面向单细胞分析的SICM 新型成像模式与定量激励方法研究

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题名	面向单细胞分析的SICM 新型成像模式与定量激励方法研究
作者	李鹏
学位类别	博士
答辩日期	2015-05-27
授予单位	中国科学院沈阳自动化研究所
授予地点	中国科学院沈阳自动化研究所
导师	李广勇 ; 刘连庆
关键词	单细胞分析扫描离子电导显微镜同相电压调制模式电容补偿技术激励定量化分析
其他题名	A Novel Imaging Mode and Quantitative Stimulus of SICM for Single Cell Analysis
学位专业	机械电子工程
中文摘要	一切生命活动的关键问题均可在细胞中寻求到最终答案，因为细胞是生命体的结构与功能的基本单元。人类对细胞的研究随着实验研究手段的更新而日益发展。目前，单细胞层次的科学研究对生物学基础问题研究、重大疾病早期诊断及个性化医疗等领域发挥着重大作用，代表了细胞研究最新的发展方向与研究热点。扫描离子电导显微镜(Scanning ion conductance microscope: SICM)由于可实现无损、高分辨率、液态环境下的成像，以及可提供简单、高效及细胞低损伤的生物/化学物质激励，成为极具发展潜力的单细胞分析工具。但目前现有SICM成像仍存在系统漂移、噪声、扫描速度慢等问题，实现活体细胞的高质量、高效观测仍面临巨大挑战。此外，SICM激励仍存在缺乏定量化分析的问题，无法满足面向活体细胞的精准、高效的生物化学物质激励的需求。本论文针对基于SICM的高质量、高效的单细胞成像研究所面临的问题以及单细胞定量化物质激励研究所面临的巨大挑战，在系统地研究分析SICM作业机理的基础上，重点开展了以下研究工作：(1) 新型成像模式研究：针对SICM传统成像模式存在的抗干扰能力差、扫描速度慢等问题，本研究创新性地提出了基于交流激励的同相电压调制成像方法，有效解决了直流模式与跳跃模式的抗干扰能力差问题；同时，该方法具有比距离调制模式更高的调制频率，有效提高了成像效率。(2) 微弱信号优化方法：SICM新型成像模式中存在信噪比低、成像质量较差、调制频率受限等问题。本研究提出了基于电容补偿的信号优化方法，有效解决了信噪比低、成像质量较差、调制频率受限等问题；同时优化了工作距离参数以及系统带宽参数。实验表明：这种优化方法使得成像效率与成像质量均有了进一步提升。(3) 激励定量化方法：目前尚未有有效手段实现SICM定量化的生物/化学物质激励。本研究提出了基于库尔特计数原理的精密操控方法，能够实现对激励量的精密操控，通过调整驱动方式的极性控制激励量的运动方向，通过同步监测电脉冲信号的个数表征激励量的多少。以聚苯乙烯小球与腺病毒颗粒为生物/化学物质代表样本的激励实验表明了该方法的有效性，且操控精度可精确到单颗粒级别。(4) 实时反馈、高速闭环的系统构建技术。本研究攻克了微弱信号检测与系统去噪等难题，完成了硬件集成、实时系统构建以及软件开发等研究工作，最后利用该实验平台开展了大量实验研究，验证了本文提出的新型成像模式与定量化激励方法的正确性与优越性。本论文通过理论分析、系统构建、及实验研究工作，丰富了SICM的成像机制，提高SICM抗干扰能力与速度，拓展了SICM的功能和应用范围。SICM在单细胞分析方面的独特优势，将进一步推动单细胞分析技术的发展。
索取号	Q503/L33/2015
英文摘要	The key to every biological problem must finally be sought in the cell. That is be-cause that cell is the basic structural and functional unit of life. Cell biology is continuously developing with the updated tools. Recently, Single-cell analysis, which plays important roles in the basic research of biology, early diagnosis of major diseases and personalized medicine, represents the hot topic and future direction of cell biology re-search. Scanning ion conductance microscope (SICM) is a single cell analysis tool with great potential in two aspects: one is single cell imaging due to features including the force-free, non-invasive，work in liquid，high resolution imaging, and the other is single cell stimulus due to the simple, effective and low-invasive advantages using the nano-pipettes. However, thus far, SICM imaging is still suffering from large system drift, high noise and low scanning speed. Thus, it is challenging to realize living cell imaging with high quantity and high efficiency using SICM. Furthermore, the lack of quantitative stimulation using SICM limits its ability to meet the desire of precise and efficient bio-chemical stimulus on living cells.This dissertation is aiming at the problems of realizing live cell imaging with high quantity and high efficiency and at overcoming the challenge of lack of quantitative stimulation on living cells using SICM. The key research work in this dissertation is based on the fundamental principle of SICM, and summarized as follows:(1)A new imaging mode was presented. Traditional modes of SICM suffer from system instability and low scanning speed. In-Phase Bias modulation (IPBM) mode based on AC driving was introduced, effectively overcomes the instability problem of DC mode and hopping mode; meanwhile, this new mode allowed the system to work at a higher modulation frequency than distance modulation mode, thus increasing the potential for higher scan speed.(2)A signal optimization method was introduced. IPBM mode has weaknesses such as a low signal to noise ratio, and limited modulation frequency. We proposed a capacitance compensation method and effectively solved those problems. Meanwhile, it can optimize the probe –sample separation and expand the bandwidth. Experimental results demonstrated that both imaging efficiency and imaging quality were further enhanced.(3)Quantification of active stimulation was introduced. Quantitative biochemical stimulation was not yet realized. Coulter counters technique in SICM will provide the method of measuring the quantity quantization of the stimulation. Polarity of driving controls the direction of stimulation; electrical pulses signal characterizes the quantity of stimulation. Experimental results with polystyrene and adenovirus particle show the effective of this method and with this method control accuracy can be go up to single particle level.(4)The SICM based platform including Real-time feedback, high–speed close loop was established. Key problems such as weak signal detection and noise elimination are solved; hardware integration, real-time system implementation and software development were finished. Finally, with this platform a lot of experimental results were carried out to verify the correctness and the validity of the new imaging mode and quantitative stimulation method.In summary, through sophisticated theoretical analysis, reliable system implementation and plentiful experimental results, the work of this dissertation enriches the imaging mechanism of SICM, improves the system stability and scans speed, and expands the application scope of SICM. Furthermore, the advantages of SICM will promote the development of single cell analysis.
语种	中文
产权排序	1
页码	120页
内容类型	学位论文
源URL	[http://ir.sia.ac.cn/handle/173321/16799]
专题	沈阳自动化研究所_机器人学研究室
推荐引用方式 GB/T 7714	李鹏. 面向单细胞分析的SICM 新型成像模式与定量激励方法研究[D]. 中国科学院沈阳自动化研究所. 中国科学院沈阳自动化研究所. 2015.