Orbital Tracking of Single Fluorescent Particles on a Commercial Confocal Microscope

Abstract

Single Particle Tracking (SPT) is a super-resolution technique used to determine the position of fluorescent particles with nanometer precision. The localization is generally obtained by analyzing the spatial distribution of fluorescence intensity emitted by the particle. In fact, the center of the distribution can be determined with an uncertainty which is much lower than the size of the distribution itself. In the orbital tracking method the position of a particle is obtained analyzing the distribution of intensity along a circular orbit scanned around the particle. In combination with an active feedback this method allows tracking of particles in 2D and 3D with millisecond temporal resolution[1]. More recently, the use of orbital tracking to perform imaging has also been proposed[2].The orbital tracking and the other 3D SPT feedback methods are generally implemented on homebuilt microscopes which are not yet commercially available. On the other hand, commercial setups offer the advantage of a user-friendly software interface and pre-calibrated hardware components. It would be of interest to implement a SPT setup based on a feedback approach with minimal modification of a commercially available microscope. Here we explore this idea using a widely used confocal laser scanning microscope, the Zeiss LSM 510, in combination with an external piezoelectric stage scanner. We discuss advantages and limitations of this implementation of the orbital tracking method and the potential application to live cell experiments.