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Huygens STED optical Option


STED images (from STimulated Emission Depletion microscopes) can be deconvolved with Huygens with truly stunning results. Latest data shows a FWHM resolution of 22nm is achievable (see below).

In 1994, Stefan Hell presented the STED theory by which the diffraction limit in microscopy can be circumvented (1). A STED microscope much resembles a confocal microscope, but has an additional laser of which the focus is deformed into a doughnut shaped distribution. The wavelength of this laser is chosen so that it stimulates emission of the excited fluorophores to a wavelength which is filtered out, thereby depleting the fluorophores in the outer region of the diffraction limited excitation spot. By increasing the intensity of the depletion beam, the outer regions are depleted more, reducing the size of the remaining spot of excited fluorophores (2).

The unique feature of the STED microscope is that it does not have a bandwidth limit, i.e. a barrier beyond which object details are not imaged. While other super resolution systems are still hampered by this wavelength dependent limit, a STED microscope moves happily beyond it.

The Z-resolution which is a very important part of biological research is relatively less. By applying the Huygens STED deconvolution option you will get a huge increase in contrast and Z resolution.

Leica Microsystems is manufacturer of commercially available STED microscopes. Per July 2012, every LEICA Microsystems STED microscope is equipped with Huygens STED deconvolution. Since 2014, from Huygens version 4.5 onwards STED deconvolution can also be applied to images from Leica STED 3X systems which have initially a better Z sampling.

As always, Huygens deconvolution reduces noise and blurring, and takes depth-dependent spherical aberration correction into account. More information on how to deconvolve STED images in Huygens can be found on STED deconvolution.

A poster on Huygens STED deconvolution, presented at a 2013 meeting of the Edinburgh Super-Resolution Imaging Consortium (ESRIC) can be viewed here. In close collaboration with several Huygens customers, we published an article in Microscopy Today (Volume 21:6, page 38-44) entitled: "Huygens STED Deconvolution Increases Signal-to-Noise and Resolution towards 22 nm."

You can visit our STED Gallery for more STED deconvolution examples.

References
1. Hell S.W. and Wichmann J. (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Optical Letters 19(11): 780-782.
2. For a more extensive introduction and references, see our article "Huygens STED Deconvolution Increases Signal-to-Noise and Resolution towards 22 nm". Microscopy Today (Volume 21:6, page 38-44)].


Single XY plane of pulsed-STED data before (left) and after deconvolution (right) of the complete z-stack with Huygens. Structure (240 nm ring diameter) was stained with primary and secondary antibody conjugated to Alexa488. Data used by permission from Dr. Grazvydas Lukinavicius, EPFL, Lausanne, Switzerland
Single XY plane of pulsed-STED data before (left) and after deconvolution (right) of the complete z-stack with Huygens. Structure (240 nm ring diameter) was stained with primary and secondary antibody conjugated to Alexa488. Data used by permission from Dr. Grazvydas Lukinavicius, EPFL, Lausanne, Switzerland


Axial MIP projection of the complete STED z-stack, before (left) and after deconvolution with Huygens (right). Data used by permission from Dr. Grazvydas Lukinavicius, EPFL, Lausanne, Switzerland
Axial MIP projection of the complete STED z-stack, before (left) and after deconvolution with Huygens (right). Data used by permission from Dr. Grazvydas Lukinavicius, EPFL, Lausanne, Switzerland



Gated STED single plane image and confocal data of a centriole stained with primary and secondary antibody conjugated to Chromeo488. Raw and Huygens deconvolved images are shown. The intensity profile was fitted to Lorentz distribution. Data used by permission from Dr. Grazvydas Lukinavicius, EPFL, Lausanne, Switzerland
Gated STED single plane image and confocal data of a centriole stained with primary and secondary antibody conjugated to Chromeo488. Raw and Huygens deconvolved images are shown. The intensity profile was fitted to Lorentz distribution. Data used by permission from Dr. Grazvydas Lukinavicius, EPFL, Lausanne, Switzerland



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