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Super-resolution techniques In 2014, the Nobel Prize in Chemistry was awarded to researchers who
developed various super-resolution microscopy techniques using novel chemistries, hardware and software to accurately determine the location and activity of individual molecules and structures without direct visual observation. Some of the most commonly used super-resolution tech- niques are STED, PALM, STORM and SIM (Table 1).
STED Stimulated emission depletion (STED) microscopy is a powerful determin- istic technique that allows images to be captured at resolutions well below the theoretical diffraction limit. In STED, a high-intensity pulsed laser is used to excite the sample. Immediately thereafter, a doughnut-shaped, red-shifted pulse follows. Excited fluorophores exposed to the second STED beam are instantly returned to their ground state by stimulated emission and do not fluoresce, allowing the collection of image data from the remaining excited fluorophores that reside in a small subresolution region at the center of the depleted area. STED’s complexity is its great- est challenge, making it relatively expensive and potentially unavailable to many researchers. In addition, the excitation pulse can damage some samples. Finally, collecting image data can be time-consuming when scanning a large field of view. However, with small fields of view, STED is quite fast.
PALM and STORM In contrast to STED, which is deterministic, several other techniques rely on localization of fluorescent structures within the sample. Stochastic optical reconstruction microscopy (STORM) and photoactivated local- ization microscopy (PALM) use fluorescent probes that switch between fluorescent and dark states. Fluorochrome positions can be determined with high precision based on calculating the center positions of each of the fluorescent spots. Through multiple image captures, with each image capturing random subsets of the fluorophores, a super-resolution image is reconstructed from the accumulated position data. Localization meth- ods such as PALM and STORM offer high spatial resolution (20–50 nm), but have low temporal resolutions ranging from 30 seconds to 30 minutes, depending on the application, sample and fluorophores used.
SIM Structured illumination microscopy (SIM) involves excitation with repeat- ing patterns that interact with the structure of the sample to produce moiré patterns that are used to extrapolate images at higher resolution than would be achievable through traditional unstructured widefield illumination. SIM provides twice the resolution potential of comparable widefield illumination systems and, depending on the implementation, imaging can be performed at relatively high frame rates, making SIM a useful technique for imaging fast-changing events within living samples. In most practical applications, resolution ranges from 100 to 140 nm.
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