Interferometric scattering (iSCAT) microscopy for high fidelity tracking at microseconds timescales

Łukasz Bujak; Marek Piliarik

doi:10.1117/12.2321086

5 September 2018 Interferometric scattering (iSCAT) microscopy for high fidelity tracking at microseconds timescales

Łukasz Bujak, Marek Piliarik

Proceedings Volume 10726, Nanoimaging and Nanospectroscopy VI; 1072615 (2018) https://doi.org/10.1117/12.2321086
Event: SPIE Nanoscience + Engineering, 2018, San Diego, California, United States

Abstract

Novel methods aiming at understanding complex biophysical processes allow revealing the dynamics and behaviour in extreme detail down to a single protein. Developments of fluorescence-based super-resolution microscopy and nanoscopic tracking techniques helped to reach a spatial resolution in length scales below 10 nm. These advances rely on the efficient collection of fluorescence at single-molecule levels. However, complex photophysics and saturation of fluorescent labels limit the temporal resolution to milliseconds timescales. To overcome the spatiotemporal limitations of fluorescent-based techniques we are employing interferometric scattering microscopy (iSCAT). iSCAT is an optical microscopy technique which allows for the detection and localization of extremely low scattering signals. It is based on interference of light scattered on the particle with a reference wave, e.g. light partially reflected at a glass coverslip. The sensitivity of iSCAT was previously proven in detection experiments with small nanoparticles as well as unlabelled single proteins. Here, we show that scattering labels can be imaged and localized with a nanometer precision and a few microseconds temporal resolution. We investigate the limits of fast tracking of scattering labels and identify pitfalls of high-speed collection for which the tracking fidelity drops rapidly due to fluctuations in the label position.

Citation Download Citation

Łukasz Bujak and Marek Piliarik "Interferometric scattering (iSCAT) microscopy for high fidelity tracking at microseconds timescales", Proc. SPIE 10726, Nanoimaging and Nanospectroscopy VI, 1072615 (5 September 2018); https://doi.org/10.1117/12.2321086

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