Fluorescence microscopy of parathyroid and thyroid tissues for localization of autofluorescent substances using near-infrared wavelengths.

Objective: The parathyroid gland emits autofluorescence with a peak at 822 nm when excited using near-infrared light at 785 nm; this observation of autofluorescence using a near-infrared detection device is useful for identifying the parathyroid gland during surgery. We aimed to clarify the localization of autofluorescent substances in parathyroid and thyroid tissues by observing them under a fluorescence microscope through filters that selectively pass specific near-infrared wavelengths.

Methods: Four cases of parathyroid and three cases of thyroid were examined under a fluorescence microscope. The frozen, formalin-fixed paraffin-embedded, and unfixed, unstained sections of parathyroid were observed through filters that selectively pass specific near-infrared wavelengths. Images were acquired at excitation 775 ± 50 nm and absorption 845 ± 55 nm in five randomly selected fields of view, avoiding tumor and inflammatory areas. Autofluorescence was measured as the ratio of fluorescent area to tissue area using hybrid cell counting.

Results: Autofluorescence was observed in all sections. In the parathyroid tissue, the frozen sections showed significantly more autofluorescence than the formalin-fixed paraffin-embedded sections, and in the thyroid tissue, although no significant difference was observed, the frozen sections showed more autofluorescence than the formalin-fixed paraffin-embedded sections. In addition, the single unfixed, unstained section showed stronger autofluorescence than the frozen sections, although no significant difference was found. The areas of autofluorescence in the parathyroid and thyroid tissues were thought to be the Golgi area and lipofuscin, respectively.

Conclusion: Fluorescence microscopy of parathyroid and thyroid tissues revealed the localization of autofluorescent substances in each tissue.