2.2. Injection of Fluorescent Dyes and Fluorescence Observation

Pericardium (PC) meridian and the acupoints were identified on the forearm from the wrist to the elbow by two TCM physicians (Drs Zhao and Hu) according to “Nomenclature and location of acupuncture points” (GB/T 12346-2006) issued by the Standardization Administration of China. Acupuncture point locations were reached by consensus and further refined or confirmed with the use of a low-electrical impedance locator device (Acupuncture Meridian Locator WQ6F30; Beijing, China) [14, 33]. The acupuncture points were demarcated with the use of two triangular stickers or circled with a marker pen aligned orthogonally to the meridian.

Both fluorescein sodium and ICG dyes were used. In 12 subjects, 0.1 mL of 20% fluorescein sodium (Wuzhou Pharmaceutical Co., Ltd., Guangxi, China; Product batch number: 181102) was injected intradermally at the left PC6 (Neiguan acupoint), and in a subset of 5 individuals, the right PC6 was also injected at a later date. Extra care was placed in ensuring that the injections were placed intradermally and not subcutaneously. When the fluorescence disappeared completely, some participates also received the 2^nd or the 3^rd injection at PC6. To assess a different fluorescent dye and other PC points, 0.1 mL of 5 mg/mL ICG (Weicai Pharmaceutical Co., Ltd, Liao Ning, China) was injected at PC5 (Jianshi), PC6, or PC7(Daling) in 6 subjects. An adjacent control for PC6 located midway between PC6 and LU7 (approximately 1 cm radial to PC6) was tested with fluorescein injection in 7 subjects.

Fluorescence of fluorescein sodium was observed on the forearm with a laser beam of wavelength 455 nm and illumination power of approximately 0.5 mW. Photographic images were obtained and recorded with a long-pass filter of 500 nm to filter out the illumination light. ICG dye was excited with 780 nm laser and recorded with a mobile phone with a long-pass filter greater than 780 nm or with a PDE imager (model C9830; Hamamatsu Photonics, Japan). This imager utilizes a LED light source (760 nm) and a CCD camera capturing photons with wavelengths greater than 820 nm [34]. Depending on the rate of migration of the fluorescent tracer, images were obtained at intervals sufficient to capture the dynamic progression/clearance of the dye and for as long as 10 hours (fluorescein) or more than 2 days (ICG). These images were obtained in a dark room with all other light sources turned off.