(678d) Two-Photon Microscopy for Deep-Tissue Imaging of Dopaminergic Neuromodulation in the Brain

Non-invasively imaging the brain neuronal structure and neuro-chemical activity stands to inform our understanding of psychiatric and neurological disorders and their pharmacological therapies. However, the high optical density of brain tissue makes imaging disease-relevant regions at its center a challenge using conventional fluorophores and microscopy techniques, particularly for intact brains in living animal models. We present a microscopic approach that could enable whole-brain, through-skull, and continuous imaging of neuromodulation. To this end, we have combined two imaging modalities that have emerged in parallel with opposing imaging strengths and weaknesses: infrared microscopy, and two-photon microscopy.¹ Recently, functionalized single walled carbon nanotubes (SWNTs) have emerged as promising near-infrared (NIR) fluorescent nanosensors for the sensitive and rapid detection of dopamine, a neuromodulatory neurotransmitter of clinical importance to brain function and disease.² SWNT nanosensors fluorescence in the NIR-II window (1000-1700 nm), where absorption and scattering of light is minimal, making them optimally suited for non-invasive imaging in brain tissue. However, to overcome the challenge of absorption and scattering of excitation photons in deep-tissue imaging, we show that intrinsically-near-infrared fluorescent SWNT nanosensors can be efficiently imaged with two-photon excitation (in lieu of one-photon excitation) through photoexcitation by a NIR-II femtosecond pulsed erbium fiber laser. We measure an effective, average quantum yield of 0.2% and a two-photon cross section of 216,000 GM. Furthermore, we demonstrate that two-photon photoexcitation improves the imaging quality of nanosensors in brain-tissue phantoms at depths approaching 2 mm and confirm that non-linear excitation preserves the molecular recognition of dopamine. Finally, we show that this imaging paradigm can be expanded to a laser-scanning system paired with a NIR photomultiplier tube, enabling layer-by-layer 3D imaging, motivating future efforts to achieve real-time imaging of dopamine neurotransmission in awake and behaving animals.

1 J. T. Del Bonis-O’Donnell, R. H. Page, A. G. Beyene, E. G. Tindall, I. R. McFarlane and M. P. Landry, Adv. Funct. Mater., 2017, 27, 1702112.

2 A. G. Beyene, I. R. McFarlane, R. L. Pinals and M. P. Landry, ACS Chem. Neurosci., 2017, 8, 2275–2289.