title: Strategies to reduce photobleaching, dark state transitions and phototoxicity in subdiffraction optical microscopy
creator: Staudt, Thorsten M.
subject: ddc-540
subject: 540 Chemistry and allied sciences
description: In all subdiffraction fluorescence microscopy techniques, the theoretically infinite attainable resolution is, in practice, limited by the photobleaching of fluorophores. Repetitive scans of the sample required for e.g. three dimensional recordings, increase photobleaching, dark state transitions and, in case of living cells, phototoxicity. To advance such experiments all possibilities to reduce the photobleaching must be explored. In this thesis, various chemical and physical approaches to tackle photobleaching are studied within the context of stimulated emission depletion (STED) microscopy, which is the first and most prominent method for subdiffraction imaging. The STED setup constructed for this purpose allows for the fast adaptation to new fluorescent dyes, and relies on a novel adaptive spectral and phase filter technique. Furthermore, the optical setup facilitates a gentle exposure strategy, in which the time that the dye is irradiated is significantly reduced. Three-dimensional images can therefore be recorded, and the palette of applicable dyes can be expanded to the blue-green regime by the so far unemployed coumarin derivatives, which are known to be prone to photobleaching. The label itself is another vantage point from which photobleaching limitations in subdiffraction microscopy can be circumvented. For the first time, light-driven modulation of the fluorescence from Mn-doped ZnSe quantum nanocrystals has been established through excited-state absorption (ESA). This enables a new type of far-field fluorescence microscopy with diffraction-unlimited resolution based on quantum dots, which are well known for their superior photostability. The correct sample embedding in the refractive index matching is also of high importance, if spherical aberrations and light scattering are to be minimized to optimize the fluorescence collection. For this purpose, an embedding medium, 2,2´-thiodiethanol (TDE) is introduced, which, by being miscible with water at any ratio, allows for refractive index matching up to that of immersion oil and making high resolution recordings deep within the sample feasible.
date: 2009
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/9848/1/staudtupload.pdf
identifier: DOI:10.11588/heidok.00009848
identifier: urn:nbn:de:bsz:16-opus-98487
identifier:   Staudt, Thorsten M.  (2009) Strategies to reduce photobleaching, dark state transitions and phototoxicity in subdiffraction optical microscopy.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/9848/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng