(direct) Stochastic Optical Reconstruction Microscopy ((d)STORM) is a new superresolving technique which enables resolution beyond the classical diffraction limit by localizing fluorescent single molecules with high precision. This requires special fluorescent dyes which switch between a fluorescent and a non-fluorescent state. During measurement, a small number of dye molecules are continuously activated in a stochastic manner, allowing precise position estimation. After photobleaching or switching back into the dark state, the next cycle of activation and detection follows. The combination of the gathered molecule positions results in a superresolved reconstruction of the structure of interest. Since this method requires longer acquisition times than classical fluorescence microscopy, the samples usually have to be fixed.
We have three different setups for (d)STORM microscopy at our disposal. The first one is a commercial Nikon Eclipse Ti-E TIRF microscope which combines the high lateral resolution of STORM with the vertical resolution of TIRF microscopy.
The second setup is a customized Nikon Eclipse Ti-E microscope which employs so-called astigmatic detection (B. Huang, W. Wang, M. Bates, and X. Zhuang. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 2008, 319(5864):810-813.). Here, estimation of the axial coordinate is enabled by an additional cylindrical lens in the beam path which leads to a z-dependent ellipticity of single molecule images. This is used to estimate the axial coordinate with high precision, allowing 3D imaging.
The third setup is a custom-built modular microscope with simultaneous detection via two objectives, offering even higher resolution. Here, two different options are implemented for 3D imaging, firstly astigmatic detection (K. Xu, H. P. Babcock, and X. Zhuang. Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. Nat. Methods 2012, 9(2):185–188) and secondly interferometric detection (G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl. Acad. Sci. U.S.A. 2009, 106(9):3125–3130, as well as D. Aquino, A. Schönle, C. Geisler, C. v Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner. Two-color nanoscopy of three-dimensional volumes by 4pi detection of stochastically switched fluorophores. Nature methods 2011, 8(4):353–359). The latter method features exceeding precision, it is however only suitable for thin and homogeneous samples.