before three research groups independently demonstrated similar optical reconstruction methods with a lateral sub-diffraction optical resolution of better than 20 nm. While Eric Betzig and collaborators, now with the Howard Hughes Medical Institute at the HHMI Janelia Farm Research Campus, termed this method “Photoactivated Localization Microscopy” (PALM), S. T. Hess and colleagues from the University of Maine called it “Fluorescence Photoactivation Localization Microscopy” (FPALM).

The importance of breaking the diffraction limit is demonstrated by the award of the Nobel Prize in Chemistry 2014 to Eric Betzig (UC Berkeley, LBNL, and HHMI Janelia Research Campus), Stefan W. Hell (MPI for Biophysical Chemistry and University of Göttingen; German Cancer Research Center, MPI for Medical Research, and University of Heidelberg) and W.E. Moerner (Stanford University) for the development of super-resolved fluorescence microscopy and the recent Breakthrough Prize in Life Sciences 2019 awarded to Xiaowei Zhuang for the development of Stochastic Optical Reconstruction Microscopy (STORM).

These photoswitching techniques all have in common that they exploit the temporal separation of single fluorescent emitters, even if the molecules cannot be isolated in space due to the diffraction limit: Multiple localizations of single molecules obtained in a series of images are used to reconstruct a super-resolved image based upon the positions obtained from the localization algorithm.

All of these methods overcome the problem of resolving structures with sizes below the optical diffraction limit and which consist of fluorescent molecules by utilizing special properties of photoactivatable proteins or photo-switchable fluorophores that make it possible to tell if the fluorescence emission was due to a single molecule or not. FPALM uses fluorescent proteins such as the photoactivatable green fluorescent protein (GFP), while PALM also makes use of photoswitchable proteins. The first fluorophores that were used for STORM were carbocyanine fluorophores, which exhibit photoswitching properties in the presence or absence of an activator molecule. The latter method, where the bare fluorophore is used in a special buffer system, is therefore called directSTORM (dSTORM)1,2. Fluorophores are either switched between a fluorescent bright (“on”) and a non-fluorescent dark (“off”) state upon illumination with light of different wavelengths (PALM/STORM) or they are photoactivated and subsequently photobleached (FPALM).

Due to their technically relatively simple implementation, wide-field single-molecule based localization approaches such as PALM, STORM, and dSTORM are currently widely used for super-resolution imaging. It is even possible to gain an improved axial resolution by introducing astigmatism due to a cylindrical lens into the detection optics or by shaping the detected point spread function (PSF) of a single emitter with adaptive optics. The distortions that this causes to the PSF can be characterized and enables one to gain a super-resolved image with up to several microns depth. Further applications of photoswitching microscopy are comprised of studying slow dynamics in living cells anchored to a substrate and quantitative high-resolution fluorescence imaging, e.g. studies that count the number of biomolecules and their structural organization in small subcellular structures, e.g. organelles or the cell membrane. Super-resolution optical microscopy techniques are also attracting attention in the fields of chemistry and material science, e.g. by resolving the morphology of microgel particles via dSTORM measurements.