Experimental determination of the linear correlation between in vivo TV fluorescence intensity and vascular and tissue FITC-Dx concentrations

A novel in vivo calibration procedure was developed to determine the microvascular and tissue concentration of fluorescein isothiocyanate-labeled dextrans (FITC-Dx) in the hamster cheek pouch, using intravital fluorescence microscopy with manually controlled TV camera gain and threshold value. Two FITC-dextrans (70,000 and 150,000 MW) were used as tracers. Five minutes after the tracer was administered, selected venules (diameter 20-50 μm) were videotaped, and intravascular gray levels were obtained by digital image processing. Simultaneously, arterial blood samples were taken to measure vascular FITC-Dx concentrations with a spectrofluorometer. The gray levels and the concentrations were used to produce a calibration curve for the vascular FITC-Dx concentration. A similar calibration curve for the interstitial FITC-Dx concentration was obtained by first video recording interstitial space areas saturated with the tracer. After flushing out the tracer in the vessels, the hamster cheek pouch was then cut, weighed, and homogenized. The interstitial FITC-Dx concentration was finally measured with a spectrofluorometer. The gray levels and the concentrations were used to produce a calibration curve for the interstitial FITC-Dx concentration. The gray level was found to vary linearly with both the FITC-Dx vascular concentration (range 0.4-3.0 mg/ml) and the interstitial FITC-Dx concentration (0.12-1.50 mg/ml) in the hamster cheek pouch.