Confocal Nanoscopy Goes Multicolor


Despite all the great insights obtained with conventional fluorescence microscopy, it is easy to become frustrated when studying subcellular structures, as the biological entities of interest are often significantly smaller than the diffraction limit. Under conventional far-field fluorescence microscopes, which are incapable of resolving objects closer to one another than about 200 nm, details of interest are lost in a blur. Several methods of far-field superresolution microscopy overcome this fundamental obstacle and allow morphological details to be studied far beyond the diffraction limit. Stimulated Emission Depletion (STED) imaging1, as confocal laser scanning microscopy, moves a spot of excitation light over the sample, detects the emitted fluorescence and generates pixel by pixel the image of the observed optical section.

To achieve superresolution, the diffraction-limited excitation spot is overlaid with a donut-shaped point spread function of a second laser: the STED laser. A process called stimulated emission prevents dyes from emitting fluorescence anywhere in the focal volume except for the very center of the donut. The amount by which the effective focus can be shrunk, i.e. the resolution that can be achieved, depends on the intensity of the STED laser as well as on the dye. With the commerically available STED-microscopes, sub 60 nm lateral resolution is routinely achieved with e.g., Atto 647N. Confocal and STED microscopy are a perfect match and can be readily implemented in the same setup. With the available commercial realizations the user can toggle between confocal and STED resolution by a single mouse click.



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