As shown in Figure , RhQ treated HeLa cells also exhibit similar behavior where the fluorescence signal initially appears on the cell surface and then the intracellular fluorescence signal gradually increases. Using time-lapse imaging, we could clearly observe this behavior with live HeLa cells through fluorescence changes within 20 min (Figure , Movie S2b). While, for the RhQ-treated CHO-K1 cells, no fluorescence signal was observed on the cell surface for the first 20 min. With only an intracellular fluorescence signal, which became significantly stronger as the time increased (Figure ). These dynamic changes of fluorescence in live CHO-K1 cells were tracked using time-lapse imaging (Figure , Movie S2c). Thus, RhQ was an effective probe for tracing cell surface protein vicinal dithiols within 20 min, which could be attributed to the relatively slow interaction between RhQ and vicinal dithiols of VDPs. Where it requires about 90 min for the complete interaction between RhQ and rBSA to occur (Figure ), conversely, our previous probe NPE only requires 10 min. Fortunately, the slow interaction between RhQ and the protein vicinal dithiols allows RhQ to have sufficient time to interact with cell-surface protein vicinal dithiols before it enters into the cells to interact with intracellular protein vicinal dithiols. From these results, it is apparent that the vicinal dithiols of VDPs are present on the cell surface and are closely related to the types of cell lines. In addition, the fluorescence signal from the MCF-7 cell surface is enhanced compared to the fluorescence signal from HeLa cells. This result indicates that abundant protein vicinal dithiols are presented on the MCF-7 cell surface. Given that that cell surface thiols may play significant role in enhancing cellular uptake, significant interest in protein vicinal dithiols has developed since they may mediate the entry of viruses (such as HIV virus) into cells. However, detailed evidence concerning the role of cell-surface protein vicinal dithiols in regulating biological processes remains unclear. For instance, although thiol/disulfide exchange is a prerequisite for HIV to enter cells, the detailed behavior of protein vicinal dithiols in HIV-1-mediated fusion and infection still require further investigation. Significantly, the use of fluorescent probe RhQ, could enable the visualization of the dynamics and existence of cell surface protein vicinal dithiols within different cell lines, which could expand our understanding of cell surface protein vicinal dithiols as well as encourage the use of cell surface protein thiols to improve cellular delivery.