Optical Characterization of Sodium Fluorescein In Vitro and Ex Vivo

Ran Xu; Wanda Teich; Florian Frenzel; Katrin Hoffmann; Josefine Radke; Judith Rösler; Katharina Faust; Anne Blank; Susan Brandenburg; Martin Misch; Peter Vajkoczy; Julia Sophie Onken; Ute Resch-Genger

doi:10.3389/fonc.2021.654300

Optical Characterization of Sodium Fluorescein In Vitro and Ex Vivo

Front Oncol. 2021 May 10:11:654300. doi: 10.3389/fonc.2021.654300. eCollection 2021.

Authors

Affiliations

¹ Department of Neurosurgery, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, and Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.
² Division Biophotonics, Federal Institute for Materials Research and Testing (BAM), Berlin, Germany.
³ Department of Neuropathology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.
⁴ German Cancer Consortium (DKTK), Heidelberg, Germany, partner site Charité Berlin, Berlin, Germany.
⁵ Berlin Institute of Health (BIH), Berlin, Germany.

Abstract

Objective: The utilization of fluorescein-guided biopsies and resection has been recently discussed as a suitable strategy to improve and expedite operative techniques for the resection of central nervous system (CNS) tumors. However, little is known about the optical properties of sodium fluorescein (NaFl) in human tumor tissue and their potential impact on ex vivo analyses involving fluorescence-based methods.

Methods: Tumor tissue was obtained from a study cohort of an observational study on the utilization of fluorescein-guided biopsy and resection (n=5). The optical properties of fluorescein-stained tissue were compared to the optical features of the dye in vitro and in control samples consisting of tumor tissue of high-grade glioma patients (n=3) without intravenous (i.v.) application of NaFl. The dye-exposed tumor tissues were used for optical measurements to confirm the detectability of NaFl emission ex vivo. The tissue samples were fixed in 4%PFA, immersed in 30% sucrose, embedded in Tissue-Tek OCT compound, and cut to 10 μm cryosections. Spatially resolved emission spectra from tumor samples were recorded on representative slides with a Confocal Laser Scanning Microscope FV1000 (Olympus GmbH, Hamburg, Germany) upon excitation with λ_exc = 488 nm.

Results: Optical measurements of fluorescein in 0.9% sodium chloride (NaCl) under in vitro conditions showed an absorption maximum of λ_{max abs} = 479 nm as detected with spectrophotometer Specord 200 and an emission peak at λ_{max em} = 538 nm recorded with the emCCD detection system of a custom-made microscope-based single particle setup using a 500 nm long-pass filter. Further measurements revealed pH- and concentration-dependent emission spectra of NaFl. Under ex vivo conditions, confocal laser scanning microscopy of fluorescein tumor samples revealed a slight bathochromic shift and a broadening of the emission band.

Conclusion: Tumor uptake of NaFl leads to changes in the optical properties - a bathochromic shift and broadening of the emission band - possibly caused by the dye's high pH sensitivity and concentration-dependent reabsorption acting as an inner filter of the dye's emission, particularly in the short wavelength region of the emission spectrum where absorption and fluorescence overlap. Understanding the ex vivo optical properties of fluorescein is crucial for testing and validating its further applicability as an optical probe for intravital microscopy, immunofluorescence localization studies, and flow cytometry analysis.

Keywords: NaFl; brain tumor; confocal; fluorescein-guided surgery; optical probe; pH sensing; sodium fluorescein; spectroscopy.