Seminar: "Microbial single-particle tracking microscopy: CRISPR-Cas and beyond"
Speaker: Dr. Koen Martens
Venue: VU LSC R103
Single-particle tracking (spt) microscopy is a powerful method to visualise and track individual proteins and elucidate protein-DNA interactions in vivo with high spatio-temporal accuracy (<10 ms, <30 nm). Using spt, I investigated the (d)Cas9 target search in Lactococcus lactis, and showed that dCas9 is screening protospacer-adjacent motifs (PAMs) 40% of the time, averaging just 17 ms per binding event. CRISPR-dCas target search is a system in equilibrium, indicating that experimental time can be indefinite. However, many biological systems are out-of-equilibrium, such as DNA repair: here, the biological mechanism of interest is resolved within 10 minutes, much faster than experimental time in spt. I will present instrument design and computational approaches to approach in vivo spt with much higher throughput: intelligent, autonomous ("self-driving"), multi-field-of-view single-molecule microscopy allows for more information per experiment, while new computational approaches (relative distance analysis; TARDIS) enables at least 10-fold higher single-molecule density in spt experiments.
About the speaker: Koen works in the lab of Ulrike Endesfelder (Rhenish Friedrich Wilhelm University of Bonn, Institute for microbiology and biotechnology). He studies biophysical processes in living cells with advanced single-molecule localization microscopy. Specifically, he is focusing on dynamical protein interaction quantification in individual cells on the nanometer spatial scale and millisecond temporal scale. To this end, he has experience in computational quantitative single-molecule microscopy, microscope hardware development, and prokaryotic genomic manipulation.