双光子吸收（TPA）荧光成像由于其无与伦比的时空分辨率，在诊断和生物医学领域具有极高的潜力。然而，目前可用的TPA探针的适应性和适用性严重受到体内有限的光致发光的挑战，这是由于不可控的聚集导致荧光猝灭，以及在恶劣的生理环境中不可避免的光氧化，通常导致染料退色。在这里，我们描述了近红外（NIR）靛蓝染料基纳米探针（NPs）的显著增强的TPA荧光成像能力，这可以通过光氧化增强发射机制来解释。在光氧化过程中生成的单线态氧使色素分子二聚形成产生增强TPA荧光发射的TPA中间体。所得到的NPs具有均匀的尺寸分布，优秀的稳定性，比一种常用的TPA探针罗丹明B（RhB）具有更有利的TPA截面和抗退色能力。这些基于靛蓝染料的TPA NPs的性质促进了它们在实时可视化血液循环和肿瘤积累，甚至在体内细胞成像方面的应用。在这些近红外靛蓝染料基纳米聚集体中观察到的光氧化增强发射机制为更先进的TPA荧光探针的设计和开发开辟了一条途径。© 2023. Springer Nature Limited.

Two-photon absorption (TPA) fluorescence imaging holds great promise in diagnostics and biomedicine owing to its unparalleled spatiotemporal resolution. However, the adaptability and applicability of currently available TPA probes, which act as a critical element for determining the imaging contrast effect, is severely challenged by limited photo-luminescence in vivo. This is particularly a result of uncontrollable aggregation that causes fluorescence quenching, and inevitable photo-oxidation in harsh physiological milieu, which normally leads to bleaching of the dye. Herein, we describe the remarkably enhanced TPA fluorescence imaging capacity of self-assembling near-infrared (NIR) cyanine dye-based nanoprobes (NPs), which can be explained by a photo-oxidation enhanced emission mechanism. Singlet oxygen generated during photo-oxidation enables chromophore dimerization to form TPA intermediates responsible for enhanced TPA fluorescence emission. The resulting NPs possess uniform size distribution, excellent stability, more favorable TPA cross-section and anti-bleaching ability than a popular TPA probe rhodamine B (RhB). These properties of cyanine dye-based TPA NPs promote their applications in visualizing blood circulation and tumoral accumulation in real-time, even to cellular imaging in vivo. The photo-oxidation enhanced emission mechanism observed in these near-infrared cyanine dye-based nanoaggregates opens an avenue for design and development of more advanced TPA fluorescence probes.© 2023. Springer Nature Limited.