Exosomes and other extracellular vesicles (EVs) are key contributors to cell-to-cell communication. EVs can cross biological barriers such as the blood-brain barrier and be internalized within the cell with a high degree of specificity. For these reasons, they are of significant interest as therapeutic agents, drug delivery vehicles and diagnostic biomarkers.
EVs typically have a hydrodynamic diameter of 30-2000 nm; for the exosomes sub-group, this value is thought to be 30-120 nm, which is smaller than the theoretical best resolution for an optical microscope. To fully understand exosomes requires instrumentation that is both sensitive enough to detect them and has sufficient resolution to visualize them.
The Nanoimager offers 20 nm resolution imaging in fixed cells and single-molecule sensitivity. It can visualize the uptake and genesis of exosomes in multiple colors, to see how exosome markers and contents interact with other cellular pathways and compartments, including lysosomes, the multi-vesicular body, the cytoskeleton or the plasma membrane.
The Nanoimager is further capable of tracking single molecules both in cells and in solution by single-particle tracking (SPT). The single-molecule sensitivity of the Nanoimager makes detection of fluorescently-labeled vesicles trivial. The Nanoimager use SPT to both count and measure the size of molecules or vesicles in solution.
The Nanoimager was used to study the uptake of exosomes in a HeLa cell. As a control, the GFP-labeled exosomes were first imaged in solution using SPT to measure their size. The size histogram indicated that the exosomes were 80-100 nm in diameter with some larger particles present.
The exosomes were next added to HeLa cells and visualized diffusing in solution before entering the cell. The colorbar in the image indicates a dramatic drop in diffusion once exosomes bound to or entered the cell. Further experiments will be undertaken with multiple colors to understand which molecules are involved in the uptake process and to measure the timescale for this process.
In summary, the Nanoimager offers: -Super-resolution multi-color imaging with 20 nm resolution to understand the interaction of exosomes with other cellular components. -Tracking of vesicles inside cells and in solution (3D single-molecule dual color SPT). -Single-molecule sensitivity for both organic dyes and fluorescent proteins.