Quantum dot labeling based on near-field optical imaging of CD44 molecules

The lateral organization of membrane proteins and lipids domains has a direct impact on many cellular processes, but generally these domains are too small to be resolved by diffraction-limited resolution of fluorescence microscopy. Here, we use quantum dot (QD) labeling based on near-field optical imaging, to study the nanoscale organization of hyaluronan receptor CD44 molecules of fixed mesenchymal stem cells (MSCs) in air, with a optical resolution down to 50 nm. The photostability and high luminance of QD evidently improve the signal-to-noise ratio and reproducibility of near-field optical data. Importantly, the blinking-intensity analysis was proposed to identify single QD, providing a calibration to relate intensity to numbers of antibody for the first time. Additionally, the fluorescence-topographic imaging enables us to investigate the topographic location pattern. Our results demonstrate that CD44 molecules on MSCs are enriched into nanosized domain and they predominantly locate on the peak of the membrane protrusions, which may contribute to clarify the underlying mechanism of functions ascribed to these molecules.