| Method |
P.4 bottom paragraph: "Typically the normalized bright-field optical microscope-based single-cell absorption spectra and corresponding single-cell microscope images of HL7702, L929, HeLa, and A549 cells were recorded by the method (Fig. 4a), using bromothymol blue as a pH indicator and randomly selected bare A549 single cells (indicator-free) as a blank sample. Here, the single-cell absorption spectra were all recorded solely from an intact single cell (acting as a sample container) and used to obtain the averaged pH value of a single whole-cell. The UV-Vis [Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry] absorption peaks of single HL7702, L929, HeLa, and A549 cells were clearly observed without interference from the intracellular substances of the cell. The peaks were distinguishable with a gradual red-shift, which means that their pH values were decreased gradually according to Fig. 3a. The results were quite reproducible for each cell type, making the method feasible and reliable for accurate single-cell pH detection. Incidentally, [investigators] found experimentally that there is always a fixed ~17 nm red-shift deviation of the indicator absorption peak obtained by the microscope-based spectroscopic method (Fig. S5) as compared with that taken by traditional UV-Vis spectrometer, which may stem from the differences in methodology and the surface-confinement effects that may exist in the microscopic method. Therefore, for accurate single-cell pH detection the absorption spectra of the bromothymol blue-dyed single-cells were corrected accordingly." |
| Comments |
p.5 top paragraph: "Figure 4b shows the histogram of the pH distribution for single HL7702, L929, HeLa, A549, and 4T1 cells, respectively, detected by the method after the calibration. For each cell type, [investigators] detected the absorption spectra of at least twenty randomly selected single cells to ensure reproducibility and reliability of the method. As shown in Fig. 4b, the diversity of cell pH for different cell types was revealed, with the averaged pH values of single HL7702, L929, HeLa, A549, and 4T1 cells in this study measured to be 7.23 ± 0.2, 7.0 ± 0.18, 5.75 ± 0.10, 5.5 ± 0.15, and 5.32 ± 0.13, respectively. The measured average pH value of the intact single HeLa cell is very consistent with the previously reported value (5.7 ± 0.2)[ ref 5]." HL7702=human liver cell line, L929=cell from mouse C3H/An connective tisue, HeLa=human cervical cancer cell, A549=adenocarcinomic human alveolar basal epithelial cells, 4T1=cell from mouse mammary tumor |