基于高分辨率多波长光声微流控技术的细胞分型研究

癌症已成为对人类威胁最大的疾病之一，近年来癌症的发病人数与致死率迅速增长。晚期癌症致死率高的主要原因是恶性肿瘤发生了扩散与转移。因此，对肿瘤转移的早期诊断是提高治疗效果、降低死亡率的有效手段。循环肿瘤细胞作为恶性肿瘤的“标志物”之一，对其进行检测可以在较早阶段诊断肿瘤转移，因此在肿瘤检测中意义重大。传统的循环肿瘤细胞检测方法利用免疫磁珠对细胞进行特异性捕获，并借助磁场实现对肿瘤细胞的富集，然而该方法难以区分各类肿瘤细胞。传统光学成像可以在血液中直接检测循环肿瘤细胞，在肿瘤检测中具有巨大的应用潜力。然而考虑到血液对光具有很强的散射效应，因此为了提高成像精度和识别准确率，该方案仍旧需要对血液进行稀释等繁琐的预处理。此外，大部分光学成像方法依旧难以实现对肿瘤细胞的特异性识别。

光声成像作为一种新型无损的医学影像技术，融合了光学的高分辨率成像与声学的高穿透性。尤其是光学分辨率光声显微成像技术的出现，使得光声成像技术成功地应用于细胞等微观结构的观测与研究。然而，目前的光声成像技术仍然难以直接检测循环肿瘤细胞，主要存在问题是：（1）用于动态细胞成像的光声系统的分辨率普遍较低，难以直观地对循环肿瘤细胞进行观测与分辨；（2）目前高重复频率的脉冲激光器的波长数目较少，导致光声成像系统对循环肿瘤细胞的分型能力受限。因此，为了解决以上问题，本文设计并搭建了高分辨率光声微流控成像系统并开展了肿瘤细胞的检测与分型研究，证明了该系统能够对血液中循环肿瘤细胞进行检测，并为光声成像用于临床的血液检测提供重要的前期基础。本文的主要研究内容包括：

（1）本文首先设计并构建了多波长光声细胞成像系统，将成像分辨率提升至1.5 μm，使得系统能够对细胞进行高分辨率成像。系统的激发光模块采用了532 nm 与770 nm的脉冲激光器，重复频率为50 kHz。基于多个激发光提供的发色团信息，该光声成像系统实现了对无标记的黑色素瘤细胞与血细胞的分辨。然后，利用外源标记的方法引入了三种发色团，来进一步检验该系统的分辨能力。根据发色团之间的吸收差异，本文提出了一种基于光声信号幅值大小、光声信号的频率分布以及发色团吸收系数比等的多参数发色团识别方法。利用该方法，在两个波长激发光的情况下，成功实现了对四种不同类型细胞的分型。

（2）本文随后设计并构建了多波长光声微流控细胞成像系统，实现了动态细胞的分型和血液中黑色素瘤细胞的检测。在多波长光声细胞成像系统的基础上，结合微流控通道，实现了对通道内流动细胞的高分辨率成像。此外在光声系统引入了超声驻波场以解决离焦与欠焦区域的细胞导致的成像质量与分辨能力下降的问题。在进一步优化超声波的频率、微流控通道的尺寸和激发光能量的基础上，该系统能够以极高灵敏度检测血液中黑色素瘤细胞。该工作为光声成像用于血液检测提供了重要前期基础。

（3）本文之后设计并构建了光声微流控细胞消除系统，实现了对血液中肿瘤细胞的准确消除。在多波长光声微流控细胞成像系统的基础上，结合信号幅值、细胞尺寸以及光声信号比值等参数实现了对肿瘤细胞的实时识别，并通过触发高功率激光对识别的肿瘤细胞进行了实时消除。实验证明对于血细胞与黑色素瘤细胞的混合样品，光声微流控细胞消除系统对肿瘤细胞的消除效率高于95%。对于血液中的黑色素瘤细胞，该系统的消除效率为85%。该工作为肿瘤细胞的诊疗一体化提供了重要的参考。

（4）本文最后设计了光声亚细胞结构成像系统，分辨率提升至0.42 μm，实现了对肿瘤细胞中亚细胞结构的观测。首先该系统对黑色素瘤细胞中的黑色素小体进行了高分辨率的成像。另外，基于纳米结构标记，系统实现了对细胞内微管、线粒体以及网格蛋白包被小窝等亚细胞结构的光声成像。最后采用多波长照明机制，完成了对同一个细胞内多种亚细胞结构的观测与成像，并定量地分析了亚细胞结构之间的内在联系，为肿瘤细胞在亚细胞结构层面上的分型提供了新的分辨手段。

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