双光子荧光探针被广泛研究应用于生物医学成像和治疗,深入了解这类探针在生物组织中激发分布特征及其相对于单光子探针的成像优势和劣势对探针的合理选择和应用具有指导意义.然而,由于实际测量难以避开众多干扰因素的影响,因此不能准确反映其固有特征.本文选取典型生物和荧光激发参数,利用生物仿真定量分析,并通过双光子和单光子的激发成像对比,获取了双光子荧光的基本激发特征.结果发现,在相同平圆光束激发下,双光子激发由于激发效率低且需要双光子吸收,再加上较高的激发阈值,虽然激发光在组织穿透深度方面存在优势,但在组织内衰减速率快、有效穿透深度比高量子效率的单光子荧光激发浅.同时,深度方向的快速衰减会导致横向激发光能的非均匀性分布,这种非均匀性对双光子荧光影响更大.仿真分析进一步表明:在生物组织耐受的前提下提高双光子激发的功率更有利于荧光成像;增加光照半径可以减弱横向激发的不均匀性,从而改善双光子荧光成像的效果.本研究结果为双光子荧光激发、成像的应用提供了基础数据参考,本仿真方法也为快速研究光与生物组织的相互作用提供了借鉴.%Two-photon fluorescent probes have been widely studied for biomedical imaging and treatment. Understanding the excitation characteristics of this type of probes in biological tissue and knowing the compared advantages and disadvantages with single-photon probes are useful for the probe selection and application. However, the actual measurement is difficult to reveal their inherent characteristics due to too many interference factors existing. In this study, following the selected typical parameters, imaging of the two types of excitation probes in tissue is simulated. It is found that, during irradiation with same power of the flat light beams, two-photon probes have more rapid intensity attenuation and lower tissue penetration than single probes, due to their low excitation efficiency and high excitation threshold, as well as the two-photon absorption. In addition, the rapid attenuation along the depth can result in unevenness distribution of light. The unevenness affects the fluorescence of two-photon probes more when comparing to single-photon probes. Simulation analysis further indicates that enhancing the two-photon power in biological tissue tolerance range can improve the fluorescence imaging of two-photon probes more;and increasing the irradiation area can weaken the in-homogeneity of lateral excitation for better imaging effect. The results provide the basic data for two-photon fluorescence excitation and imaging. The simulation method also provides a reference for the rapid study of the interaction between light and biological tissue.
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