PhD defence by Masih Fahim

On Monday 5 February at 13:00, Masih Fahim will defend his PhD thesis "PSF distortion and mislocalization by dielectric nanoparticles in single-molecule microscopy".

Place: bldg. 341 auditorium 21 & zoom: https://dtudk.zoom.us/meeting/register/u5YucO-qqj8oGtL1aA5LVQkZ2QEJD31jowZ5

Main supervisor: Associate Professor Rodolphe Marie
Co-supervisor: Associate Professor Jonas Nyvold Pedersen

Members of assessment committee:
Senior Scientist Lars Lindvold, DTU Elektro
Professor Guillermo Acuna, University Fribourg
Associate Professor Daniel Würstner

Chairperson:
Associate Professor Line Hagner Nielsen, DTU Health Tech

Abstract:

Polymer nanoparticles are particles 1000 times smaller than the width of a human hair. They have interesting applications in the field of bio-sensing and drug delivery. To allow such applications, the nanoparticles are often decorated with biomolecules such as DNA and antibodies. The particles’ surface properties such as the number and distribution of biomolecules, are crucial to their performance. Traditional characterization methods rely on ensemble averages analyzing many particles at the same time and do not provide the distribution of molecules on particles nor particle-to-particle differences. Individual biomolecules can be imaged with fluorescence microscopy, however the conventional light microscopes cannot distinguish molecules that are less than 200 nm apart.

Recent advances in single-molecule localization microscopy (SMLM) allow the visualization and quantification of molecules on nanoparticles. With SMLM, individual biomolecules can be resolved on nanoparticles by strategically tagging molecules of interest with light-emitting fluorophores that randomly shift between 'on' and 'off' states. The ensuing blinking fluorophores is captured in a camera movie, with only a few emitting molecules in each frame. This results in isolated bright spots in each image – so-called diffraction-limited spots – enabling precise localization of each fluorophore in the image with, in principle, nanometer precision.

The interaction of the emitted light with the nanoparticle itself however distorts the image, affecting accurate localization.

In my thesis, I tried to visualize and utilize distortions caused by interactions with polymer micro- and nanospheres. I demonstrated that mislocalization occurs even for particles as small as 200 nm. By increasing the bead size to 1 μm and employing a newly developed analytical model to extract the molecule position, I successfully visualized and analyzed distortions in experimental data. My findings allow for the 3D localization of molecules on dielectric particles from a single-plane image, providing valuable insights for optimizing the conjugation of molecules to nanoparticles.