Ex vivo tissue fluorescence microscopy

A subset of biopsies (n = 7) from inside the primary tumor border were processed by frozen section for fluorescence microscopy. Frozen biopsies were collected from n = 4 low-dose patient specimens and n = 3 high-dose patient specimens. Frozen biopsies were cut serially into 8 μm frozen tissue sections and placed on glass microscopy slides for confocal fluorescence microscopy before histological staining. Frozen sections were wrapped in tin foil to limit light exposure and stored at − 80 °C until fluorescence microscopy was performed. Serial sections were cut from each biopsy for confocal microscopy, H&E, and additional staining, as required. Slides were imaged with the Nikon A1R resonance scanning confocal fluorescence microscope (Nikon Instruments Inc. Melville, NY, U.S.A.) using 405 nm excitation, a 525/50-nm emission filter for background tissue autofluorescence (AF) and a 600/50 + 685/70-nm emission filter for PpIX fluorescence. Resonance mode was used to scan frozen tissue sections rapidly (30 or 420 frames/second), to optimize image acquisition settings and minimize photobleaching of PpIX fluorescence. Fluorescence images were acquired using Galvano scanning at 1024 × 1024 pixels.

Immediately following fluorescence microscopy, frozen sections were fixed in formalin and stained with H&E. Two additional serial sections of the same sample were stained using standard protocols with Masson’s trichrome (MT) to identify connective tissues and Oil Red O (ORO) to identify adipocytes. This enabled spatial correlation between fluorescent tissue features in the frozen section and tissue-specific staining for histology. Nikon NIS Elements C software (Nikon Instruments Inc. Melville, NY, USA) was used to convert fluorescence microscopy images to TIF format and Aperio ImageScope was used to export corresponding images of H&E, MT, and ORO-stained tissues to JPEG image format. Fluorescence H&E, MT, and ORO-stained tissue section images were examined by the study pathologist (S.J.D.).