*1.1. Contrast-Enhanced Fluorescence and Autofluorescence*

Exogenous contrast-enhanced fluorescence can be induced via the use of fluorescent dyes. For intraoperative imaging, indocyanine green (ICG), a sterile tricarbocyanine dye, is the most commonly utilized fluorophore. ICG absorbs light at excitation wavelengths of 790–805 nm and re-emits light at 835 nm [23]. Other NIR fluorescent agents, such as methylene blue, are used less frequently than ICG due to their potential toxicity [24].

In contrast, autofluorescence is based on intrinsic biomolecules that act as endogenous fluorophores. Light emission occurs in the ultraviolet, visible, or NIR spectral range when biological molecules are excited with light at an appropriate specific wavelength [25].

Intrinsic tissue fluorescence can be perceived as a background disturbance signal when cells and tissues are labeled with exogenous fluorophores. However, tissue autofluorescence alone has shown great promise when used for research and diagnostic purposes [25]. In particular, the PGs are characterized by exceptional autofluorescence in the NIR range, as discovered by tissue spectral analysis [26,27]. A research group at Vanderbilt University identified the ideal spectral range, with excitation light at 785 nm producing maximal autofluorescence from thyroid and parathyroid tissue within the NIR range (820 nm), with the parathyroid signal ranging from 1.2 to 25 times higher than thyroid, and all surrounding tissues [28] with brown fat and lymph nodes that are known false NIRAF positives in non-parathyroid tissues [13]. This group further explored real-time intraoperative autofluorescence imaging of the PGs [27,29]. The specific molecule acting as an intrinsic fluorophore has not yet been identified.

Multiple NIR imaging systems are commercially available for clinical use or for research purposes. Some of these devices are optimized for pure autofluorescence imaging, whereas others enable both autofluorescence and ICG contrast-enhanced fluorescence imaging. All autofluorescence devices can detect ICG, but the reverse is usually not true [29]. As the field of potential applications is expanding, customizable systems with a choice of different wavelengths and multispectral fluorescence imagers are being explored [30]. For example, hyperspectral imaging showed distinct spectral signatures of the thyroid glands and PGs compared with the surrounding tissue during neck exploration in a patient [12].

Postoperative complications after thyroid surgery may include temporary or permanent hypoparathyroidism, recurrent laryngeal nerve injury or palsy, chyle fistula, Horner's syndrome, injury to motor nerves in the neck, and hematoma or seroma formation [5,31]. Hypoparathyroidism is the most common complication after total or near-total thyroidectomy due to the accidental resection or devascularization of PGs. Fluorescence imaging has the potential to revolutionize thyroid surgery by significantly reducing hypoparathyroidism, especially for thyroid cancer necessitating an enlarged dissection.
