双光子自发荧光（TPAF）成像能够以高时空分辨率提供精确的细胞代谢信息，使其成为一种有前途的活检工具。该技术受到光学系统或数据处理的复杂性的极大阻碍。在此，对激发波长进行了优化，以同时激发黄素腺嘌呤二核苷酸和烟酰胺腺嘌呤二核苷酸，并消除意外的 TPAF。在优化的单波长激发下无需额外校准即可稳健地获得光学氧化还原比（ORR）图像。以肝细胞癌和转移瘤为例，对TPAF法的体外、离体和体内活检进行了系统的研究和比较。结果表明，所提出的 TPAF 方法简化了光学系统，提高了 ORR 的鲁棒性，并能够实现早期癌症诊断，与以前的方法相比显示出显着的优势。

Two-photon autofluorescence (TPAF) imaging is able to offer precise cellular metabolic information with high spatiotemporal resolution, making it a promising biopsy tool. The technique is greatly hampered by the complexity of either the optical system or data processing. Here, the excitation wavelength was optimized to simultaneously excite both flavin adenine dinucleotide and nicotinamide adenine dinucleotide and eliminate the unexpected TPAF. The optical redox ratio (ORR) images were robustly achieved without additional calibration under the optimized single-wavelength excitation. The in vitro, ex vivo, and in vivo biopsy by the TPAF method were systematically studied and compared using hepato-cellular carcinoma and metastasis as examples. It was demonstrated that the proposed TPAF method simplified the optical system, improved the robustness of ORR, and enabled early-stage cancer diagnosis, showing distinguished advantages as compared with previous methods.