direct Stochastic Optical Reconstruction Microscopy
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 resolution of better than 20 nm. While Eric Betzig and collaborators, now with the Howard Hughes Medical Institute at the 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). Using a somewhat different approach using organic fluorophores, Xiaowei Zhuang and collaborators from Harvard University developed STORM “Stochastic Optical Reconstruction Microscopy”.
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 so far optically unresolvable structures consisting of fluorescent molecules by utilizing special properties of photoactivatable proteins or 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, 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 in a special buffer system is used, is therefore called directSTORM (dSTORM). 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. Further applications of photoswitching microscopy are comprised of dynamic studies in living cells and quantitative high-resolution fluorescence imaging, e.g. studies that count the number of biomolecules and their structural organization in small subcellular structures or membranes.
Publications in this area from our group
Heilemann, M., van de Linde, S., Schüttpelz, M., Kasper, R., Seefeldt, B., Mukherjee, A., Tinnefeld, P., Sauer, M.: Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angewandte Chemie - International Edition 47 (33): 6172-6176, 2008.
Wolter, S., Schüttpelz, M., Tscherepanow, M., van der Linde, S., Heilemann, M., Sauer, M.: Real-time computation of subdiffraction-resolution fluorescence images. Journal of Microscopy 237 (1): 12-22, 2010.
Mönkemöller, V., Schüttpelz, M., McCourt, P., Sørensen, K., Smedsrød, B., Huser, T.: Imaging fenestrations in liver sinusoidal endothelial cells by optical localization microscopy. Physical Chemistry Chemical Physics 16 (24): 12576-12581, 2014.