Advanced multiplexing techniques in single molecule localisation microscopy

by Nazar Oleksiievets
Doctoral thesis
Date of Examination:2021-05-17
Date of issue:2022-05-13
Advisor:Prof. Dr. Jörg Enderlein
Referee:Prof. Dr. Jörg Enderlein
Referee:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Marcus Müller
Referee:Prof. Dr. Alexander Egner
Referee:Prof. Dr. Timo Betz
Referee:Prof. Dr. Stefan Luther
Persistent Address: http://dx.doi.org/10.53846/goediss-9196

 

 

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Abstract

English

DNA 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 bio-imaging.
Keywords: Fluorescence Microscopy; Super-resolution microscopy; Single Molecule Localisation Microscopy (SMLM); Fluorescence Lifetime Imaging (FLIM); DNA point accumulation for imaging in nanoscale topography (DNA-PAINT); Exchange-PAINT; Fluorescence Lifetime PAINT (FL-PAINT); Multiplexing; Metal-induced Energy Transfer (MIET)
 
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