3D Real-Time Orbital Tracking Microscopy in Zebra Fish Embryos

Abstract

Single particle tracking makes it possible to follow individual molecules and biomolecular complexes in living systems as they perform their biological function. This provides a wealth of information regarding where the molecules reside, how they are transported and with whom they interact.There are several methods for performing 3D single-particle tracking. Our approach is to use orbital tracking with dual-plane detection. In this method, the laser is rotated about the particle of interest. When the particle moves from the center of the orbit, a modulation of the signal is detected and the new lateral position of the particle can be determined. Two confocal pinholes allow simultaneous detection of two planes slight above and below the focus of the laser to allow determination of the axial position of the particle. Using a feedback loop, the center of the orbit is repositioned on the particle of interest. The 3D orbital tracking microscope is controlled by an FPGA so that tracking can be performed in real time with nanometer resolution independent of any latency difficulties from the controlling computer. In addition, a wide-field setup has been mounted such that the surrounding environment is measured simultaneously.Cells, and in particular neuron cells, are dependent on the proper functioning of mitochondria. Although much is known regarding the function of mitochondria in cell culture, few experiments have been performed in living organisms. With our advances in 3D particle tracking, we are now able to follow individual mitochondria as they are transported in neuronal cells in developing zebra fish embryos. From the single particle tracking data, we can quantify the motion of mitochondria, its velocity, the number of pauses, etc. Hence, it is now possible to follow the life cycle of mitochondria in a living organism.