Endoscopic NIRAF-LSCI multimodal system with pseudo-color visualization for parathyroid autofluorescence localization and tissue perfusion assessment: preclinical validation

Postoperative hypoparathyroidism remains a prevalent complication of thyroid surgery, primarily caused by unrecognized parathyroid gland (PG) injury or devascularization. While near-infrared autofluorescence (NIRAF) enables accurate PG localization, it provides no functional perfusion information; conversely, laser speckle contrast imaging (LSCI) offers label-free real-time perfusion assessment. Herein, we validated a multimodal endoscopic system integrating NIRAF and LSCI for simultaneous PG identification and vascular evaluation, focusing on LSCI perfusion performance and the clinical practicability of its pseudo-color semi-quantitative visualization. The system was tested in 8 Sprague-Dawley rats, where rat thyroids with homologous blood supply served as perfusion surrogates due to the minute volume of rat PGs. Imaging was performed under three strictly controlled in situ conditions: intact native vascularization, partial vascular occlusion, and complete vascular occlusion. LSCI pseudo-color visualization was generated via a standardized pipeline including temporal laser speckle contrast analysis (tLASCA)-based blood flow index (BFI) calculation, baseline-anchored joint 5th-95th percentile normalization, and Jet colormap mapping. Intraoperative probe-based quantitative NIRAF was used to validate NIRAF-visual signal foci. NIRAF clearly identified PGs with a fluorescence intensity of 149.8 ± 8.7 mV, significantly higher than thyroid (93.8 ± 8.0 mV), trachea (84.6 ± 9.1 mV), and muscle (66.3 ± 10.4 mV) (all P < 0.001), achieving 100% positive predictive value (PPV) and 100% negative predictive value (NPV), with zero false-positive findings among all NIRAF-visual foci. PPV was determined using both probe-based NIRAF and histopathology as dual validation references, whereas NPV was determined using postoperative histopathology alone as the reference standard. LSCI speckle contrast increased progressively with decreasing perfusion, with speckle contrast values mapped to a continuous blue-to-red real-time pseudo-color perfusion visualization. Histopathology confirmed all fluorescent tissues as PGs. This multimodal system targets two core clinical needs for intraoperative PG protection, and its standardized pseudo-color semi-quantitative workflow provides real-time label-free perfusion visualization. It shows potential to serve as an intraoperative adjunct to guide parathyroid preservation decisions and reduce reliance on delayed hormonal test results.