Abstract
Single-particle tracking (SPT) is a powerful method for exploring single-molecule dynamics in living cells with nanoscale spatiotemporal resolution. Photostability and bright fluorescence make quantum dots (Qdots) a popular choice for SPT. However, their large size could potentially alter the mobility of the molecule of interest. To test this, we labelled B cell receptors on the surface of B-lymphocytes with monovalent Fab fragments of antibodies that were either linked to Qdots via streptavidin or directly conjugated to the small organic fluorophore Cy3. Imaging of receptor mobility by total internal reflection fluorescence microscopy (TIRFM), followed by quantitative single-molecule diffusion and confinement analysis, definitively showed that Qdots sterically hinder lateral mobility regardless of the substrate to which the cells were adhered. Qdot labelling also drastically altered the frequency with which receptors transitioned between apparent slow- and fast-moving states and reduced the size of apparent confinement zones. Although we show that Qdot-labelled probes can detect large differences in receptor mobility, they fail to resolve subtle differences in lateral diffusion that are readily detectable using Cy3-labelled Fabs. Our findings highlight the utility and limitations of using Qdots for TIRFM and wide-field-based SPT, and have significant implications for interpreting SPT data.
Highlights
The lateral mobility of plasma membrane receptors is a major determinant of their function and signalling output[1,2,3]
Mouse A20 B-lymphoma cells were allowed to adhere to coverslips coated with non-stimulatory anti-major histocompatibility complex II (MHCII) monoclonal antibodies[8, 49, 50] and Single-particle tracking (SPT) was performed on the ventral cell surface for 10 s at 33 Hz
We found that the lateral mobility of cell surface B cell receptor (BCR) is significantly reduced when labelled with Quantum dots (Qdots) and imaged by total internal reflection fluorescence microscopy (TIRFM)
Summary
The lateral mobility of plasma membrane receptors is a major determinant of their function and signalling output[1,2,3]. Qdots are semiconductor nanocrystals that allow precise localization due to their bright fluorescence[15] Their high photostability allows long tracks to be obtained, providing greater insights into phenomena such as directional motion, turning behaviour, state switching, and confinement. This makes them a popular choice for SPT8, 15–21. Directly-labelled monovalent Fab fragments have a simpler stoichiometry and their small size (1–2 nm diameter) reduces the potential for steric hindrance. They can exhibit rapid photobleaching (restricting track duration) and they are significantly dimmer than Qdots (reducing tracking precision)
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