基于非线性光学显微成像的脑肿瘤边界术中精确诊断方法研究

The infiltrating growth of brain tumor results in an unclear tumor margin, making it difficult to be completely resected and easily leading to the injury of normal brain function. The precise identification of brain tumor margin is very crucial to the success of operation. Nonlinear optical microscopic imaging of endogenous optical biomarkers provides a non-invasive, intravital, label-free, high-resolution biopsy method, is promising to solve this problem. However, since lacking intravital long-term comparative analysis and systematically quantitative investigation of endogenous optical biomarkers of the normal and cancerous brain tissues, intraoperative diagnosis of brain tumor margin based on nonlinear optical microscopy is still immature so far. Therefore, we developed a fluorescence-spectrum and fluorescence-lifetime resolved multi-dimensional nonlinear optical microscope in our previous study, and demonstrated in preliminary that there are several differences in the endogenous optical characteristics between normal and cancerous brain tissues. In this research, we will further develop a brain tumor model with a chronic cranial window and complete methods for integrating and analyzing the multi-dimensional nonlinear optical information (fluorescence intensity, spectrum, and lifetime in three-dimensional space). Based on these works, we intend to realize the intravital, orthotopic, long-term imaging and quantitative characterization of brain tumors, so that to reveal quantitative indicators for the precise identification of brain tumor margin. As a result, this research will potentially provide guidance for surgeons to retain normal brain functions as far as possible on the premise of complete brain tumor resection, and thus improving patients’ life quality after operation. This will contributes to the reducing of recurrence and mortality of brain tumors.

脑肿瘤大多呈浸润性生长，无明显边界，导致手术难以彻底切除且容易伤及脑功能，精确识别其边界是手术成功的关键。基于检测内源性光学标志物的非线性光学成像能够实现非侵入式的活体、无标记、高分辨组织活检，有望解决这一问题。但是，目前缺乏对正常和癌变脑组织光学标志物的活体长时程对比研究和系统性量化分析，基于非线性光学成像的脑肿瘤边界诊断方法尚不成熟。为此，我们前期研发了一套具备荧光光谱和荧光寿命分辨功能的新型非线性光学成像系统，利用该系统我们初步证实正常和癌变脑组织的确存在诸多内源性光学特征差异。在此基础上，本项目拟进一步通过建立慢性颅窗模型和多维光学信号（三维空间，荧光强度、光谱和寿命）整合分析方法来实现脑肿瘤的活体、原位、长时程成像研究和量化表征，从而揭示精确识别其边界的定量指标。本研究对在彻底切除脑肿瘤的前提下，尽可能保留正常脑功能，进而提高患者术后生存质量，降低复发率和死亡率具有重要指导意义。

脑肿瘤具有浸润性生长、无明显边界的特点，手术难以彻底切除且极易损伤脑功能，精确识别肿瘤边界是其手术成功的关键。荧光光谱和寿命分辨的非线性光学显微成像技术能够在活体内，以亚细胞水平的分辨率，探测内源荧光强度、光谱和寿命的三维分布，实现组织形态结构和代谢状态的同时表征，因而有潜力揭示正常组织和肿瘤组织的差异，实现脑肿瘤边界的精确识别。然而，目前相关的探索性工作缺乏对正常和癌变脑组织的活体长时程对比研究和系统性量化分析，基于非线性光学成像的脑肿瘤边界诊断方法尚不成熟。为此，我们基于慢性颅窗技术创建了一种适用于脑肿瘤边界非线性光学成像研究的活体、原位模型，发展了一套用于挖掘脑肿瘤识别指标的多维光学信息整合分析方法，进而结合我们前期搭建的荧光光谱和寿命分辨的非线性光学显微成像系统，建立了针对脑肿瘤边界光学特征研究的无标记、高分辨、原位、长时程成像平台。利用该平台，基于生物组织内源性光学标志物和神经胶质瘤这一载体，我们以揭示正常脑组织和脑肿瘤组织的差异为目标，从细胞、组织、活体等多个层面，对脑肿瘤进行了全方位的成像和分析。基于以上研究，我们提取出一套有潜力用于在体高分辨识别脑肿瘤边界的定性、定量指标。这套指标包括定性的组织形态学特征，以荧光光谱比率为核心的定量荧光光谱特征和以蛋白绑定NADH的荧光寿命为核心的定量荧光寿命特征。这些指标有望为脑肿瘤的手术切除提供有效的辅助信息，对于提高脑肿瘤患者术后生存质量，降低脑肿瘤的复发率和死亡率具有重要意义。相关研究成果发表SCI论文 6篇，获授权发明专利 1项，申请发明专利 4项。