Superresolution microscopies

E. Betzig, A. Harootunian, A. Lewis, and M. Isaacson, Near-Field Diffraction by a Slit - Implications for Superresolution Microscopy, Appl. Opt. 25, 1890 (1986)... 

Superresolution microscopy methods have revolutionized optical microscopy within approx, the last two decades [79]. These methods can be separated in localization-based methods [80], which exploit the possibihty to localize the isolated emission patterns of single molecules with high accuracy, and in methods which restrict the volume of excitation by stimulated emission. The latter can be combined with FCS resulting in STED-FCS (stimulated-emission-depletion FCS) [81, 82], where the excitation volume is minimized by an intensive donut-shaped STED laser pulse which depopulates basically all excited states except for a central volume of sub-diffraction size. This way, the spatial resolution Ad in lateral direction can be reduced to... 

Different fluorescence microscopy techniques are commonly used to explore the organization of the microtubule cytoskeleton. The resolution of these techniques is, however, limited by diffraction to approximately 250 nm, which makes them not suitable for nanoscale mapping of microtubule properties. Superresolution microscopy techniques that rely on single molecule localization (Single Molecule Localization Microscopy SMLM) combine high protein specificity, multi-color imaging, and a resolution in the order of 5-50 nm, making it an ideal tool to study the neuronal cytoskeleton and its properties. 

For many fluorescence-based imaging techniques, such as confocal laser scanning microscopy, spinning disk confocal microscopy, 4pi microscopy, stimulated emission depletion (STED) microscopy, and other superresolution microscopy techniques, we refer to the indicated literature. 

In another superresolution approach. Rust et al. have utilized controlled photoswitching of small molecule emitters for superresolution demonstrations [134] (STORM, for Stochastic Optical Reconstruction Microscopy). This method uses a Cy3-Cy5 emitter pair in close proximity in the presence of thiols that show a novel property restoration of Cy5 s photobleached emission can be achieved by brief pumping of the Cy3 molecule. In this way, the emission from a single Cy5 is turned on and off, again and again, to measure its position accurately multiple times. In the Moerner laboratory, a covalently bound Cy3-Cy5 dimer has been prepared as shown in Fig. 2.12C [135]. This molecule has the advantage that the need for random close associations of the Cy3 and... 

However, finding out the position of an object with arbitrary precision is not the same as resolution, which is about separating similar objects at small distances. Localization per se cannot provide superresolution. This is also why, although it had been known and used for decades [109,110] and even routinely applied to single molecules [125,126], localization alone has not provided nanoscale images. (Note that in spite the use of localization in the 1980 s and earlier, near-field optical microscopy still seemed to be the only way to attain nanoscale resolution up to the early 1990 s.) Resolution clearly requires a criterion to discern objects or molecules, the simplest of which is bright vs. dark. ... 

Watanabe, T., Iketaki, Y., Omatsu, T., Yamamoto, K. and Fujii, M. (2005) Two-point separation in far-field superresolution fluorescence microscopy based on two-color fluorescence dip spectroscopy, Part I Experimental evaluation. Appl. Spectrosc., 59, 868-872. 

Presently, methods are being developed that focus on the use of superresolution confocal Raman microscopy (CRM), electron back-scattered diffraction (EBSD), and, in combination with high-resolution XRD, to identify and measure the stress distributions of structures and defects that control... 

Takasaki KT, Ding JB, Sabatini BL (2013) Live-cell superresolution imaging by pulsed STED two-photon excitation microscopy. Biophys J 104 770-777... 

Rieger B, StaUinga S (2014) The lateral and axial localization uncertainty in superresolution light microscopy. Chemphyschem 15 664-670... 

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