STED-microscopy ( eng. Stimulated Emission Depletion Microscopy - microscopy based on the suppression of spontaneous emission) is a type of fluorescence microscopy that reaches resolution beyond the diffraction limit by selective quenching of fluorescence [1] . The method was developed by Stefan Hell in 1994 [2] and demonstrated in 1999. Awarded the Nobel Prize in Chemistry in 2014 [3]
In 1986, V. A. Okhonin ( Institute of Biophysics, Siberian Branch of the USSR Academy of Sciences ) patented the idea of a STED microscope [4] . This patent was apparently unknown to Hell and Wichmann in 1994.
Principle of the method
Under ordinary confocal luminescent microscopy, the test substance is optically excited, and its fluorescence is detected by the receiver. The spatial resolution of the method is limited by the diffraction limit of the order
Where Is the wavelength , and - aperture .
To overcome it, STED microscopy uses a second laser with a longer wavelength to stimulate radiative transitions in the material along the edges of the focal spot. Upon irradiation with a ring laser, the transition from an excited level to a certain vibrational level is forcedly emitted. Thus, depletion of the population of the excited level occurs at the edges of the spot, and luminescence at the wavelength suppressed, allowing for better resolution in the central area.
Notes
- ↑ Westphal, V .; SO Rizzoli, MA Lauterbach, D. Kamin, R. Jahn, SW Hell (2008). Science 320 : 246-249.
- ↑ SW Hell, J. Wichmann (1994). Breaking the diffraction resolution limit by stimulated emission: Stimulated-emission-depletion fluorescence microscopy. Optics Letters 19 (11): 780–782.
- ↑ SW Hell, Nanoscopy with Focused Light , Nobel Lecture in Chemistry, 12/08/2014.
- ↑ V. Okhonin, Institute of Biophysics, Siberian Branch of the Academy of Sciences of the USSR, Method for Researching the Microstructure of a Sample , Patent Number: 1374922, Priority 04/10/1986, Published: 07/30/1991, USSR Patent Database. It is cited in US patents: US 5394268 A (1993) and US RE38307 E1 (1995). From the description of the invention: "The essence of the invention lies in the fact that they excite the luminescence of a sample placed in the field of several standing light waves, causing quenching of luminescence due to forced transitions from the luminescent state to short-lived states everywhere, except for small neighborhoods of points at which the transitions to the short-lived state of the field component (the driving component of the field) of the standing waves also vanishes. "
Links
- Overview at the Department of Nano Biophotonics at the Max Planck Institute for Biophysical Chemistry.
- Brief summary of the RESOLFT equations developed by Stefan Hell.