Scientific Organisers: Stefanie Reichelt, Alex Sossick, Nick Barry, Alessandro Esposito and Kirti Prakash
The meeting will begin at 1pm UK Time.
As part of the 'Imaging ONE WORLD' series, the focus of these lectures is on microscopy and image analysis methods and how to apply these to your research. Almost all aspects of imaging such as sample preparation, labelling strategies, experimental workflows, ‘how-to’ image and analyse, as well as facilitating collaborations and inspiring new scientific ideas will be covered. Speakers will be available for questions and answers. The organisers, CRUK CI core facility staff, Gurdon Institute, MRC-LMB, MRC Cancer Unit and NPL will be able to continue the discussion and provide advice on your imaging projects.
Speaker
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Johannes Holbein
Wageningen U&R
After obtaining a Ph.D. in Physics (2008, Martin Luther University Halle-Wittenberg, Germany), Johannes Hohlbein worked as a postdoc in the ‘Gene Machines’ group of Achilles Kapanidis at the University of Oxford. Since 2012 he is working in the Laboratory of Biophysics at Wageningen University & Research (The Netherlands), first as an assistant professor and currently as a tenured associate professor. His lab looks into the amazing world of DNA-protein interactions utilising methods of single-molecule fluorescence spectroscopy and super-resolution microscopy.
Speakers Abstract
Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex biological samples. The number of truly accessible microscopy frameworks that are suitable for demanding applications such as single-particle tracking photo-activation localisation microscopy (sptPALM), however, has remained low. We therefore developed the miCube: a versatile super-resolution capable fluorescence microscope, which combines high spatiotemporal resolution, good adaptability, and easy installation. We further enabled ultrafast data analysis using a phasor-based localisation algorithm and I will show how phasor-based analysis can be used in combination with adaptive optics.
In the second part, I will present our work on CRISPR-Cas. Type II, CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM). To quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we introduced Monte-Carlo diffusion distribution analysis (MC-DDA) which revealed that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17±4ms per binding event. Using heterogeneous dCas9 expression, we determined the number of cellular target-containing plasmids and derived the copy number dependent Cas9 cleavage. Furthermore, we could show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimisation of the CRISPR-Cas toolbox.