INSTRUMENTAL TECHNIQUE PRESENTATION

Stimulated Emission Depletion Microscopy (STED)

KRISHNADAS06-12-2014

Eric Betzig Stefan W. Hell William E. Moerner

The Nobel Prize in Chemistry 2014 was awarded jointly to Eric Betzig, Stefan W. Hell and William E. Moerner

"for the development of super-resolved fluorescence microscopy"

Howard Hughes Medical Institute, Ashburn, VA, USA

Max Planck Institute for Biophysical Chemistry

Stanford University

STED Principles

ChemPhysChem 2012, 13, 1986 – 2000

Confocal Laser Scanning Microscope

The light intensity of the focal spot distributes according to the point spread function (PSF)

The resoltuion of CLSM is determined by the size of the PSF: The smaller the focal spot is, each pixel in the acquired image will be corresponding to a smaller region in the sample, and thus the image will be less blurred.

Ernst Abbe pointed out that the PSF size has a lower limit proportional to λ/NA (circular aperture) due to diffraction.

http://www.anes.ucla.edu/sted/principle.html;

Point spread function (PSF) and diffraction limiting

ChemPhysChem 2012, 13, 1986 – 2000

Spontaneous and stimulated emission

http://www.anes.ucla.edu/sted/principle.html

A fundamental mode Gaussian beam mode has a spherical phase front.

A Spiral phase plate has a thickness which varies circumferally ‘around’ the plate, but is uniform radially. The plate is made of a dielectric material which is transparent.

Hence a fundamental mode beam passing though the plate undergoes a phase-change which introduces a ‘spiral’ element into its phase front. By choosing the appropriate plate geometry we can turn the beam phase front into a “spiral staircase” pattern.

Spiral phase plates (SPP)

http://www.standrews.ac.uk/~www_pa/Scots_Guide/MMWave/QO/components/spir.html

Generation of optical vortex

An optical vortex (also known as a screw dislocation or phase singularity) is a zero of an optical field, a point of zero intensity

In an optical vortex, light is twisted like a corkscrew around its axis of travel.

Because of the twisting, the light waves at the axis itself cancel each other out.

When projected onto a flat surface, an optical vortex looks like a ring of light, with a dark hole in the center.

This corkscrew of light, with darkness at the center, is called an optical vortex.

Microtubules of a Caco-2 cell stained with Atto647N

ChemPhysChem 2012, 13, 1986 – 2000

Schmidt, R.; Wurm, C. A.; Punge, A.; Egner, A.; Jakobs, S.; Hell, S. W. Nano Lett. 2009, 9, 2508-2510

Wildanger, D.; Patton, B. R.; Schill, H.; Marseglia, L.; Hadden, J. P.; Knauer, S.; Schönle, A.; Rarity, J. G.; O’Brien, J. L.; Hell, S. W.; Smith, J. M. Adv. Mater. 2012, 24, 309–313

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