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The Warsaw Voice » The Polish Science Voice » February 23, 2012
Physics
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Student Builds Super-Microscope
February 23, 2012   
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Optical microscopes are still unrivaled in analyzing biological samples. However, their low resolution continues to be a problem.

This has improved only in recent years with the advent of STED microscopes. A device of this type, one of the first in Poland, has been constructed by a student researcher at the University of Warsaw’s Faculty of Physics.

There are many imaging methods with a nanometer-level of resolution known to science, for example electron or atomic force microscopy. These methods require special preparation of the samples and make it possible to observe only the surface itself. When it comes to samples of biological origin, not infrequently living ones, optical microscopes are still unrivaled. One of their strengths is that scientists can observe the spatial structure of the sample. A major drawback, however, is low resolution.

An optical microscope makes it possible to discern details no smaller than half the wavelength of the light illuminating the sample. This limitation is due to diffraction, which makes it impossible to focus the beam of light into a point. As a result, if we use a red light source with a wavelength of 635 nanometers, for example, we can at best see details around 300 nanometers in size.

In 1994, Stefan W. Hell from the Max-Planck-Institut für Biophysikalische Chemie in Göttingen, Germany, proposed a theoretical way to overcome the diffraction limitations in optical microscopes by means of stimulated emission depletion (STED). Five years later he built the first super-resolution STED fluorescence microscope.

In standard fluorescence confocal microscopy, a laser beam scans a biological sample and locally excites dye molecules introduced into the sample earlier. Upon excitation, the molecules begin to emit light. The light is passed through a filter and recorded by a detector located behind a confocal aperture. Due to the size of the aperture, light from out-of-focus planes is eliminated, increasing the contrast of the image. The dye itself is selected in such a way that it accumulates in those parts of a living cell that are of interest to researchers.

In STED microscopy, an additional laser beam, called a depletion beam, is used. The beam has such a wavelength that it induces stimulated emission in the dye molecules it illuminates. Molecules that have lost energy as a result of stimulated emission are no longer able to fluoresce. Therefore, their light will not pass through the filter in front of the detector, and they will not be visible on the recorded image.

The essence of the STED method is based on the special shape of the depletion beam. If such a beam is properly synchronized in time and space with the illuminating beam, fluorescence will occur primarily in the area of the sample located in the center of the depletion beam.

The confocal microscope using the STED system was built at the University of Warsaw’s Faculty of Physics, making use of commercially available components. The greatest problem was to ensure that both laser beams overlapped. “In order to observe the STED effect, both beams need to be ideally aligned—the depletion beam needs to closely overlap with the center of the excitation beam,” says Joanna Oracz, the student researcher behind the project, which was the subject of her master’s thesis.

The prototype microscope has a resolution of about 100 nm, more than twice as high as that of a standard confocal microscope. Work is under way to increase the resolution. “The advantage of our microscope is the possibility of controlling all parameters and studying the physics of the optical phenomena occurring,” says Oracz, now a Ph.D. student at the Ultrafast Phenomena Lab of the University of Warsaw’s Institute of Experimental Physics.

The aim is to reach a resolution of about 60 nm, enough to observe details as minute as dendritic spines of neurons.

According to Prof. Czes³aw Radzewicz, head of the Ultrafast Phenomena Lab at Warsaw University’s Institute of Experimental Physics, it would not have been possible to construct such a sophisticated device without collaboration with other scientific institutions. For example, the researchers used experience gained during the construction of a confocal microscope at the Laser Center of the Polish Academy of Sciences’ Institute of Physical Chemistry, and the University of Warsaw’s Faculty of Physics. The samples were dyed at the Nencki Institute of Experimental Biology, part of the Polish Academy of Sciences.
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