Visual screening and efficacy evaluation of high-performance fluorescent probe DAF-FM in esophagitis cancer transformation

Figure 1 Imaging of a rat model of esophagitis. A: Hematoxylin-eosin staining of esophageal tissue in the normal control group (left) and esophagitis model group (right); B: Quantitative statistical chart of inflammatory cell count per field of view (normal group: 8.2 ± 1.5, esophagitis group: 35.6 ± 4.2); C: Quantitative statistical chart of epithelial defect area (normal group: 0 mm², esophagitis group: 0.12 ± 0.03 mm²); D: Quantitative statistical chart of submucosal edema thickness (normal group: 0.02 ± 0.01 mm, esophagitis group: 0.08 ± 0.02 mm); E: Quantitative statistical chart of ulcer number per rat (normal group: 0, esophagitis group: 2.3 ± 0.5). Scale bar: 250 μm; data are expressed as mean ± SD, n = 10 per group, ^bP < 0.01.

Figure 2 Concentration-dependent and time-dependent fluorescence intensity of DAF-FM probe with nitric oxide. A: Fluorescence images of DAF-FM incubated with different concentrations of nitric oxide (NO) donor DEANO (0 μM, 50 μM, 150 μM, 300 μM) for 60 minutes; B: Fluorescence intensity-time curve (0–60 minutes) of DAF-FM at different NO concentrations [0 μM: From 52 ± 3 to 55 ± 4 arbitrary units (a.u.); 300 μM: From 223 ± 9 to 458 ± 15 a.u.]; C: Fluorescence intensity-concentration scatter plot of DAF-FM at 60 minutes (R² = 0.98, P < 0.05). Scale bar: 10 μm; data are expressed as mean ± SD, n = 3 per group.

Figure 3 Cholecystokinin-8 assay for cytotoxicity of DAF-FM probe. A: Absorbance values at 490 nm of esophageal cancer cells treated with different concentrations of DAF-FM (0 μM: 1.28 ± 0.05; 50 μM: 1.05 ± 0.04); B: Cell survival rate-concentration curve (control group: 100% ± 2.1%, 50 μM group: 82.3% ± 4.1%). Data are expressed as mean ± SD, n = 6 replicate wells per group, P < 0.05 vs control group.

Figure 4 Targeted colocalization of DAF-FM probe with lysosomes. A: Merged images of DAF-FM (red) with Lyso-Tracker Green (lysosome, green), Mito-Tracker Green (mitochondria, green), and BODIPY493/593 (liposome, green); B: Bar chart of Pearson colocalization coefficients (lysosome: 0.82 ± 0.03, mitochondria: 0.21 ± 0.04, liposome: 0.18 ± 0.03). Scale bar: 10 μm; data are expressed as mean ± SD, n = 3 per group, ^bP < 0.01 vs mitochondria/Liposome groups.

Figure 5 Flow cytometry analysis of DAF-FM probe cytotoxicity. A: Flow cytometry dot plots (FL1-H: FITC, SSC-H: Side scatter) of esophageal cancer cells treated with 0 μM, 50 μM, 150 μM, 300 μM DEANO; B: Bar chart of apoptotic rate (0 μM DEANO: 4.8% ± 0.9%, 50 μM DEANO: 5.2% ± 1.1%, P > 0.05); C: Bar chart of the proportion of living cells (0 μM DEANO: 94.5% ± 1.2%, 50 μM DEANO: 92.3% ± 1.5%, P > 0.05). Data are expressed as mean ± SD, n = 3 per group.