nils_walter_microscope

Nils G. Walter

By | | No Comments

Nature and Nanotechnology likewise employ nanoscale machines that self-assemble into structures of complex architecture and functionality.  Fluorescence microscopy offers a non-invasive tool to probe and ultimately dissect and control these nanoassemblies in real-time.  In particular, single molecule fluorescence resonance energy transfer (smFRET) allows us to measure distances at the 2-8 nm scale, whereas complementary super-resolution localization techniques based on Gaussian fitting of imaged point spread functions (PSFs) measure distances in the 10 nm and longer range.  In terms of Big Data Analysis, we have developed a method for the intracellular single molecule, high-resolution localization and counting (iSHiRLoC) of microRNAs (miRNAs), a large group of gene silencers with profound roles in our body, from stem cell development to cancer.  Microinjected, singly-fluorophore labeled, functional miRNAs are tracked at super-resolution within individual diffusing particles.  Observed mobility and mRNA dependent assembly changes suggest the existence of two kinetically distinct assembly processes.  We are currently feeding these data into a single molecule systems biology pipeline to bring into focus the unifying molecular mechanism of such a ubiquitous gene regulatory pathway.  In addition, we are using cluster analysis of smFRET time traces to show that large RNA processing machines such as single spliceosomes – responsible for the accurate removal of all intervening sequences (introns) in pre-messenger RNAs – are working as biased Brownian ratchet machines.  On the opposite end of the application spectrum, we utilize smFRET and super-resolution fluorescence microscopy to monitor enhanced enzyme cascades and nanorobots engineered to self-assemble and function on DNA origami.

Artistic depiction of the SiMREPS platform we are building for the direct single molecule counting of miRNA biomarkers in crude biofluids (Johnson-Buck, A. et al. Kinetic fingerprinting to identify and count single nucleic acids. Nat Biotechnol 33, 730-732 (2015)).

Artistic depiction of the SiMREPS platform we are building for the direct single molecule counting of miRNA biomarkers in crude biofluids (Johnson-Buck, A. et al. Kinetic fingerprinting to identify and count single nucleic acids. Nat Biotechnol 33, 730-732 (2015)).