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. 2019 Jun 12;9:8550. doi: 10.1038/s41598-019-44873-1

Figure 3.

Figure 3

In vitro fluorescence and opposing optoacoustic binding signal of fluorescent ICG-pHLIP (green) and dark quencher QC1-pHLIP (cyan) to murine breast cancer cell line 4T1. (a) ICG-pHLIP (5 µM) is a turn-on fluorescent probe with zero fluorescence in cell-free media. Fluorescence was immediately observed when pHLIP inserts into the lipid bilayer of plasma membrane at low pH found at the surface of 4T1 cells (n = 9, scale bar from 0 to 65535). Optoacoustic signal is inversely proportional to the number of cells incubated with ICG-pHLIP, whereas it increases proportionally for cells incubated with QC1 pHIP. (b) Quantification of ICG-pHLIP fluorescence signal increases as the number of cells increases. QC1-pHLIP has negligible signal. (c) For ICG-pHLIP, overall optoacoustic instensities (a.u) decreases as the number of cells incubated with ICG-pHLIP (5 µM) is increased. (d) For QC1-pHLIP, optoacoustic signal increases as the number of cells incubated with QC1-pHLIP (5 µM) is increased. (e) Spectrally unmixed signal shows ICG-pHLIP is a signal-off optoacoustic agent and complicates quantification, whereas (f), QC1-pHLIP is a signal-on probe, provides clear quantifiable signal with high significance. Ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (unpaired t-test).