The primary objective of this study was to optimize an intraoperative imaging system utilizing indocyanine green (ICG), the only voltage-sensitive dye approved by the FDA for clinical use. The study also aimed to observe optical properties of ICG in solvents of varying polarities and visualize stimulated neonatal cardiomyocyte rat cultures. Voltage-sensitive dyes alter their optical properties based on changes in membrane potential, making them excellent fluorophores for recording the firing activity of neurons and myocytes and ultimately visualizing scarring and nervous tissue inside the heart to detect cardiac arrhythmias.

ICG fluoresces when exposed to near-infrared light, emitting light at a slightly longer wavelength. The overlap in excitation and emission spectra results in reabsorption of light from the camera, so bandpass (BP) and long-pass (LP) filters were installed into the camera and LED light source. Specifically, a 750/800 nm BP filter was installed to the LED light, and two 830 nm LP filters and one 790 LP filter was installed to the camera. The entire setup was placed on a vibration isolation table, and additional precautions were taken, including the use of a black plastic sheet to eliminate red light reflections and recording in a dark room to maximize the signal-to-noise ratio. A dark frame was also taken prior to recording to subtract background noise. The camera operates at a 640 Hz frame rate, capturing 5000 images in 2 ms at a 13 cm working distance, with a 3.5 by 3.5 cm field of vision.

To investigate the optical properties of ICG in various solvents, dilutions were prepared in ethanol, octanol, Tyrode’s solution, and distilled water. The absorbance spectra were measured using a UV-vis spectrophotometer, revealing absorbance peaks at 779.4, 780.2, 788.2, and 796 nm for water, Tyrode’s, ethanol, and octanol, respectively. The findings indicated that solvents with higher polarity, such as water and Tyrode’s, had shorter wavelength peaks, while less polar solvents like ethanol and octanol had longer peaks.

In the next phase, recordings of stimulated myocyte cultures were taken by the imaging system. To stain the cultures, ICG was first dissolved in trace water and diluted 100x in Tyrode’s solution. After a 20-minute staining period, the ICG-Tyrode’s solution was replaced with pure Tyrode’s solution. The cultures were then stimulated by wire electrodes to induce contractions. Although the camera captured fluorescence and bleaching from ICG, it failed to detect any fluorescence changes because the confluent cells were not prepared to contract synchronously, which is necessary to have a total action potential large enough to visualize.

The results suggest that a larger sample, like a live muscle tissue or frog’s heart, is recommended to visualize action potentials. The current system is well-suited for future experiments on frog hearts, which are cost-effective and spontaneously beat in Tyrode’s solution. Frog hearts are easily stained by injecting ICG into the aorta and are an ideal model for visualizing cardiac action potentials, providing a crucial next step in the visualizing action potentials.