Spatiotemporal mapping of the position and orientation of nano-machinery inside complex and dynamic cellular environments is essential for the detailed understanding of many bio-physical processes. For the genuine observation of such biomolecular dynamics with high signal to noise ratio and reduced disturbance from the labeling probes, reduction in the size of nano-bio labels and simplification of techniques for their observation are important. Here we achieve this using polarized dark field scattering micro-spectroscopy (PDFSMS), in its simplest form so that it is deployable in several experiments. We not only locate tiny gold nanorods (GNRs) of size 30 (length) × 10 nm (diameter) inside HEK293 cells but also demonstrate mapping of their in-situ polarization patterns using a novel method. Real time observations of rotating GNR with DFSMS and PDFSMS are used to resolve in-plane and out-of-plane rotational modes of GNR. We have shown that PDFSMS itself can provide complete information about the state of GNR. A step ahead, we demonstrate the application of PDFSMS to track three dimensional rotational dynamics of transferrin-conjugated GNRs inside live HEK293 cells. These first-time observations of the three dimensional intracellular rotational dynamics of tiny GNRs using PDFSMS present a new landmark in single particle scattering spectroscopy.
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