A Fixed Cytomter Chip For Identification Of Cell Populations And Real-time Monitoring Of Tumor Single Cells

Cytometry,a fundamental method to measure cell populations and obtain phenotypes from a large number of cells,greatly benefits the clinical diagnosis and biological research.For example,the identification of lymphocyte population by cytometry is helpful to diagnose autoimmune diseases,immunodeficiency diseases,viral infections and malignancy.Beyond the identification of cell populations,cytometry is also used to investigate cell morphological information and their responses toward different stimulants such as ultraviolet(UV)for the molecular mechanisms related to apoptotic pathways.Flow cytometry and hemocytometry have been commonly used in the fields of current cytometric technology.flow cytometry allows for quantifying large numbers of cells rapidly and classifying of cell populations accurately.However,it involves a high price of bulky equipment and requires competent personnel to operate.while hemocytometry,another cytometric technology,is limited by its simple function,although it has the advantages of small size,low cost and simple operation.Both of them are not suitable for real-time monitoring of the morphological changes of single cells.Microfluidic cytometry has been considered as a promising technology and applied on identifying cell populations.However,few chips have both functions of identifying cell populations and real-time monitoring cells.If a multifunctional cytometry chip which integrates functions of identification of cell populations and real-time monitoring could be designed,it will be used by more researchers.Herein,we designed a fixed cytometer chip which had two functional modes,one was to identify cell populations(I-mode).Another functional mode was to real-time monitor of single-cell morphological changes under gradient UV radiation(M-mode).In I-mode,tumor cells were fixed and arranged in the orderly single-cell arrays by optimally designed micropillars.And cell populations with varied fluorescent labels were easily identified by conventional fluorescent microscope with less signal interference from cell overlapping and simple image analysis.Moreover,similar to the flow cytometry,cell populations were well identified from the scatter plot of two-color fluorescence intensities,which provided more information than the simple cell counts by one fluorescent parameter.In M-mode,the chip was applied to real-time monitor of leukemia tumor single cell apoptosis under gradient UV radiation.we developed a stair-like UV shield to form a gradient UV radiation on varied cell trapping arrays region.The heights of the ink layers on steps were varied,thus the UV shield could attenuate to form different UV radiation intensities on chip.the dynamic apoptotic morphologies of a large number of tumor single cells were monitored under this shield in real-time by time-lapse imaging.Owing to the regular single-cell arrays and removable UV shield,we easily tracked numerous tumor single cells according to their locations at specific rows and lines.And the chip will provide an efficient and easy-to-use platform for the study of phototherapy and light-induced tumor diseases.In addition,we integrated eight parallel channels on a 60 mm × 30 mm chip,and the chip could achieve scalable single-cell capture and analysis capability.Overall,this cytometer chip was multifunctional,easy-to-handle,low-cost,universal and scalable.We believe it will be an efficient and alternative cytometer tool for the cell biology research and clinical diagnosis.