Intravenous application of fluorescein for confocal laser scanning microscopy: evaluation of contrast dynamics and image quality with increasing injection-to-imaging time

Background: Confocal laser scanning microscopy (CLSM) is reported to be a promising tool for in vivo histopathology during an endoscopy. The most commonly used florophore is fluorescein sodium given intravenously. However, so far, there are no objective data on contrast dynamics and image quality over time after injection of the substance. Objective: We aimed to investigate the ideal time period for the best CLSM imaging when using florescein sodium. Design: The proximal stomachs of 4 anesthetized pigs were examinee via standard endoscopes with a portable confocal miniprobe. After injection of 1% florescein intravenously (0.1 mL/kg both weight), consecutive sequences were recorded after 1, 2, 3, 5, 8, 20, 30, 40, 50, and 60 minutes. After the procedure, histograms, which represented signal intensity of 20 images (at respective time points), were analyzed from representative regions of interest (ROI). The mean (standard deviation [SD]) pixel Value of the pixel values in the ROI were noted. A signal-to-noise ratio (SNR), defined as the ratio between the mean and the SD, was calculated. In addition, 5 images per time period were put into a random order and then blindly evaluated by 2 endoscopists. Results: A total of 560 histograms from selected ROIs were analysed. The SNR decreased significantly after 8 minutes, with the lowest values being reached after 30 minutes. subjective evaluation of still images also demonstrated a continuous decrease, with the lowest image quality after 30 minutes. A slight increase of image quality after 40 minutes occured because of extramucosal leakage of fluorescein, with fluorophore deposits in the crypt orifices. Conclusions: In the pig model, flourescein-based CLSM of the GI tract delivered the best contrast and image quality within the first 8 minutes after injection of the fluorophore. These data might facilitate standardization of the procedure for research purposes but may also help when applying this new technology in a daily routine manner.