PhD defence by Teun A.P.M. Huijben

On Wednesday, 29 November, Teun A.P.M. Huijben will defend his PhD thesis "Quantitative analysis methods for single-molecule localization microscopy: molecular orientation, nanoparticles, and counting".

Time: 13:00

Place: Building 341, auditorium 22 & zoom: https://dtudk.zoom.us/meeting/register/u5Uuce2sqTkoHN1tbfvb_2XghO4EfQb4D5OR

Please be aware that the PhD defense may be recorded - This will also be informed at the beginning of the PhD defense.

Supervisor: Associate Professor Rodolphe Marie

Co-supervisor: Associate Professor Jonas N. Pedersen, DTU Health Tech

Assessment committee:
Associate Professor Martijn Wubs, DTU Electro

Professor Jonas Ries, University of Vienna, Austria

Associate Professor Susana Rocha, KU Leuven, Belgium

Chairperson:
Professor Anders Kristensen, DTU Health Tech

Abstract:
Nanoparticles (NPs) are particles with a size in the nanometer regime. They have many applications in the biomedical field as agents in biosensing, drug delivery and photothermal therapy. The function of these NPs critically depends on the number and distribution of functional molecules on their surface. In principle, light microscopy is the ideal method for investigating the surface functionalization of NPs, but the NP scatters light and thereby distorts the microscopy image, leading to significant errors in the obtained image.

One such microscopy technique is single-molecule localization microscopy (SMLM). SMLM is a powerful technique that allows scientists to visualize and study individual molecules within a cell or other biological sample. SMLM achieves nanometer resolution by strategically labeling the molecules of interest with a light-emitting fluorophore that stochastically transitions between the ‘on’- and the ‘off’-state. The resulting blinking fluorophores are typically imaged over thousands of camera frames, where in each frame only a few fluorophores emit light. Consequently, each fluorophore creates an isolated diffraction-limited spot in the image, allowing localization of the fluorophore with nanometer precision through fitting with a model for the point-spread function (PSF).

In my thesis, I set out to develop quantitative analysis methods for SMLM to study the number and distribution of functional molecules on the surface of nanoparticles. In the first part of the thesis, I extend SMLM to localize fluorophores on the surface of NPs. SMLM is, in principle, the ideal method for investigating the surface functionalization of NPs, but the NP distorts the PSF, leading to significant localization errors. To address this problem, I developed the first fully analytical model for the PSF when the fluorophore is located close to an NP. This model makes it possible to extract the position of functional groups on the surface of the NPs with 5 nm precision. In the second part, I present a method for quantifying the number of molecules within an assembly of molecules, based on the intensity fluctuations of the signal over time.

Taken together, I developed multiple quantitative approaches for characterizing the position and number of fluorophores in localization microscopy, which will be widely applicable in the field of nano- and biotechnology.