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chi20190524上海光机所黄惠杰557594Bioaerosols are ubiquitous in nature, including bioactive microorganisms (bacteria, fungi, algae and viruses, etc.), plant debris (pollen, leaf fragments, etc.), non-bioactive insect limbs, putrid bioheaps and various metabolites. If the concentration of bioaerosols in the atmosphere exceeds the threshold, the health of human beings, animals and plants will suffer from huge threat. Moreover, the detection of suspended microorganisms is particularly important in sterile pharmaceutical workshops, hospital operating rooms and other occasions. In the area of defense and security, since the attack of the anthrax bioterrorism happened in the United States in 2001, countries have begun to pay attention to the threat posed by toxic and harmful aerosols. Therefore, the research and development detection of technology and instruments, which can achieve the monitoring of the industrial sterile environment and the early warning of emergencies, to ensure national security, people's health is great significance. At present, a lot of research activities on intrinsic fluorescence detection technology have been studied in other countries and some instruments have been developed. However, our country started relatively later in this field, and it is urgent for us to make research activities on relevant technology and application. This paper mainly focuses on the research of the biological particle optical detection technology and application, including the development of fluorescence particle counter, the improvement of gas path, the accurate measurement of microbial concentration, the research of practical application algorithm, and the research of new methods to distinguish microbial aerosol from non-microbial aerosol. (1) In order to protect the optical cavity and improve the saturation measurement concentration of the instrument, and reduce the overlapping error, many equipment and products in other countries will have a gas sheath flow device. So far, there is few relevant research in our country. On the basis of the existing theory and experimental results, we use the discrete model in the Ansys to analyze the impact of key parameter such as the ratio of sample gas flow and sheath gas flow, outlet Angle, and the best compressed position, and then combined with the actual processing technology to get the optimal design parameters. The results show that under the optimal design parameters, when the sample gas and sheath gas flow ratio of 1:3, to be able to sample gas compression of 1 mm to 0.34nm. On this basis, we developed a real gas sheath flow device, and proposed a new performance test method for the gas sheath flow device. According to this method, the measured results of the gas sheath flow device were basically consistent with the simulation results, which not only showed good compression effect but also verified the feasibility of the hydrodynamic analysis method for the gas sheath flow device. (2) Fluorescence particle counter based on elastic light scattering technology and laser induced intrinsic fluorescence technology is an effective method to distinguish biological particles from non-biological particles. In this paper, a single channel fluorescence particle counter with 405nm semiconductor laser as the light source is developed. The standard polystyrene microsphere (PSL) and standard fluorescence microsphere (B800) were used as test samples to present a calibration method of particle counting and fluorescence technology. (3) At present, many bioaerosol research focus on about the identification of bioaerosol species, and ignores the accurate measurement of bioaerosol concentration. So far, the recognized detection method of microbial aerosol is the culture method. As a new technology, fluorescence particle counter must firstly seek the corresponding relationship between the measurement results of fluorescence particle counter and the measurement results of culture method before practical application. A comparative experiment was designed with test samples which are staphylococcus aureus, escherichia coli and agrobacterium aeruginosa. At the same time, staphylococcus aureus was used as the test sample to design the bacterial fluorescence detection experiments under different states. The results showed that the bacterial fluorescence under dormant state was very weak, which was difficult to detect, resulting in the omission of fluorescence particle counter. The above experimental results have laid a foundation for the detection of microbial aerosol by fluorescence particle counter in the actual environment. (4) Due to the existence of many pseudo - biological particles in the air, it has great influence on the detection of biological particles of fluorescence particle counter. In order to make the application of fluorescent particle counter in real environment, it is necessary to study the algorithm to eliminate influence of background value. Firstly, the empirical algorithm was studied, and a practical empirical algorithm was obtained after a lot of data analysis and evaluation of various situations. However, the empirical algorithm has no basis of algorithm model, and the actual environment is diverse and complex, so it is difficult to adapt to the new environment, and it is also so difficult to deal with some accidental situations that cause false alarm and omission. In order to solve these problems, the Kalman filtering algorithm is introduced into the field of aerosol instrument counting for the first time. Combining with the characteristics of aerosol environment, we get the particle counting Kalman filtering algorithm and fluorescence counting Kalman filtering algorithm. In the meanwhile, we design a series of experiments to evaluate the practicability of the new algorithm. The results show that Kalman filtering algorithm can be fully applied to the field of aerosol particle counting. (5) Although the fluorescence particle counter has been well used at present, it is still unable to distinguish biological particles from non-biological particles. Considering that microbial particles are the living entities of biological particles, they are essentially different from other biological particles. Based on the extension of laserinduced fluorescence technology, we study the method of distinguishing microbial aerosols from non-microbial aerosols by photobleaching. A new device was developed to obtain the spectrum of microbial aerosols. After analyzing the spectrum changes before and after photobleaching, we got two characteristics: the percentage of main peak shift and the percentage of fluorescence intensity change. According to the above two characteristics, microbial aerosols and non-microbial aerosols can be distinguished.2019atalunwen21961462959392Laser induced fluorescence technology; Gas sheath flow apparatus; Kalman filtering algorithm; Photobleaching; Bioaerosol classificationBiological particle optical detection technology and application research生物粒子光学探测技术及应用研究生物气溶胶在自然界中无处不在,包括具有生物活性的微生物(细菌、真菌和病毒等)、植物残骸(花粉、树叶碎片等) 和没有生物活性的昆虫肢体及各种代谢物。当大气中生物气溶胶的浓度超过某一阈值时,会引起各种疾病,对人类和动植物的健康造成威胁。不仅如此,悬浮微生物的控制在无菌制药车间、医院手术室等场合也尤为重要。在国防安保领域,自从 2001 年美国发生的炭疽生物恐怖袭击以后,各国开始关注有毒有害生物气溶胶带来的威胁。因此,研究与发展可实时、灵敏地监测空气中微生物粒子的探测技术与仪器,实现对工业无菌环境的监测和突发事件的预警,对保障国家安全、人们健康具有重大的意义。 目前为止国外对基于生物气溶胶本征荧光的检测技术开展了大量的研究并开发出一些实用化仪器,而国内在这方面起步较晚,急需开展相关的技术与应用研究。本论文主要围绕生物粒子光学探测技术从气体鞘流器研制、 荧光粒子计数器的研制、微生物浓度的精确测量、实际应用算法研究与荧光非微生物粒子的排除方法等五个方面开展研究: (1) 介绍气体鞘流器的研制。在国外相关产品中为了保护光学腔及提高仪器的饱和测量浓度,减少重叠误差确保单颗粒测量,很多都带有气体鞘流器。目前国内在这方面研究甚少,我们根据已有的理论基础和实验成果,利用 Ansys 软件包 Fluent 中的离散型模型,仿真分析样气和鞘气的流量比、 出口倾斜角、 及最佳压缩点到光敏感区的距离等关键参数对气体鞘流器压缩效果的影响,然后结合实际加工工艺得到最佳设计参数,结果表明在此最佳设计参数下,能够把直径1mm 的样气压缩到 0.34mm,此时样气和鞘气的流量比为 1:3。在此基础上研制了气体鞘流器实物,提出一种新的气体鞘流器性能测试方法,利用此方法得到的气体鞘流器实测结果与仿真结果基本吻合,具有良好的压缩效果,同时验证了气体鞘流器流体动力学分析方法的可行性。 (2) 介绍荧光粒子计数器的研制。以弹性光散射技术和激光诱导本征荧光技术为基础的荧光粒子计数器是区分生物粒子和非生物粒子的有效手段。本文研制了以 405nm 半导体激光器为光源、单通道接收的荧光粒子计数器。以标准聚苯乙烯小球(PSL)及标准荧光小球(B800)为测试样品,提出了一种粒子计数及荧光粒子计数的标定方法,且设计了相关实验来测试荧光粒子计数器的性能,结果表明荧光粒子计数器具有良好的粒子计数效率及荧光探测能力。 (3) 介绍以实际微生物为测试样品,对比荧光粒子计数器测量结果与培养法测量结果,得到两种方法测量结果的对应关系。目前对于生物气溶胶研究的主要关注点是生物气溶胶的种类辨别,但是生物气溶胶的浓度测量同样重要。在某些特定场合,只有生物气溶胶浓度超过一定界限才会影响人的健康,此时生物气溶胶的浓度必须严格控制。荧光粒子计数器作为一种测量生物粒子浓度的新技术,在实际应用之前必须经过严格的微生物粒子探测能力的测试。目前公认标准的微生物气溶胶探测方法是培养法,因此以培养法为对照方法, 寻求荧光粒子计数器对实际微生物测量结果的规律。以金葡萄球菌、大肠杆菌及绿脓杆菌为测试样品,设计了对比实验,通过结果分析获得了两种方法的线性关系。同时以金葡萄球菌为测试样品,设计了不同状态下的细菌本征荧光探测实验,结果表明休眠状态下的细菌本征荧光很弱,难以被荧光粒子计数器探测而导致漏报。以上实验结果为荧光粒子计数器在实际环境中对微生物气溶胶的探测打下了基础。 (4)介绍 Kalman 滤波算法在荧光粒子计数器实际测量中的应用。由于在自然界中很多非生物粒子也会被激发出荧光,而荧光粒子计数器只以有无荧光来初步判别是否是生物粒子,所以会存在很多干扰荧光信号,我们称那些能被激发出荧光的非生物粒子为伪生物粒子。本文以能够使荧光粒子计数器很好地在实际环境中应用为目的,开展了算法研究来消除正常空气中本身存在的伪生物粒子的影响。最初研究的是经验算法,经过大量数据分析及各种情况的评估,得到了比较实用的经验算法。但是经验算法并没有数学模型基础, 并且实际环境情况复杂多样, 在新的环境中很难适应, 对于一些偶然情况也难以处理, 容易出现误报和漏报。为了解决这些缺陷,本文首次在气溶胶仪器计数领域引入 Kalman 滤波算法,结合气溶胶环境的特点,给出了粒子计数 Kalman 滤波算法及荧光粒子计数Kalman 滤波算法,并且设计了一系列实验评估算法的实用性。实验表明 Kalman滤波算法可以很好的应用到气溶胶粒子计数领域。 (5)主要介绍荧光非微生物粒子的排除方法。虽然目前荧光粒子计数器得到了很好的应用,但是依然不能区分微生物粒子和非微生物粒子。考虑到微生物粒子是生物粒子中具有生命活性的个体,和其他生物粒子有着本质区别。作为激光诱导本征荧光技术的拓展研究,本文基于光漂白特征开展了区分微生物气溶胶和非微生物气溶胶的方法研究。搭建了一套实验装置来获取微生物气溶胶的本征荧光光谱,分析光漂白前后的本征荧光光谱变化,得到两个区分微生物气溶胶和非微生物气溶胶的特征:荧光主峰偏移百分比及荧光强度变化百分比。根据以上两个特征,我们可以辨别出微生物气溶胶和非微生物气溶胶。激光诱导本征荧光技术;气体鞘流器;Kalman滤波算法;光漂白现象;生物气溶胶分类中国科学院上海光学精密机械研究所鲁晨阳光学工程博士
中文题目: 生物粒子光学探测技术及应用研究
外文题目: Biological particle optical detection technology and application research
作者: 鲁晨阳
导师姓名: 黄惠杰
学位授予机构: 中国科学院上海光学精密机械研究所
答辩时间: 20190524
Laser induced fluorescence technology; Gas sheath flow apparatus; Kalman filtering algorithm; Photobleaching; Bioaerosol classification
学位级别: 博士
正文语种: chi
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