For assignment purposes

1. Novel Method of STED by Stefan W. Hell
Abhishek Das
Optical microscopy has been a revelation in the field of microbiology
when it was first introduced in the 17th century to study small organisms like
bacteria and yeast cells, since its use made it possible to study the smallest
cells without damaging the cells. But the wavelength of light set a limit to
the ability of the optical microscopes to resolve closely-spaced objects. Ernst
Abbe quantified this so called diffraction limit by stating that one cannot see
objects sized less than half the wavelength of light using such microscopes
which proved a major deterrent for scientists trying to study the processes
inside the cell.
In search for better resolution microscopy, many scientists toiled
hard to break the Abbes limit. Among them, a German physicist, Stefan Hell
is credited with the invention of a few methods that enabled us to circumvent
Abbes diffraction limit. One of such methods is the novel idea of Stipulated
Emission Depletion microscopy or STED. Hell was one of the first few to
have conceived an idea of a microscope using fluorescence, a phenomenon in
which certain substances became luminous due to exposed light, to find a
way around the universal Abbes limit.
The method developed by Hell in 1994 makes use of the principle
of two spatially and temporally overlapped light pulses; the first one exciting
some fluorescent molecules to glow while the second one(known as depletion
pulse) causing the molecules around the periphery of the glowing fluorescent
molecules to become dark. The general idea is to optically drive the electronic
transition from a higher energy excited state to the lower energy ground state
through stimulated emission of radiation to turn off the fluorescence ability
of the fluorescent molecules. The second beam has a doughnut or annular
shape for better efficiency of specific depleting the molecules of excited energy
state. The STED principle is to to turn all molecules within the excitation
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2. area except the inner center of the focus into the dark state by applying an
annular STED. An image is then created by targeted scanning of the sample
with the given light sources which is of a much higher resolution than an
optical microscope. It has been seen via experiments that the resolution can
improved just by increasing the power of the STED beam. According to
Stefan Hell, The STED microscope collects light from the multitude of small
volumes to create a large whole.
Thus this method became a ground-breaking discovery in modern
science as it helped scientists to study the interaction between individual
molecules inside of the cell and other biophysical processes. This combined
with 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 Prize
in Chemistry in 2014 ”for the development of super-resolved fluorescence mi-
croscopy.
References
[1] Stefan W. Hell and Jan Wichmann Breaking the diffraction resolution
limit by stimulated emission: stimulated-emission-depletion fluorescence
microscopy. 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: A
concept for breaking the diffraction resolution limit. Applied Physics B,
May 1995, Volume 60, Issue 5, pp 495-497
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