Optical microscopes are still second to none when it comes to analyzing biological samples. However, their low resolution, improved only in recent years in STED microscopes, continues to be a problem. A device of this type, one of the first in Poland, has been constructed by a student of the Faculty of Physics, University of Warsaw.
Due to diffraction limit, optical microscopes will never be able to discern details smaller than 200 nanometres -- it was believed only a dozen or so years ago. In recent years, scientists have managed to overcome this limit and build super-resolution devices, including, for example, STED confocal microscopes. A prototype device of this type has recently been built at the Faculty of Physics, University of Warsaw (FUW), as part of Joanna Oracz's MA thesis. As of next year, the new microscope will be used not only for research in the field of optics but also to analyze biological samples.
There are many imaging techniques with a resolution of the order of nanometres (billionths of a metre) known to science, for example, electron or atomic force microscopy. These techniques require special preparation of samples and make it possible to observe only the surface itself. When it comes to samples of biological origin, not infrequently living ones, optical microscopy is still second to none. One of its advantages is the possibility of observing the spatial structure of the sample. A major disadvantage, however, is a low resolution.
An optical microscope makes it possible to discern details no smaller than half the wavelength of the light illuminating the sample. This limit is due to diffraction, which makes it impossible to focus the beam of light onto a point. As a result, if we use a red light source with a wavelength of 635 nanometres, we can, at best, see details around 300 nanometres in size.
In 1994 Stefan W. Hell from the Max-Planck-Institut fόr biophysikalische Chemie in Gφttingen proposed a theoretical way to overcome the diffraction limit in optical microscopy 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 researches.
An additional laser beam -- depletion beam -- is used in STED microscopy. Given its wavelength, the beam induces stimulated emission in dye molecules it illuminates. Molecules that have lost energy as a result of stimulated emission are no longer able to fluoresce. Therefore, their light (similarly to the light from stimulated emission) 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 lies in the fact that the depletion beam is donut-shaped. If a beam of this shape is properly synchronized in time and space with the illuminating beam, fluorescence will occur first and foremost in the area of the sample located in the centre of the depletion beam.
"Thanks to the second beam, the area of the sample emitting light as a result of fluorescence is distinctly smaller than the diameter of laser beams. The effect is as if the illuminating beam were better focused, meaning that we can scan the sample with a higher resolution," explains Joanna Oracz, adding that when she began working on her device a year ago, there was only one STED microscope in Poland, purchased for a million and a half euros.
The confocal microscope with a STED setup was built at the Faculty of Physics, University of Warsaw, using commercially available elements. 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 minimum of the depletion beam needs to closely overlap with centre of the excitation beam," says Oracz.
The prototype microscope at FUW has a resolution of about 100 nm, over two times higher than that of a standard confocal microscope. Works are still underway 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," stresses Oracz, currently a PhD student at the Ultrafast Phenomena Lab of the Institute of Experimental Physics FUW. The aim is to reach a resolution of about 60 nm. It would make it possible to observe details as minute as dendritic spines of neurons.
"It would not have been possible to construct such a sophisticated device without collaboration with other scientific institutions," stresses Prof. Czesław Radzewicz, head of the Ultrafast Phenomena Lab of the Institute of Experimental Physics FUW. The scientists relied, among other things, on the experience gained during the construction of a confocal microscope at the Laser Centre of the Institute of Physical Chemistry, Polish Academy of Sciences and the Faculty of Physics, University of Warsaw. The samples were dyed at the Nencki Institute of Experimental Biology, Polish Academy of Sciences.
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