基于飞秒光场调控的单分子相干成像及其在癌症诊断中的应用研究

The loss of coherence information has been regarded as the origin of cancer. Although single-molecule fluorescence imaging has become one of the most important trends in cancer diagnosis, the ultrafast dynamics at the femtosecond scale and the corresponding coherence time of single molecules cannot be obtained by traditional fluorescence imaging. Consequently, the early diagnosis of cancer as well as the diagnostic accuracy are still unsatisfactory. In this project, we will prepare the single-molecule coherent superposition state with high coherence property by a pulse shaping, which can product an ultrafast pulse pair with given central wavelength, pulse shape and relative phase. By utilizing an electro-optic modulator with high operation frequency to manipulate the relative phase difference between the pulse pair, we can periodically modulate the fluorescence intensity of the single molecule. On the basis of modem technique, the fluorescence interference arising from background and auto-fluorescence of biological tissue can be effectively suppressed. Consequently, the modulation magnitude of the single molecule under different time delay can be determined in short time integration. In particular, the ratio of signal to noise can be improved by two or three orders of magnitudes by this method. Combing the theoretical model of the light-matter interaction between femtosecond pulses and a single molecule, the coherence time of the single molecule can be readily demonstrated at the time scale of millisecond. Then, we can obtain the coherence microscopy of a biological sample at the time scale of minutes through point-by-point scanning. In this project, we will also study the coherence time of a single molecule varied as different laser wavelength, pulse shape, modulation frequency, time delay and others. The comparison of coherence imaging between normal cells and cancer cells will be performed, and the fluctuation of coherent times will also be investigated. On the basis of the results mentioned above, a cancer diagnostic protocol based on the coherence microscopy will be proposed and checked on various normal and cancer cells. The single-molecule coherence time microscopy based on the modulation of femtosecond laser pulse in this project provides a new insight into the nano-probe and cancer diagnosis.

相干信息的丢失被认为是引起细胞癌变的重要原因，尽管单分子荧光成像已成为癌症诊断最重要的趋势之一，但传统方法无法有效提取单分子在飞秒量级的超快动力学过程及其相干信息，因此在癌症的早期诊断及诊断准确率上仍有明显不足。本项目通过脉冲整形器获得具有特定中心波长、波形以及相位的飞秒脉冲，制备具有高相干性的单分子相干叠加态；利用高频电光调制器对飞秒脉冲对相对相位进行快速调控，实现对单分子荧光辐射强度的周期性调制；基于调制解调技术，抑制背景和自发荧光的影响，获得单分子在不同延迟条件下的调制强度，可以将信噪比提高2-3个量级；结合飞秒激光与单分子相互作用的理论模型，快速有效提取单分子的相干时间，实现单分子相干成像。研究飞秒波长、波形、调制频率、延迟时间等对获取单分子相干时间的影响；研究正常细胞和癌变细胞上单分子相干时间的差异性；提出基于相干成像的癌变诊断方案，提高癌症诊断准确性，实现早期癌症诊断。

癌症已成为严重威胁我国国民健康的重大公共健康问题，研究癌细胞的代谢过程可以阐述癌症的发病机理，对癌症的预防以及治理具有重要的指导意义。目前单分子荧光成像技术已经成为无损癌症诊断最重要的趋势之一，然而传统荧光成像不仅难以克服细胞自荧光的影响；而且无法有效获得单分子的超快动力学过程及其相干信息，因此在癌症的早期诊断及诊断准确率上仍有明显不足。本项目首先从理论上阐述了利用飞秒双脉冲制备、调控单分子相干叠加态进而确定其量子相干超快动力学的可行性，并通过定义相干可视度来量化单分子的退相干过程。然后在实验上构建了单分子相干成像的实验装置，基于调制解调技术实现了对背景光子和自发荧光干扰的抑制(在干扰噪声与信号强度之比大于100的情况下实现了成像)，将单分子量子相干光谱的抗噪声能力提升了2个数量级，量子相干成像对比度提高了4个数量级。最后在毫秒量级获得单个分子的相干时间，实现了细胞层面的量子相干成像与温度成像。基于量子相干成像研究了癌变细胞和正常细胞相干可视度的差异性，其统计值存在显著性差异，满足医学基本判断标准，可用于判别正常细胞和癌变细胞。基于量子相干成像与温度的关联性，项目组进一步发现癌变细胞的线粒体温度要比正常细胞线粒体温度高2.07 ℃，细胞质温度要高0.55 ℃，说明癌变细胞比正常细胞释放了更多的能量。项目组还研制了单分子量子相干光谱仪，在山西医科大学第一附属医院、太原中心医院等开展了癌症诊断合作。这些研究成果为发展基于单分子的微纳探针和医学诊断提供了新的思路和实验方法。.项目组在Phys. Rev. Lett., ACS Nano, Nano Lett.等国内外期刊发表论文21篇，其中一区论文9篇；申请发明专利6项，授权5项。项目负责人及项目组成员与瑞典隆德大学Ivan教授等研究团队实现互访20人次；参与国际国内会议14人次。项目组研制了高性能的单分子量子相干光谱仪，“新型量子相干光谱技术与应用”获2020年度山西省科学技术奖技术发明一等奖。通过本项目的实施，项目负责人2020年晋升为教授，2022年获得了基金委优秀青年项目资助。同时项目组培养了博士生6名，硕士生4名，其中已毕业博士生4名，硕士生2名，已毕业研究生已在太原理工大学、山西医科大学等单位开展独立工作。