Novel Method of STED by Stefan W. Hell

Abhishek Das

Optical microscopy has been a revelation in the field of microbiologywhen it was first introduced in the 17th century to study small organisms likebacteria and yeast cells, since its use made it possible to study the smallestcells without damaging the cells. But the wavelength of light set a limit tothe ability of the optical microscopes to resolve closely-spaced objects. ErnstAbbe quantified this so called diffraction limit by stating that one cannot seeobjects sized less than half the wavelength of light using such microscopeswhich proved a major deterrent for scientists trying to study the processesinside the cell.

In search for better resolution microscopy, many scientists toiledhard to break the Abbes limit. Among them, a German physicist, Stefan Hellis credited with the invention of a few methods that enabled us to circumventAbbes diffraction limit. One of such methods is the novel idea of StipulatedEmission Depletion microscopy or STED. Hell was one of the first few tohave conceived an idea of a microscope using fluorescence, a phenomenon inwhich certain substances became luminous due to exposed light, to find away around the universal Abbes limit.

The method developed by Hell in 1994 makes use of the principleof two spatially and temporally overlapped light pulses; the first one excitingsome fluorescent molecules to glow while the second one(known as depletionpulse) causing the molecules around the periphery of the glowing fluorescentmolecules to become dark. The general idea is to optically drive the electronictransition from a higher energy excited state to the lower energy ground statethrough stimulated emission of radiation to turn off the fluorescence abilityof the fluorescent molecules. The second beam has a doughnut or annularshape for better efficiency of specific depleting the molecules of excited energystate. The STED principle is to to turn all molecules within the excitation

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area except the inner center of the focus into the dark state by applying anannular STED. An image is then created by targeted scanning of the samplewith the given light sources which is of a much higher resolution than anoptical microscope. It has been seen via experiments that the resolution canimproved just by increasing the power of the STED beam. According toStefan Hell, The STED microscope collects light from the multitude of smallvolumes to create a large whole.

Thus this method became a ground-breaking discovery in modernscience as it helped scientists to study the interaction between individualmolecules inside of the cell and other biophysical processes. This combinedwith colour and 3-D video rate-imaging helped in determining various cell-growth mechanisms and has extensive uses in biological and medical sciences.

For such dedicated work and path-breaking discovery Stefan W.Hell (along with Eric Betzig and W. Moerner) was conferred the Nobel Prizein Chemistry in 2014 ”for the development of super-resolved fluorescence mi-croscopy.

References

[1] Stefan W. Hell and Jan Wichmann Breaking the diffraction resolutionlimit by stimulated emission: stimulated-emission-depletion fluorescencemicroscopy. Optics Letters Vol. 19, Issue 11, pp. 780-782 (1994) doi:10.1364/OL.19.000780

[2] S. W. Hell, M. Kroug Ground-state-depletion fluorscence microscopy: Aconcept for breaking the diffraction resolution limit. Applied Physics B,May 1995, Volume 60, Issue 5, pp 495-497

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