During thyroid surgery fast and reliable intra-operative pathological feedback has the potential to avoid a two-stage procedure and significantly reduce health care costs in patients undergoing a diagnostic hemithyroidectomy (HT). However, with current techniques, intra-operative assessment of histopathological characteristics is not always reliable within the operation time. Therefore, there is an urgent need for techniques that provide the surgeon with better pathological feedback during the operation. As a step towards this purpose, the potential of higher harmonic generation (HHG) microscopy, which combines second harmonic generation (SHG), third harmonic generation (THG), and multiphoton excited autofluorescence (MPEF) is explored. We used a compact, portable HHG microscope to record images of freshly excised healthy tissue, benign nodules (follicular adenoma) and malignant tissue (papillary carcinoma, follicular carcinoma and spindle cell carcinoma). The SHG/THG/MPEF images were generated on unprocessed tissue within minutes, typically, a 10 by 10 mm² area was acquired within 10 minutes. The SHG/THG/MPEF images show relevant morphological thyroid structures in good accordance with the histology images. The thyroid follicle architecture, cells, cell nuclei (THG), collagen organization (SHG) and the distribution of thyroglobulin and/or thyroid hormones T3 or T4 (MPEF) could be visualized. We conclude that SHG/THG/MPEF imaging is a promising tool for clinical intraoperative assessment of thyroid tissue. More patients need to be included, to assess the feasibility of HHGM to determine capsular or vascular invasion, features essential for intra-operative thyroid cancer diagnosis.
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