Real-time optical diagnosis for surgical margin in low rectal cancer using multiphoton microscopy

Abstract

Multiphoton microscopy (MPM), based on advances in the field of nonlinear optics and femtosecond lasers, has been shown to provide detailed real-time information on tissue architecture and cell morphology in live tissue. The purpose of this study was to evaluate the feasibility of using MPM to make real-time optical diagnoses for surgical margins in low rectal cancers. Thirty fresh, unfixed, and unstained full-thickness surgical margins of low rectal cancers underwent MPM examination and then went through intraoperative frozen procedures and routine pathological procedures. MPM images were compared with the gold standard hematoxylin-eosin (H-E) stained images. MPM images were acquired by two channels: broadband autofluorescence from cells and second harmonic generation (SHG) from tissue collagen. Peak multiphoton signal intensity was detected in mucosa excited at 800 nm. There were significant differences between negative surgical margins and positive surgical margins under MPM examination. In negative surgical margins, MPM revealed regular tissue architecture and cell morphology, including a typical foveolar pattern with central, round crypt openings, and glands lined by epithelial and goblet cells. SHG signals could be detected around the glands. In positive surgical margins, MPM demonstrated irregular tubular structures, reduced stroma, and cellular and nuclear pleomorphisms. Cancer cells were characterized by an irregular size and shape, enlarged nuclei, and an increased nuclear-cytoplasmic ratio. SHG signals were significantly decreased in positive surgical margins compared with negative surgical margins. MPM images were comparable to H-E stained images. We demonstrated the feasibility of using MPM to make real-time optical diagnoses for surgical margins in low rectal cancer. With the miniaturization and integration of colonoscopy or probes, MPM has the potential to provide real-time noninvasive optical diagnosis for surgical margins in low rectal cancer in the near future.