微流体衍射相位显微成像及其在寄生虫测量中的应用

顾鑫, 黄伟, 杨立梅, 等. 微流体衍射相位显微成像及其在寄生虫测量中的应用[J]. 光电工程, 2019, 46(12): 190046. doi: 10.12086/oee.2019.190046
引用本文: 顾鑫, 黄伟, 杨立梅, 等. 微流体衍射相位显微成像及其在寄生虫测量中的应用[J]. 光电工程, 2019, 46(12): 190046. doi: 10.12086/oee.2019.190046
Gu Xin, Huang Wei, Yang Limei, et al. Microfluidic diffraction phase microscopy and its application in parasites measurement[J]. Opto-Electronic Engineering, 2019, 46(12): 190046. doi: 10.12086/oee.2019.190046
Citation: Gu Xin, Huang Wei, Yang Limei, et al. Microfluidic diffraction phase microscopy and its application in parasites measurement[J]. Opto-Electronic Engineering, 2019, 46(12): 190046. doi: 10.12086/oee.2019.190046

微流体衍射相位显微成像及其在寄生虫测量中的应用

  • 基金项目:
    国家自然科学基金资助项目(61505240);苏州市应用基础研究计划(SYG201414);中国科学院青年创新促进会人才资助项目(2015258)
详细信息
    作者简介:
    通讯作者: 黄伟(1983-),男,博士,副研究员,主要从事激光生物成像的研究。E-mail:whuang2008@sinano.ac.cn
  • 中图分类号: O436.1; TH742

Microfluidic diffraction phase microscopy and its application in parasites measurement

  • Fund Project: Supported by National Natural Science Foundation of China (61505240), the Applied Basic Research Programs of Suzhou City (SYG201414), and the Youth Innovation Promotion Association of CSA (2015258)
More Information
  • 本文提出了一种将衍射相位显微技术与微流体芯片相结合的方法对水源性寄生虫进行定量测量。结合干涉技术与光学显微镜搭建了衍射相位显微成像系统,实现对寄生虫的高灵敏度实时测量。基于光刻工艺,设计和制作了U型捕获结构双层微流体芯片,实现高通量的单个寄生虫捕获。将与聚二甲基硅氧烷(PDMS)折射率相同的聚蔗糖水溶液通入微腔,消除U型捕获结构边缘衍射在相位成像时产生的显著干扰噪声。利用不同直径的标准聚苯乙烯微球验证了该系统的准确性,最大相位值误差不超过3%。采用上述系统测量了100个贾第鞭毛虫包囊和100个隐孢子虫卵囊,然后从干涉图中重构出两虫的相位图。通过对定量相位图的分析得出两虫的形态学参数与定量的光体积差分布,定量的数据为了解其生理特性提供了依据。微流体衍射相位显微成像系统结构简单,稳定性好,测量精度高,在对单个微生物进行实时监测和无标记定量研究方面具有巨大的潜力。

  • Overview: Quantitative phase imaging (QPI) is developed based on light microscopy as an advanced modality for label-free biomedical optical imaging. Diffraction phase microscopy (DPM) is demonstrated as a novel QPI technique, which combines many of the best attributes of current QPI methods. In the quantitative phase imaging process, the suspended sample is usually placed between a coverslip and a glass slide for observation imaging. The sample is easy to aggregate and form clusters and have weak Brownian motion in this case, which can introduce unexpected noise interference in the imaging area.

    In this paper, we propose a method of using DPM combined with microfluidic chip to quantitatively measure Giardia Lamblia (G. Lamblia) cysts and Cryptosporidium Parvum (C. Parvum) oocysts. The DPM system is placed at the output port of conventional light microscope. The DPM interferometer is created using a diffraction grating in conjunction with a 4f lens system. A three-dimensional structure of a microfluidic chip is fabricated using polydimethylsiloxane (PDMS). The chip consists of parallel arrays of U-shaped trapping structures, which contained between 4 and 5 traps over its width. Each row of traps is placed at the gap from the previous row to allow the sample to be fully trapped. A double-layered structure is designed and fabricated to increase trap efficiency and reduce pressure in the chip. Ficoll solution with the same refractive index as polydimethylsiloxane (PDMS) is introduced into the microfluidic chamber to eliminate significant artifacts in phase imaging originating from diffraction at the edges of trapping structures. The accuracy of the system is verified using standard polystyrene microspheres of different diameters, and the error of maximum phase shift does not exceed 3%. The microfluidic phase imaging system can be accurately used for quantitative phase imaging. 100 G. Lamblia cysts and 100 C. Parvum oocysts are measured using this system. The phase maps of the parasites are obtained from the interferograms. The morphological parameters and quantitative optical volume difference distribution of the two kind of waterborne parasites are obtained by analyzing the quantitative phase maps. Quantitative data provides the basis for understanding their physiological characteristics. The microfluidic diffraction phase microscopy system has simple structure, good stability and high measurement accuracy, and has great potential for real-time monitoring and label-free quantitative studies of single microorganism.

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  • 图 1  微流体衍射相位显微成像系统示意图。(a)微流体芯片三维结构图;(b)双层结构侧视图;(c)双层结构俯视图

    Figure 1.  Schematic of microfluidic quantitative phase imaging system. (a) Three-dimensional structure diagram of microfluidic chip; (b) Side view of double-layered structure; (c) Vertical view of double-layered structure

    图 2  聚苯乙烯微球定量相位成像。(a)去离子水中捕获聚苯乙烯微球明场图;(b)去离子水中捕获聚苯乙烯微球干涉图;(c)去离子水中捕获聚苯乙烯微球定量相位图;(d)折射率匹配液中捕获聚苯乙烯微球明场图;(e)折射率匹配液中捕获聚苯乙烯微球干涉图;(f)折射率匹配液中捕获聚苯乙烯微球定量相位图

    Figure 2.  Quantitative phase imaging of polystyrene microspheres. (a) Bright field image of trapped polystyrene microsphere in deionized water; (b) Interferogram of trapped polystyrene microsphere in deionized water; (c) Phase image of trapped polystyrene microsphere in deionized water; (d) Bright field image of trapped polystyrene microsphere in refractive index matching solution; (e) Interferogram of trapped polystyrene microsphere in refractive index matching solution; (f) Phase image of trapped polystyrene microsphere in refractive index matching solution

    图 3  (a) 不同直径聚苯乙烯微球的相位延迟;(b)像素点处相位值波动直方图

    Figure 3.  (a) Phase shift of polystyrene microspheres of different diameters; (b) Histograms of pixel-wise phase fluctuations

    图 4  贾第鞭毛虫包囊捕获阵列。(a)在PBS中;(b)在折射率匹配液中

    Figure 4.  Trapping array with trapped G. Lamblia cysts. (a) In PBS; (b) In refractive index matching solution

    图 5  水源性寄生虫定量相位成像。(a)折射率匹配液中捕获贾第鞭毛虫包囊干涉图;(b)折射率匹配液中捕获贾第鞭毛虫包囊定量相位图;(c)折射率匹配液中捕获隐孢子虫卵囊干涉图;(d)折射率匹配液中捕获隐孢子虫卵囊定量相位图

    Figure 5.  Quantitative phase imaging of waterborne parasites. (a) Interferogram of trapped G. Lamblia cyst in refractive index matching solution; (b) Phase image of trapped G. Lamblia cyst in refractive index matching solution; (c) Interferogram of trapped C. Parvum oocyst in refractive index matching solution; (d) Phase image of trapped C. Parvum oocyst in refractive index matching solution

    图 6  贾第鞭毛虫包囊和隐孢子虫卵囊的形态学测量统计图

    Figure 6.  Statistical analysis of morphological measurements of G. Lamblia cyst and C. Parvum oocyst

    图 7  贾第鞭毛虫包囊和隐孢子虫卵囊的光体积差分布图

    Figure 7.  OVD of G. Lamblia cyst and C. Parvum oocyst

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出版历程
收稿日期:  2019-01-25
修回日期:  2019-05-09
刊出日期:  2019-12-01

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