Biology is a fundamental scientific field which has made significant progress over the course of recent centuries and with the help of modern microscopy techniques, major discoveries are still being made today. The time span of processes such as protein dynamics ranges from slow to extremely fast. That is why high temporal resolution has recently become one of the desired parameters in biological experiments. The improvement of ultrafast image acquisition technology can help us to achieve higher temporal resolutions than before and detailed biological processes of rapid nature can now be observed. With these possibilities comes a desire to determine the noise characteristics of ultrafast cameras to set the limitations in localization precision in tracking of biological objects and their labels, which is the focus of this manuscript.
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.