Contrast-enhanced photoacoustic imaging of live lobster nerve cord

Photoacoustic imaging provides optical contrast with good penetration and high spatial resolution, making it an attractive tool for. noninvasive neural applications. We chose a commercial dye (NK2761) commonly used for optical imaging of membrane potential to enhance photoacoustic images of the live lobster nerve cord. The abdominal segment of the nerve cord was excised, stained and positioned in a custom neural recording system, enabling electrical stimulation and recording of compound action potentials. Photoacoustic and pulse echo images were also collected using a commercial ultrasound scanner and a 10-MHz linear probe. A wavelength-tunable pulsed laser source (Surelite™, 5 ns, ∼15 mJ, 30 mJ/cm²) operating at 20 Hz produced photoacoustic waves. Longitudinal photoacoustic scans of a 25-mm segment of the excised nerve cord, including ganglionic and axonal processes, were collected and displayed every 7 seconds. Without the contrast agent, an average of 10 scans produced a peak photoacoustic signal 6 dB over background noise. An additional 29 dB was obtained after the nerve was submerged in the dye for 20 minutes. The gain decreased to 23 dB and 14 dB at 810 nm and 910 nm, respectively - consistent with the dye's optical absorbance measured using a portable spectrometer. The contrast-enhanced photoacoustic signal had a broad spectrum peaking at 4 MHz, and, after high pass filtering, images approached 200-μm spatial resolution. The hybrid imaging system, which provided several hours of electrical stimulation and recording, represents a robust testbed to develop novel photoacoustic contrast for neural applications.