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 endoscopy. The most commonly used fluorophore is fluorescein sodium applied intravenously (iv). However, so far, there are no objective data on contrast dynamics and image quality over time after injection of the substance. We therefore aimed to investigate the ideal time period for best CLSM imaging using this substance.

Material and Methods:

The proximal stomach of 4 anaesthetized pigs was examined via standard endoscopes with a portable confocal miniprobe. After injection of 1% fluorescein iv (0.1ml/kg body weight) consecutive sequences were recorded after 1, 2, 3, 5, 8, 20, 30, 40, 50 and 60 minutes. Post procedural, histograms of 20 images (at respective time points) were analyzed from representative regions of interest (ROI). Mean pixel value and standard deviation of the pixel values in the ROI were noted. A signal-to-noise ratio (SNR) was calculated defined as the ratio between the mean and standard deviation. In addition, 5 images per time period were put into a random order and blindly evaluated by two endoscopists.

Results:

A total of 560 histograms from selected ROIs were analyzed. SNR decreased significantly after 8 minutes with lowest values reached after 30 minutes. Subjective evaluation of still images also demonstrated a continuous decrease with lowest image quality after 30 minutes. A slight increase of image quality after 40 minutes occurred due to extramucosal leakage of fluorescein with fluorophore deposits in the crypt orifices.

Conclusion:

Fluorescein based CLSM of the gastrointestinal tract delivers 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 applying this new technology in a daily routine manner.

Keywords: confocal laser scanning microscopy, fluorescein, dynamics, injection-to-imaging time, endomicroscopy