激光的生物学效应
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摘要
激光自从上世纪中期被发明后，它的生物学效应就一直是一个为人们所关注的课题。随着激光技术的不断发展，激光在医学和生物学中的应用也已经日渐成熟，在很多领域都已经取得了巨大的成就。但是，由于生物系统的复杂性，激光的一些生物效应(如弱激光的生物刺激作用等)的机理至今还不是很清楚。
激光诱发的荧光光谱是研究生物组织成分最常用的方法和手段之一，它得到的通常是大量生物组织分子宏观统计的综合信息。本文中，我们应用光谱分析的方法对弱激光治疗中的一种形式——弱激光血管内照射治疗的机制进行了实验研究，提出和验证了激光照射改善血液的携氧能力是血管内照射治疗的机理之一。
为了进一步研究单个细胞及生物大分子的生物行为，我们进行了光镊技术及其应用的研究。所谓光镊就是利用光的压强效应，由一束高度会聚的激光形成的光学势阱，利用它可以对单个细胞、生物大分子等微小物体实现非接触、无损伤操作。在本课题研究中，一方面，我们建立和完善了一套国内领先的可以进行纳米量级位移及pN量级力测量的光镊系统，对光镊系统本身的一些相关技术做了进一步的研究，提出了在低频响条件下用布朗运动测量光阱刚度的方法，研究了光镊系统中照明条件对样品池中温度及四象限测量系统的影响，并设计制作了用于微管实验的流动样品池，实现了对池内温度的控制。另一方面，对于光镊技术在生物学中的应用我们也进行了较深入的研究。用光镊技术研究微管的力学性质，是国家基金委资助的大项目之一，相关结果在国际上尚未见有报道。目前，我们已经成功地实现了单根微管的体外合成，在聚苯乙烯小球表面进行微管抗体蛋白的包被，并用光阱捕捉小球对微管进行了粘接。同时，我们还对实验中出现的微管断裂现象进行了详细的研究。在细胞膜力学性质的研究中，我们用细胞膜拉丝的方法，测定了单个白细胞、乳腺癌细胞在各种条件下的动态和静态膜丝力，首次建立了细胞膜质库的数学物理模型，对膜丝形成的力学机理进行了较详细的探讨。
这些工作的完成，为我们进一步开展微区光谱，单分子荧光及生物分子间作用规律的研究打下了良好的基础。
山西大学博士学位论文
论文共分九章。第一章介绍了弱激光治疗、光镊技术的发展及应用现状。第
二、三章应用光谱分析的方法研究了弱激光血管内照射治疗的机理。第四至九章
的内容是对光镊技术及其在生物学上应用的研究。结论部分对本文的工作进行了
总结，对未来的工作进行了展望。
本论文工作的创新点:
1、应用光谱分析的方法，从物理学的角度宏观地研究了激光与生物组织的
作用规律，探讨了临床上弱激光照射治疗的机理。
2、建立了一套先进的光镊系统，对光镊系统中的一些关键技术进行了详细
的研究。提出了用布朗运动在低频响及低采样频率下测量光阱刚度的方法;对光
镊系统中的照明系统进行了改造，研究了照明条件对样品池中温度和四象限探测
器输出的影响;研制了用于微管实验的流动样品池。
3、应用光镊这一现代的光学手段在微观上对单个细胞及微管蛋白等生物
大分子的生物行为进行了较深入的研究。我们测定了白细胞、乳腺癌细胞在各种
条件下的膜丝力，建立了细胞膜质库的物理模型，从理论和实验两方面研究了膜
丝形成的力学机制，得到了令人满意的结果:在微管聚合与解聚实验中，实现了
单根微管的体外合成，微管的光阱捕获及与小球的粘结，同时对实验中发现的微
管断裂现象进行了详细的研究。
With the development of laser technology, the bio-application of laser has made great progress in many fields.
Laser irradiation therapy, especially intravascular low level laser (ILLLI) therapy has been applied in clinic for a long time and has been proved to be an effective method to various diseases. However, its mechanism still remains unclear. In this dissertation, animal test and spectrum analysis were employed to investigate the mechanism of ILLLI therapy. A viewpoint that laser irradiation can improve the capability of blood carrying oxygen was brought forward based on the results.
The invention of optical tweezers is another important achievement. Because it can be used to manipulate living biological specimens without physical contact and invasion, the technology and applications attract more and more physicists and biologists' attentions. In this dissertation, the optical tweezers system, its key techniques and biological applications have been studied in detail.
On construction of optical tweezers, a dual-optical tweezers system was set up using a Ti-Sapphire laser. The optical trap steering and sample micro-manipulation systems were constructed. The displacement and force measurement systems were incorporated, and displacements of nanometers and forces of picnewtons can be detected. The influence of illumination conditions on the detector and temperature in sample cell was investigated. And a method of calibrating laser trap stiffness using Brownian motion in the condition of low sampling frequency was studied. Furthermore, in order to study the mechanisms of polymerizaton and depolymerization of tubulin under different conditions such as PH, temperature, and biological factor, a flow sample cell with temperature-control system was designed and fabricated.
In applications of optical tweezers, red blood cells, yeasts, coliforms, phagocytes, chloroplast, etc. were successfully manipulated. In force measurements, the static and dynamic tether forces of leukemia cell and mammary cancer cell membranes were
detected. The tether forces of mammary cancer membranes in different conditions were compared. And the mechanical properties in the process of tether formation were discussed. In the experiment of microtubule, the mechanics of depolymerization of tubulin after treatment is being studied using this system, and a phenomenon of microtubule breaking after being irradiated by exciting light was studied in detail.
激光诱发的荧光光谱是研究生物组织成分最常用的方法和手段之一，它得到的通常是大量生物组织分子宏观统计的综合信息。本文中，我们应用光谱分析的方法对弱激光治疗中的一种形式——弱激光血管内照射治疗的机制进行了实验研究，提出和验证了激光照射改善血液的携氧能力是血管内照射治疗的机理之一。
为了进一步研究单个细胞及生物大分子的生物行为，我们进行了光镊技术及其应用的研究。所谓光镊就是利用光的压强效应，由一束高度会聚的激光形成的光学势阱，利用它可以对单个细胞、生物大分子等微小物体实现非接触、无损伤操作。在本课题研究中，一方面，我们建立和完善了一套国内领先的可以进行纳米量级位移及pN量级力测量的光镊系统，对光镊系统本身的一些相关技术做了进一步的研究，提出了在低频响条件下用布朗运动测量光阱刚度的方法，研究了光镊系统中照明条件对样品池中温度及四象限测量系统的影响，并设计制作了用于微管实验的流动样品池，实现了对池内温度的控制。另一方面，对于光镊技术在生物学中的应用我们也进行了较深入的研究。用光镊技术研究微管的力学性质，是国家基金委资助的大项目之一，相关结果在国际上尚未见有报道。目前，我们已经成功地实现了单根微管的体外合成，在聚苯乙烯小球表面进行微管抗体蛋白的包被，并用光阱捕捉小球对微管进行了粘接。同时，我们还对实验中出现的微管断裂现象进行了详细的研究。在细胞膜力学性质的研究中，我们用细胞膜拉丝的方法，测定了单个白细胞、乳腺癌细胞在各种条件下的动态和静态膜丝力，首次建立了细胞膜质库的数学物理模型，对膜丝形成的力学机理进行了较详细的探讨。
这些工作的完成，为我们进一步开展微区光谱，单分子荧光及生物分子间作用规律的研究打下了良好的基础。
山西大学博士学位论文
论文共分九章。第一章介绍了弱激光治疗、光镊技术的发展及应用现状。第
二、三章应用光谱分析的方法研究了弱激光血管内照射治疗的机理。第四至九章
的内容是对光镊技术及其在生物学上应用的研究。结论部分对本文的工作进行了
总结，对未来的工作进行了展望。
本论文工作的创新点:
1、应用光谱分析的方法，从物理学的角度宏观地研究了激光与生物组织的
作用规律，探讨了临床上弱激光照射治疗的机理。
2、建立了一套先进的光镊系统，对光镊系统中的一些关键技术进行了详细
的研究。提出了用布朗运动在低频响及低采样频率下测量光阱刚度的方法;对光
镊系统中的照明系统进行了改造，研究了照明条件对样品池中温度和四象限探测
器输出的影响;研制了用于微管实验的流动样品池。
3、应用光镊这一现代的光学手段在微观上对单个细胞及微管蛋白等生物
大分子的生物行为进行了较深入的研究。我们测定了白细胞、乳腺癌细胞在各种
条件下的膜丝力，建立了细胞膜质库的物理模型，从理论和实验两方面研究了膜
丝形成的力学机制，得到了令人满意的结果:在微管聚合与解聚实验中，实现了
单根微管的体外合成，微管的光阱捕获及与小球的粘结，同时对实验中发现的微
管断裂现象进行了详细的研究。
With the development of laser technology, the bio-application of laser has made great progress in many fields.
Laser irradiation therapy, especially intravascular low level laser (ILLLI) therapy has been applied in clinic for a long time and has been proved to be an effective method to various diseases. However, its mechanism still remains unclear. In this dissertation, animal test and spectrum analysis were employed to investigate the mechanism of ILLLI therapy. A viewpoint that laser irradiation can improve the capability of blood carrying oxygen was brought forward based on the results.
The invention of optical tweezers is another important achievement. Because it can be used to manipulate living biological specimens without physical contact and invasion, the technology and applications attract more and more physicists and biologists' attentions. In this dissertation, the optical tweezers system, its key techniques and biological applications have been studied in detail.
On construction of optical tweezers, a dual-optical tweezers system was set up using a Ti-Sapphire laser. The optical trap steering and sample micro-manipulation systems were constructed. The displacement and force measurement systems were incorporated, and displacements of nanometers and forces of picnewtons can be detected. The influence of illumination conditions on the detector and temperature in sample cell was investigated. And a method of calibrating laser trap stiffness using Brownian motion in the condition of low sampling frequency was studied. Furthermore, in order to study the mechanisms of polymerizaton and depolymerization of tubulin under different conditions such as PH, temperature, and biological factor, a flow sample cell with temperature-control system was designed and fabricated.
In applications of optical tweezers, red blood cells, yeasts, coliforms, phagocytes, chloroplast, etc. were successfully manipulated. In force measurements, the static and dynamic tether forces of leukemia cell and mammary cancer cell membranes were
detected. The tether forces of mammary cancer membranes in different conditions were compared. And the mechanical properties in the process of tether formation were discussed. In the experiment of microtubule, the mechanics of depolymerization of tubulin after treatment is being studied using this system, and a phenomenon of microtubule breaking after being irradiated by exciting light was studied in detail.
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