Ground State Depletion Fluorescence Microscopy
Hochauflösende Fluoreszenzmikrospie durch Entvölkerung des Grundzustandes
by Stefan Bretschneider
Date of Examination:2007-12-21
Date of issue:2008-01-16
Advisor:Prof. Dr. Stefan Hell
Referee:Prof. Dr. Stefan Hell
Referee:Prof. Dr. Rainer G. Ulbrich
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EnglishMicroscopy is one of the most important analysis tools of modern science. One defining attribute of a microscope is its resolution as it specifies the minimum distance at which one can distinguish two alike objects within an image. Therefore, if the resolution is not sufficient to resolve structures within the sample, important information will be lost and prevents the gaining of new insights. Today, the best microscopes can resolve single atoms like, for example, the transmission electron microscope (TEM)  or the scanning tunneling microscope (STM) . In the life sciences, far-field light microscopy remained the most popular microscopy mode with fluorescence being its most important readout. The reason for this is that far field light microscopy is non-invasive, applicable to living cells, and the fluorescence labeling is specific and very sensitive. However, one drawback of far-field light microscopy is that the resolution is limited by diffraction. The discovery of the diffraction barrier of resolution by Abbe in 1873  has lead to the widely accepted notion that the resolution of a far-field light microscope is limited to about half of the wavelength of light, l/2. Therefore, the scanning nearfield optical microscope (SNOM)  was invented which images the sample through a small subwavelength aperture placed in close proximity to the sample so that the resolution is not limited by diffraction but the size of the aperture. But due to the fact that all scanning probe techniques like SNOM, STM, atomic force microscopy (AFM)  as well as high resolution techniques like scanning or transmission electron microscopy1 are bound to the surface, their use in life sciences is limited to special applications. Also, the samples usually have to be fixed for these techiques or even have to be embedded in epoxy or frozen or imaged under vacuum conditions. Therefore it is desireable to have a far-field microscope whose resolution is much below the diffraction limit so that one can obtain high resolution non-invasive three dimensional images of living cells. Fortunately, the fluorescence contrast mode is favorable to overcome Abbes barrier, since changing the parameters of fluorescence emission allows one to surmount the limiting role of diffraction. The first general idea to fundamentally break the diffraction barrier and promise unlimited resolution in far-field light microscopy was the RESOLFT (Reversible Saturable OpticaL (Fluorescence) Transitions) concept [7, 8, 9], which uses reversible saturable optical transitions to bypass the restrictions of diffraction. For example, stimulated emission depletion (STED) microscopy breaks the diffraction barrier by inhibiting fluorescence emission of the fluorescent marker [10, 11, 12]. By depleting the fluorescence, for example, with a donut shaped beam with an intensity zero in the center, the fluorescence spot can be effectively reduced in size below the diffraction limit. The advantage of STED microscopy is that stimulated emission is possible with almost every dye molecule but there is also a disadvantage - high resolution in STED microscopy necessitates depletion intensities as high as GW/cm2. RESOLFT and other new sub-diffraction methods such as photoactivated localization microscopy (PALM)  also make use of the possibility that dye molecules [14, 15] or fluorescent proteins [16, 17, 13] can be switched between a bright and a dark state. Here, the big advantage is that one needs only very low intensities which can be in the range of W/cm2 for switching the molecules to their dark state. However, the available switchable fluorescent proteins and dyes are limited. In this thesis the concept of ground state depletion (GSD)  is pursued, which aims to shelve the fluorophores in a metastable dark state (for example the triplet state), thereby effectively depleting the fluorophores ground state S0. Since the lifetime of these dark states is usually very long compared to the fluorescence lifetime, the necessary intensities are much smaller than those for STED microscopy. Further, nearly every dye molecule has a metastable dark state and therefore GSD microscopy promises to break the diffraction barrier by combining the advantage of generality and low intensities. For this reason GSD microscopy has a good chance to become a powerful tool for biological research for new scientific insights.
Keywords: fluroescence microscopy; diffraction limit; resolution enhancement; nanoscopy
Hochauflösende Fluoreszenzmikrospie durch Entvölkerung des Grundzustandes. Diese Arbeit demonstriert die Umsetzung eines neuen Mikroskopieverfahrens, mit dem man durch Entvölkerung des Grundzustandes die Auflösung unterhalb der Beugungsgrenze verbessert kann.
Schlagwörter: Fluoreszenzmikroskopie; Beugungsgrenze; Auflösungserhöhung; Nanoskopie