Parathyroid and thyroid tissues from rats and pigs were preserved for hematoxylin and eosin (H&E) and NIR fluorescence microscopic assessment. Tissues extracted from the animals post-intraoperative imaging were placed in 2% paraformaldehyde in PBS for 30 min before mounting in Tissue-Tek OCT compound (Fisher Scientific, Pittsburgh, PA) and flash-freezing in liquid nitrogen. Frozen samples were cryosectioned (50 μm per slice), observed by fluorescence microscopy, then stained with H&E.
NIR fluorescence microscopy for resected tissues was performed on a 4 filter set Nikon Eclipse TE300 microscope system as previously described27 (link),28 (link). The microscope was equipped with a 100 W mercury light source (Chiu Technical Corporation, Kings Park, NY), NIR-compatible optics, and a NIR-compatible 10X Plan Fluor objective lens and a 100X Plan Apo oil immersion objective lens (Nikon, Melville, NY). Images were acquired on an Orca-AG (Hamamatsu, Bridgewater, NJ). Image acquisition and analysis was performed using iVision software (BioVision Technologies, Exton, PA). Two custom filter sets (Chroma Technology Corporation, Brattleboro, VT) composed of 650 ± 22 nm and 750 ± 25 nm excitation filters, 675 nm and 785 nm dichroic mirrors, and 710 ± 25 nm and 810 ± 20 nm emission filters were respectively used to detect T700-F and T800-F signals in the frozen tissue samples.
NIR fluorescence microscopy for resected tissues was performed on a 4 filter set Nikon Eclipse TE300 microscope system as previously described27 (link),28 (link). The microscope was equipped with a 100 W mercury light source (Chiu Technical Corporation, Kings Park, NY), NIR-compatible optics, and a NIR-compatible 10X Plan Fluor objective lens and a 100X Plan Apo oil immersion objective lens (Nikon, Melville, NY). Images were acquired on an Orca-AG (Hamamatsu, Bridgewater, NJ). Image acquisition and analysis was performed using iVision software (BioVision Technologies, Exton, PA). Two custom filter sets (Chroma Technology Corporation, Brattleboro, VT) composed of 650 ± 22 nm and 750 ± 25 nm excitation filters, 675 nm and 785 nm dichroic mirrors, and 710 ± 25 nm and 810 ± 20 nm emission filters were respectively used to detect T700-F and T800-F signals in the frozen tissue samples.
