Additionally, an intriguing map of the distribution of VDPs in whole cells was developed through the analysis of selected single cells (Figure ). Specifically, while a major part of RhQ labeled fluorescence localized in the mitochondria as the time increased, the cell surface fluorescence was still very strong, suggesting the presence of cell surface protein vicinal dithiols on live MCF-7 cells (Figure ). Conversely, no fluorescence signal from the cell surface of live CHO-K1 cell was observed as the time increased. In addition, most of the RhQ labeled fluorescence signal merged well with the Mitotracker Green labeled fluorescence signal, indicating that most of the VDPs are present on the mitochondria of live CHO-K1 cells (Figure ). The regions of interest (ROI) analysis of the cells further confirmed this phenomenon (Figure and). Accordingly, the Pearson's correlation coefficient from CHO-K1 cells is higher than the values obtained from the other two cell lines (above 0.8 vs below 0.8) in addition the Pearson's correlation coefficient increased with time (Figure ). Finally, movies of the three channels (RhQ channel, Mitotracker Green channel and overlay channel) were obtained from the time-lapse images, enabling the dynamics of the protein vicinal dithiols for one single live cell to be monitored over a period of 90 min (Movie S3.1b and 3.2b). All these results indicated that RhQ could be used as an effective probe for illuminating the distribution of endogenous protein vicinal dithiols in live cells. In addition, a map of VDPs in different types of live cell lines could be clearly identified using RhQ. To further identify the presence of the protein vicinal dithiols and related VDPs on the cell surface. MCF-7 cells were first treated with DTNB (5,5'-Dithiobis(2-nitrobenzoic acid), a cell impermeable reagent that was used for blocking the cell surface thiols). Then, RhQ was loaded into the cells, and the dynamic changes of the fluorescence signal of the cells was tracked every 2 min (movie S4.1a). Significantly, minimal cell-surface fluorescence was observed when compared to the non DTNB-treated MCF-7 cells (Figure , Figure , movie S1a). Additionally, N-Ethylmaleimide (NEM) an inhibitor of cell thiols was incubated with the MCF-7 cells for 30 min, then RhQ was loaded into the cells. Negligible fluorescence signal throughout the whole cell after dynamic tracing of the fluorescence signal for 90 min (Figure , movie S4.2a). All these results indicate the presence of cell-surface protein vicinal dithiols. To assess the VDPs, associated with the cell-surface, electrophoresis was used to analyze the total cellular proteins after the cells were lysed (Figure ). Compare to the lane loaded with only RhQ-labeled cell lysis, the lane loaded with NEM-treated cell lysis displayed no fluorescence signal. This result was caused by the inhibition of cell thiols using NEM. A significant fluorescence protein band appeared at the molecular weight of 69 kDa, 53 kDa, 47 kDa, 38 kDa and 35 kDa, which indicated the presence of RhQ-labelled VDPs. The identified peptide sequences. The predicted molecular weights are shown and matched closely to those observed on the gels. For the peptide fragment ion masses searched MASCOT scores >45 are significant. The cellular distribution information of the identified proteins were from UniProt. (d) The positions of the vicinal dithiols in part of the X-ray structures of the selected proteins. The yellow ball and sticks show the thiols in the cysteine residues of the proteins. The X-ray structures of the selected proteins is from the protein data bank (PDB) data which is available from UniProt.