The resolution of a light microscope is usually limited by the physics of diffraction, with the limit being proportional to the wavelength of the light. There are, however, super-resolution optical microscopy techniques that allow resolutions above this theoretical diffraction limit.
Stimulated emission depletion (STED) microscopy is one such method. It is based on the selective deactivation of fluorophores by a beam with a “hole” in the middle, which minimizes the area of illumination at the focal point, and thus, improves the achievable resolution for a given system. STED was co-developed by Stefan W. Hell, who was awarded the Nobel Prize in Chemistry in 2014 together with Eric Betzig and William E. Moerner, “for the development of super-resolved fluorescence microscopy.”
Stefan W. Hell, Max Planck Institute for Biophysical Chemistry, Göttingen, and Max Planck Institute for Medical Research, Heidelberg, both Germany, and colleagues have developed an improved light microscopy method, called MINSTED, which resolves fluorescently labeled details with molecular sharpness. In MINSTED, the fluorescent molecules are initially isolated by randomly switching them on through an independent photochemical switching process. The fluorescence-preventing STED beam is then used to locate the fluorescent molecules individually, with its hole serving as a reference point.
According to the researchers, the method can achieve molecular resolution while effectively suppressing the background noise. The resolution can be adjusted almost continuously from 200 nm down to molecular size.
- MINSTED fluorescence localization and nanoscopy,
Michael Weber, Marcel Leutenegger, Stefan Stoldt, Stefan Jakobs, Tiberiu S. Mihaila, Alexey N. Butkevich, Stefan W. Hell,
Nat. Photonics 2021.