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Stochastic Optical Fluctuation Imaging - Labels and Applications

dc.contributor.advisorEnderlein, Jörg Prof. Dr.
dc.contributor.authorHuss, Anja
dc.titleStochastic Optical Fluctuation Imaging - Labels and Applicationsde
dc.contributor.refereeEnderlein, Jörg Prof. Dr.
dc.description.abstractengSuperresolution fluorescence imaging is an important tool for studying subcellular structures and processes. A technique with great potential is stochastic optical fluctuation imaging (SOFI), a fluorescence imaging modality that yields superresolved spatial resolution, 3D-sectioning and high image contrast by making use of independent temporal intensity fluctuations (blinking) of fluorescent labels. The compatibility of SOFI with a large range of blinking kinetics allows for the use of a great variety of labels. The inherent blinking of Quantum Dots can be utilized as well as the induced intensity fluctuations of organic dyes and fluorescent proteins, allowing for superresolution imaging in a multitude of systems. Exploiting the natural blinking of Quantum Dots for SOFI is straightforward and requires merely a basic widefield microscope and low excitation intensities. The provided optical sectioning and resolution increase are highly useful for biological applications, for example enabling the quantification of the distribution of calcium channels in the neuronal ER membrane in order to link ER structure to properties of calcium waves. Studying the organization of live cells with SOFI is enabled by reversibly switchable fluorescent proteins (RSFPs). RSFPs are a type of fluorescent proteins whose absorption and emission properties depend strongly on illumination. Light of certain wavelengths stimulates transitions between fluorescent and non-fluorescent states, thus opening up the possibility to induce statistically independent temporal intensity fluctuations. The dependence of the photoswitching kinetics on the irradiation intensities of several RSFPs was studied, and Dreiklang and rsEGFP(N205S) were identified as suitable labels. After confirming the lifetimes of the fluorescent and non-fluorescent states with calibration measurements, the induced blinking was used for SOFI on live mammalian cells. SOFI is much more robust regarding intensity fluctuation kinetics than localization methods such as dSTORM. As a result, SOFI can be applied in cases where the blinking properties render single molecule localization impossible. This is illustrated by comparing SOFI and STORM results of studies of the organization of the axon initial segment. SOFI is highly suitable for densely labeled structures as well as for samples with only sparse labeling, and for all kinds of blinking kinetics. Superresolved images can be acquired at low excitation power, limiting photodamage of the sample and the fluorophores even for prolonged data acquisition. Combined with its 3D-sectioning capability, this allows for the recording of z-stacks of superresolved images over large axial distances on a modest widefield setup. Due to its straightforward and robust nature, SOFI is therefore advantageous for a variety of
dc.contributor.coRefereeWouters, Fred Prof. Dr.
dc.subject.engstochastic optical fluctuation imagingde
dc.subject.engsuperresolution microscopyde
dc.subject.engreversibly switchable fluorescent proteinsde
dc.subject.englive-cell imagingde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

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