gms | German Medical Science

Objective: The utilization of fluorescein-guided biopsies and resection has been discussed as a suitable strategy to improve and expedite operative technique for the discrimination between contrast-enhancing and healthy tissue. However, little is known about the optical characteristics of fluorescein after biological metabolization in tumor tissue.

Methods: Tumor tissue was obtained from a study cohort of a prospective observational study on the utilization of fluorescein-guided biopsy and resection (n=6) and compared to the optical properties in vitro and tumor tissue of glioma patients (n=5) without i.v. application of fluorescein. Tissue samples were fixed in 4% PFA overnight at 4C°, immersed in 30% sucrose, embedded in OCT and cut to 10um sections. The dye-exposed tumor tissues were used for optical measurements to confirm the detectability of fluorescein emission signals in vitro. Confocal laser scanning was conducted using an APD for light detection and filters for separation of green (545±12.5nm) and red (655±20nm) emission. Spectra measurements at 488nm excitation were performed under same conditions using an emCCD camera.

Results: Optical measurements of fluorescein in 0.9% NaCl under in vitro conditions showed an absorption maximum of lmax abs=479nm as detected with spectrophotometer Specord200 and an emission maximum of lmax em=538 nm as recorded with the emCCD detection system of a custom-made microscope-based single particle setup using a 500nm long-pass filter. Under ex vivo conditions, the fluorescein tumor samples revealed a broadening of the emission band. This can be explained by the high pH sensitivity of fluorescein, that can exist in the three different protonation states "neutral", "monoanion", and "dianion", accounting for the different characteristics of fluorescein molecules located in the acid extracellular milieu of the tumor, which most likely exist mainly as monoanions, compared to fluorescein molecules sensing a more basic environment. The latter can result in the formation of the dye's dianion.

Conclusion: Biological metabolization of fluorescein within the scope of preoperative i.v. leads to a change in the optical properties of the dye, particularly a broadening of the emission band. This underlines that a profound understanding of the ex vivo optical properties of fluorescein is crucial for applications such as intravital microscopy and immunofluorescence staining protocols for localization studies.