The preservation of the parathyroid glands (PTGs) and their vascularization is crucial during thyroidectomy because damage to the PTG can result in postoperative hypoparathyroidism and hypocalcemia, which can significantly increase cardiovascular and renal morbidity and patient mortality (1, 2).

Autofluorescence has recently been introduced in thyroid surgery to facilitate the detection of PTGs (3–6). Initially, surgeons relied only on their experience and training to detect the parathyroid glands and decide which blood vessels to preserve to protect gland vascularization during thyroidectomies. Depending on the surgeon, in 30–60% of cases, autofluorescence imaging allows for the detection of the PTGs before the surgeon sees them. Although, in some cases, autofluorescence allows for better preservation of the PTG (4), it does not provide any information regarding the vascularization pathway of the glands. Identifying the blood vessels feeding the PTGs is critical because the vascularization of the PTG is unpredictable (7). Recently, fluorescence angiography using indocyanine green (ICG) during thyroidectomy has demonstrated the possibility of identifying critical blood vessels needing preservation (8). 0.1mg/kg of ICG is usually intravenously injected as a bolus. 10 to 30 seconds after injection, ICG reaches the thyroid gland and fluorescence signal can be detected by the imaging device and displayed on a screen.

The main limitation of ICG fluorescence angiography, used for detection in the range of 800–900 nm, is that once ICG has been injected, even at 0.1mg/kg, it would no longer be possible to visualize the autofluorescence of the PTGs. ICG Angiography is performed to visualize small blood vessels. Therefore, injected dose leads to high levels of fluorescence that overwrite parathyroid autofluorescence. ICG fluorescence is at least one order of magnitude brighter than PTG autofluorescence in the range of 800–900 nm. Currently, available systems use fluorescence excitation wavelengths longer than 740 nm and detect in the range 800-900nm (9). Thus, once ICG is present in the field, the PTG autofluorescence signal becomes negligible compared with the ICG signal.

Thyroidectomies are usually performed in two phases (one lobectomy per side), and the surgeon must preserve the PTGs independently on each side. If the surgeon wants to use autofluorescence on both sides of the thyroid, then ICG can only be used on the second operated side. Consequently, during total thyroidectomy using existing devices, ICG fluorescence angiography can be combined with autofluorescence on only one side of the patient.

With classical devices based on excitation wavelengths longer than 740 nm, a waiting time of about 1 hour is required between the last ICG injection and the next autofluorescence measurement. However, despite this delay, several areas remain marked by ICG because it is injected into the dissected tissue, which degrades the readability of the autofluorescence images by registering many ICG-related false positives.

Furthermore, the inability to use perfusion imaging on the two operative sides leads to (i) a potentially greater risk of damaging the vascularization of the PTGs of the first lobe and (ii) not knowing if the PTGs left in place on the first lobe are functional. This has a significant implication on how the second lobe should be dissected, especially if the PTGs on the second lobe are poorly localized. Therefore, it is crucial to visualize the autofluorescence of the PTGs in situ even after ICG injection. Here, we described an approach that allows for visualization of the PTGs by autofluorescence, even after ICG injection.

ICG (25 mg) was purchased from Diagnostic Green (Indocyanine Green for Injection Kit, 25 mg). Bovine Serum Albumin was purchased from Sigma Aldrich (Bovine Serum Albumin, 5%; reference A4628-20ML). We performed fluorescence spectrometry measurements of ICG diluted with albumin at an excitation wavelength of 685 nm. We observed that the fluorescence yield was 30–50% lower than that measured at an excitation wavelength of 750 nm. The detection was performed on a larger bandwidth of 700–900 nm. The maximum fluorescence of ICG excited at a wavelength of 685 nm was observed near 820 nm.

Next, a comparison of the emission spectra of PTG autofluorescence for excitation at 650 nm, as provided by Serra (10), with that of ICG ( Figure 1 ) showed that their peak maxima were well separated. We assumed that the autofluorescence spectra for wavelengths longer than 700 nm were similar, with excitation wavelengths of 650 and 685 nm.

To alternatively visualize PTG autofluorescence and the ICG signal on the same system, we modified a FLUOBEAM_® LX device (FLUOPTICS^© France) by replacing the 750 nm excitation laser with a 685 nm laser and also fit detection filters with a range of 700–900 nm. This system will hereafter be referred to as the LX700. The excitation power at the area of interest was 4.6 mW/cm² at a distance of 10 cm from the optical head, and we ensured that the device was operating in laser class 1 (11).

To separate the PTG autofluorescence signal from the ICG fluorescence signal using this device, we tested a retractable low-pass filter with a cut-off wavelength of 775 nm in front of the camera sensor. This led to two spectral detection windows: 700–900 nm without the filter and 700–775 nm with the filter. The experimental setup is shown in Figure 2 .

To perform the tests, we used (i) five different dilutions of ICG, i.e., from 0.05 to 1 µM (0.05 µM, 0.1 µM, 0.25 µM, 0.5 µM, and 1 µM) in a solution containing 5% of albumin described earlier, which were arranged as drops of 10 µL, and (ii) freshly resected anatomical specimens of PTGs obtained during a parathyroidectomy. Six series of ICG droplets and resected PTGs were examined. The sample setup and areas of interest for each series are shown in Figures 3 – 8 .

We measured the fluorescence signal using three possible system configurations, namely, FLUOBEAM_® LX, LX700, and LX700 with a low-pass filter at 775 nm, referred to as the LX700/775.

All acquisitions were performed under typical detection conditions for the PTGs with the FLUOBEAM_® LX, LX700, and LX700/775. The measurement distance was 10 cm, and the integration time was 40ms. Different camera amplification factors were used depending on the system configuration to provide useful, unsaturated images.

All results were scaled to a 40 ms/8.7 dB gain before computation. When the signal was saturated, the gain was reduced to obtain unsaturated data. Decreasing the camera gain does not impact the SNR.

Until recently, it was commonly accepted that the maximum autofluorescence of the PTGs is observed between 800 and 900 nm and that this autofluorescence is reduced significantly below 800 nm (3, 9, 12, 13). Thus, PTG autofluorescence used to be measured between 800 and 900 nm. Furthermore, as the excitation wavelength is between 740 and 805 nm in most commercially available systems, fluorescence is usually detected between 800 and 900 nm in these systems, e.g., PTeye (Medtronic), FLUOBEAM LX (Fluoptics), EleVision IR platform (Medtronic), and PDE (Hamamatsu). Balasubramanian et al. (14), using the FLUOBEAM® 700, indicated that PTG autofluorescence is visible with an excitation wavelength of 685 nm and an emission window between 700 and 900 nm. This autofluorescence allowed for the pre-localization of the PTGs during thyroidectomy. More recently, Serra et al. (10), using fluorescence spectrometry and excitation at 650 nm, confirmed that the PTGs emitted autofluorescence at least at 700 nm and that maximum autofluorescence was observed close to 711 nm.

In order to avoid using two excitation wavelengths, we examined a single excitation that rebalanced the measured signal levels between ICG and PTG autofluorescence. Exciting at a wavelength of 685 nm reduces the ICG contribution and increases the autofluorescence signal. Nevertheless, the contribution of PTG autofluorescence over a detection window ranging from 700 to 900 nm is negligible compared to that of ICG. Narrowing the detection window between 700 and 775 nm will reduce the contribution of ICG more than that of the PTG.

In our study, we observed that PTG autofluorescence could be visualized with a good SNR ratio upon excitation at 685 nm. Moreover, the autofluorescence signal was substantial between 700 and 800 nm.

We also demonstrated that the SNR of the autofluorescence signal obtained with an excitation at 685 nm and a detection between 700 and 775nm is similar to that obtained with the FLUOBEAM® LX (about 10 dB). This imaging approach opens new opportunities for the possibility of i) using autofluorescence alone even after injection of ICG and ii) combining images with different detection windows (700–900 nm and 700–775 nm) to split the contribution of ICG and autofluorescence.

By considering the possibility of renewing the ICG injections if necessary while observing the parathyroids by autofluorescence, the technical improvement described here removes the current main obstacle of intraoperative evaluation of the PTGs by fluorescence imaging at the end of the dissection of each lobe. Once the ICG has been injected, it would not be possible to observe the autofluorescence because the signal is dominated by ICG. However, identification by autofluorescence is crucial because it aids in PTG identification, which positively impacts the quality and function of the preserved glands (4).

By contrast, the injection, sometimes repeated, of ICG helps improve the dissection and/or intraoperative decision by helping to create a vascular mapping of the PTGs before thyroid dissection (8). It can also help improve the dissection by helping to evaluate the parathyroid perfusion at the end of the lobectomy, which would allow for the adaptation of the radicality of the thyroid resection on the second lobe in cases of total thyroidectomy (15). Both modalities are greatly facilitated by the possibility of easy and accurate visualization of the PTG with autofluorescent imaging during ICG injection.

Without the innovation described in this study, to be able to use autofluorescence on the four PTGs without endangering the recurrent nerve in case of loss of signal requiring a stop of the procedure and re-intervention in an already dissected area, the surgeon has to reserve mapping angiographies and perfusion assessment to one side only (the second) during total thyroidectomies.

In conclusion, this study demonstrates that PTG autofluorescence can be detected in the presence of ICG, in concentrations as high as 1.0 µM, with a fluorescence imaging device performing excitation at 685 nm and with detection between 700 and 775 nm. It also demonstrates that ICG can be detected at concentrations as low as 0.05 µM with the same excitation at 685 nm and a detection window between 700 and 900 nm.

The expected clinical benefits versus drawbacks (added time, expense, etc.) from using fluorescence-based parathyroid imaging combined with ICG angiography compared to either technique alone or other imaging methods must be evaluated because the potential for this combination is the key advance offered by changing the excitation and emission settings.

This feasibility study needs to be complemented by an extensive clinical assessment to evaluate the clinical benefits of using a device with excitation at 685 nm and a removable low-pass filter for different pathologies leading to thyroid and/or parathyroid surgery.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

MR and PR contributed equally to the conception, design, and data analysis of the study. All authors contributed to the article and approved the submitted version.