Publications
Direct observation of mobility state transitions in RNA trajectories by sensitive single molecule feedback tracking.
JH Spille, TP Kaminski, K Scherer, JS Rinne, A Heckel, U Kubitscheck. Nucleic Acids Research 43 (2), e14 (2014).
Abstract: Observation and tracking of fluorescently labeled molecules and particles in living cells reveals detailed information about intracellular processes on the molecular level. Whereas light microscopic particle observation is usually limited to two-dimensional projections of short trajectory segments, we report here image-based real-time three-dimensional single particle tracking in an active feedback loop with single molecule sensitivity. We tracked particles carrying only 1–3 fluorophores deep inside living tissue with high spatio-temporal resolution. Using this approach, we succeeded to acquire trajectories containing several hundred localizations. We present statistical methods to find significant deviations from random Brownian motion in such trajectories. The analysis allowed us to directly observe transitions in the mobility of ribosomal (r)RNA and Balbiani ring (BR) messenger (m)RNA particles in living Chironomus tentans salivary gland cell nuclei. We found that BR mRNA particles displayed phases of reduced mobility, while rRNA particles showed distinct binding events in and near nucleoli.
We were able to improve the 3D feedback tracking a lot to achieve single molecule sensitivity. (As in: single fluorescent emitter!) 150-200 photons detected per frame are sufficient for reliable trackign in living cells. This makes it possible to follow single oligo-labelled mRNA particles over tens of microns throughout the nucleus. That means we can observe a single mRNA for 16 seconds instead of 0.2 seconds while maintaining localization precision better than 100nm in axial direction. Careful analysis of those long trajectories revealed that particles do undergo changes in mobility even in chromatin-free space. We had suspected this before but with the new method, we can actually pinpoint where the mRNA slows down. We could observe single mRNAs probing the nuclear envelope several times, presumably sampling several nuclear pores. We also observed rRNA interacting with nucleoli and fluorescently labelled lipids covering the membrane of giant unilamellar vesicles.
The key improvement was using a template matching approach rather than iterative fitting to estimate the axial particle position from its PSF shape. After taking an image it takes only 1.2ms to bring the molecule back to focus!
I still hope that somebody will transform this into a µ-Manager plugin. Let's talk if you are interested!
