New 5-aminolevulinic acid fluorescence filter enhances glioma and background structure visualization: A controlled preclinical study

Background: The BLUE 400 S is a newly approved fluorescence filter for 5-aminolevulinic acid fluorescence- guided surgery that renders fluorescence as orange against a yellow-green background instead of the conventional color scheme of BLUE 400. Methods: Three orthotopic GL261 mouse gliomas were imaged under BLUE 400 and BLUE 400 S filters at four resection stages using both microscope camera and smartphone ocular photography. Four blinded reviewers measured the fluorescence dimension and rated boundary delineation confidence across eight pairs of images. Red-channel signal-to-background ratio (SBR) was computed from fixed-coordinate regions-of- interest. Widefield fluorescence imaging was correlated with conventional histology and in vivo confocal laser endomicroscopy (CLE) imaging. Results: BLUE 400 S provided a brighter, anatomically richer operative field across all stages. Tumor fluorescence was clearly delineated under both filters. Interrater reliability was excellent across all imaging conditions. Fluorescence dimension measurements did not differ between filters, but boundary-delineation confidence was higher under BLUE 400 S despite lower red-channel SBR. The digital camera amplified apparent fluorescence brightness and contrast relative to the ocular view at the cost of reduced internal texture within positive fluorescence and background details. CLE and histology confirmed graded correspondence between fluorescence intensity and tumor cellularity. Conclusion: BLUE 400 S improved visualization of background anatomy and rater confidence in fluorescence boundary determination while maintaining equivalent tumor fluorescence dimension measurements. These findings support its perceptual advantage over conventional BLUE 400 and should be further evaluated clinically.