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 |
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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.
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
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
(a) Phase shift of polystyrene microspheres of different diameters; (b) Histograms of pixel-wise phase fluctuations
Trapping array with trapped G. Lamblia cysts. (a) In PBS; (b) In refractive index matching solution
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
Statistical analysis of morphological measurements of G. Lamblia cyst and C. Parvum oocyst
OVD of G. Lamblia cyst and C. Parvum oocyst