Behavioral dynamics of neuroprotective macrophage polarization in neuropathic pain observed by GHz femtosecond laser two‐photon excitation microscopy

Abstract Macrophage polarization in neurotoxic (M1) or neuroprotective (M2) phenotypes is known to play a significant role in neuropathic pain, but its behavioral dynamics and underlying mechanism remain largely unknown. Two‐photon excitation microscopy (2PEM) is a promising functional imaging tool for investigating the mechanism of cellular behavior, as using near‐infrared excitation wavelengths is less subjected to light scattering. However, the higher‐order photobleaching effect in 2PEM can seriously hamper its applications to long‐term live‐cell studies. Here, we demonstrate a GHz femtosecond (fs) 2PEM that enables hours‐long live‐cell imaging of macrophage behavior with reduced higher‐order photobleaching effect—by leveraging the repetition rate of fs pulses according to the fluorescence lifetime of fluorophores. Using this new functional 2PEM platform, we measure the polarization characteristics of macrophages, especially the long‐term cellular behavior in efferocytosis, unveiling the dynamic mechanism of neuroprotective macrophage polarization in neuropathic pain. These efforts can create new opportunities for understanding long‐term cellular dynamic behavior in neuropathic pain, as well as other neurobiological problems, and thus dissecting the underlying complex pathogenesis.