Nano-Resolution in Vivo 3D Orbital Tracking System to Study Cellular Dynamics and Bio-Molecular Processes

We present a microscopy technique, orbital particle tracking, in which the scanner scans orbits around species, unlike a raster imaging technique in which the scanner scans an area one line at a time. By analyzing the fluorescence emission intensity variation along an orbit, the location of a species in the orbit can be determined with precision of a tenth of a nanometer in a millisecond time scale, and the orbit can be moved to the new location of the species through a feedback loop if any movement is detected. This technique can be extended to two scanning orbits, one above and one below the sample plane to track the sample in 3D space. It can be used in vitro or in vivo to track a motion of a sample or to understand the dynamics of the sample. Additional detectors can help reveal the correlation between events with different emission spectrums. We have performed two different experiments with the system to show the capability of the technique. In the first example, we track a transcription site to understand the relationship between transcription factor - DNA binding and RNA transcription. By labeling a transcription factor with Halo-JF646 and nascent RNA with PP7-GFP, we were able to cross correlate fluorescence intensity to discover temporal coordination between transcription factor DNA binding and resulting gene activation. In the second experiment, we tracked lysosomes in live cells to understand the nature of the transport whether it is an active transport or a free diffusion. Trajectories of a total of 24 lysosomes are recorded during the experiment. The mean squared displacement (MSD) curves of the trajectories showed some clear differences between the behaviors of the lysosomes which were attributed to the active transport along microtubules as opposed to freely diffusing lysosomes.