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Advanced multiplexing techniques in single molecule localisation microscopy

dc.contributor.advisorEnderlein, Jörg Prof. Dr.
dc.contributor.authorOleksiievets, Nazar
dc.titleAdvanced multiplexing techniques in single molecule localisation microscopyde
dc.contributor.refereeEnderlein, Jörg Prof. Dr.
dc.subject.gokPhysik (PPN621336750)de
dc.description.abstractengDNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging applications. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. This thesis presents several novel techniques for multiplexed imaging, with the main focus on the DNA-PAINT super-resolution technique. At first, I demonstrate multiplexed imaging by using three different single-domain antibodies (nanobodies). They have a three times smaller size than conventional antibodies, therefore reducing the distance between the target site and the fluorophore. The used nanobodies have a high and specific binding affinity against three common fluorescent proteins: GFP, BFP and RFP. This allows me to use conventional fluorescent protein fusion for labelling cellular structures of interest, and then to use fluorescently labelled nanobodies as imager molecules in PAINT. In addition, I introduce a novel wide-field Time-Correlated Single Photon Counting (TCSPC) camera, that was successfully used for Fluorescence Lifetime Imaging (FLIM) with single-molecule sensitivity. As an additional application of Fluorescence-Lifetime Single-Molecule Localization Microscopy (FL-SMLM), I used FL-SMLM for single-molecule metal-induced energy transfer (smMIET) imaging which is a first step towards three-dimensional single-molecule localization microscopy (SMLM) with isotropic nanometer resolution. Next, I combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT (together FL-PAINT) and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) images multiple targets simultaneously, therefore shortening the total acquisition time, and requires no fluid exchange, thus both leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. FL-PAINT can be readily combined with other DNA-PAINT based techniques of multiplexed imaging, and therefore FL-PAINT has a great potential for highly multiplexed
dc.contributor.coRefereeRizzoli, Silvio Prof. Dr.
dc.contributor.thirdRefereeMüller, Marcus Prof. Dr.
dc.contributor.thirdRefereeEgner, Alexander Prof. Dr.
dc.contributor.thirdRefereeBetz, Timo Prof. Dr.
dc.contributor.thirdRefereeLuther, Stefan Prof. Dr.
dc.subject.engFluorescence Microscopyde
dc.subject.engSuper-resolution microscopyde
dc.subject.engSingle Molecule Localisation Microscopy (SMLM)de
dc.subject.engFluorescence Lifetime Imaging (FLIM)de
dc.subject.engDNA point accumulation for imaging in nanoscale topography (DNA-PAINT)de
dc.subject.engFluorescence Lifetime PAINT (FL-PAINT)de
dc.subject.engMetal-induced Energy Transfer (MIET)de
dc.affiliation.instituteFakultät für Physikde

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