Distance and concentration significantly influence indocyanine green fluorescence intensity in near-infrared endoscopic viewing modes: an in vitro blotting paper model.

OBJECTIVE: To evaluate the effects of indocyanine green (ICG) concentration, camera-to-target distance, and their interaction on fluorescence intensity (FI) using a near-infrared (NIR) endoscopic system in dark and overlay modes. METHODS: 7 ICG dilutions (0 to 2.5 mg/mL) were applied to white blotting paper. Images were captured using a 0°, 10-mm NIR endoscopic system at distances of 2, 5, and 10 cm in dark mode (monochromic NIR signal) and overlay mode (NIR signal superimposed on white light). Fluorescence intensity (dark mode) and light intensity (LI; overlay mode) were quantified using ImageJ software (version 1.54; NIH). Data were analyzed via ANOVA and bivariate linear regression. RESULTS: In dark mode, distance significantly influenced FI, with maximum intensity at 2 cm and no signal at 10 cm. Indocyanine green concentration also significantly affected FI, with the highest FI at 0.0156 mg/mL; concentrations above 0.625 mg/mL showed quenching. In overlay mode, LI was maximized at 5 cm and decreased significantly at 2 cm. Concentration had an inverse effect in overlay mode, where 0 mg/mL (control) yielded the highest LI. Dark mode FI and overlay mode LI were significantly inversely correlated. CONCLUSIONS: In this experimental model, fluorescence and light intensities depend on camera distance and ICG concentration. Signal loss occurs at greater distances, and quenching effects are prominent in specific viewing modes. CLINICAL RELEVANCE: In this in vitro model, ICG concentration, camera-to-target distance, and viewing mode influenced fluorescence and light intensity. These findings support standardization of image acquisition and future in vivo validation.