A Large Field Of View Microscopic System Based On Microfluidic Chips For Imaging And Sorting Circulating Tumor Cells(CTCs)

At present,malignant tumors(cancers)have seriously affected the health of Chinese residents.How to detect and analyze tumor cells efficiently and minimally at an early stage is imminent.Circulating tumor cells(CTCs)are tumor cells that discretely enter the blood circulation from the tumor tissues of the original lesions in the human body.The detection of these cells can monitor and analyze tumors in an early stage,which has great significance for the treatment of cancer patients.Clinical Value.However,the sorting,observation and identification of CTCs face many difficulties,mainly in three aspects: first,the number of CTCs in the blood of patients is very small,and it is difficult to sort and enrich them;second,the number of blood cells is huge,and the cell size spans a large range.Covering tens to several microns,it is difficult to realize the full-field visualization of the sorting process and observe the cell details;third,the cells become transparent under bright-field microscopy,and it is difficult to distinguish between them.In order to solve the above problems,this paper designs and develops a large field of view microscopic imaging and sorting system based on a microfluidic chip.The microfluidic chip is used to sort and enrich CTCs,combined with microscopic imaging technology and image stitching algorithms,and finally realized In order to efficiently sort and enrich CTCs,full-field high-resolution imaging and accurate identification,the specific work content is as follows:Aiming at the problem that CTCs are scarce and difficult to be sorted and captured,this paper uses microfluidic sorting to study the relevant theories of CTCs microfluidic sorting,then uses an chip which has Fishbone microchannels to combinate with immunomagnetic bead.The selection method realizes efficient sorting and capture of tumor cells in blood samples.Aiming at the problem of obtaining high-resolution cell images under the large field of view of the chip,this paper proposes a method of combining a large field of view microscope objective with an image stitching fusion algorithm.First,the relationship between the microscopic field of view and the resolution is studied from the mechanism of microscopic imaging.Based on the theory,a high-space-bandwidth product(Space-bandwidth Product,SBP)objective lens is selected to match the appropriate photoelectric focal plane,and the design and construction Inverted transmission type bright field microscopic imaging system device.And combined with the image stitching and fusion algorithm based on SURF,the partial images of the chip obtained by the imaging system device are stitched and fused.Finally,a large field of view high-resolution microscopy imaging of the whole area of the chip sample flow is realized,and the imaging resolution is sufficient to observe individual cells.Aiming at the identificationAiming at the identification of CTCs,this paper adopts the identification method of fluorescently labeled CTCs combined with fluorescence microscopy imaging.First,this paper studies the basic theory of fluorescence microscopy imaging.Based on the transmissive brightfield microscopy imaging system,the epi-fluorescence microscopy imaging optical path is designed and built.Then the mechanism of fluorescent labeling of CTCs was studied,and FITC-labeled anti-Ep CAM antibody was selected as the label,and matched with the corresponding fluorescent filter set.Finally,the fluorescently labeled CTCs are individually displayed in the field of fluorescence microscopy imaging,realizing specific recognition of CTCs under the background of whole blood.After the development of the system device was completed,a series of verification experiments were carried out,including the imaging effect verification experiment of the hardware system,the validity verification experiment of the full-field splicing imaging,and the CTCs imaging and sorting experiment,which verified the reliability of the system device.The experimental results show that the final sorting and capture rate of this system device is 81.5%,the imaging field of view is 40mm×15mm,the resolution is 3.48μm,and the fluorescence imaging contrast is 64%,which realizes the efficient sorting and enrichment of CTCs and fullfield high-resolution imaging and accurate identification and distinguish.