Research On Key Technologies Of Single-laser Multi-color Flow Cytometry

Flow cytometry is a novel analysis technique that enables rapid measurement of cell or subcellular structures.Flow cytometers are instruments designed based on this technology,integrating multiple disciplines such as biomedical detection technology,instrumentation science and technology,and optoelectronics technology.They are widely used in cell biology,cell genetics,and other research areas.This study focuses on two key technologies of a single-laser multi-color flow cytometer: the stable formation of single-cell flow and optical system design.The aim is to solve the problems of stable single-cell flow formation and optical system design.Theoretical parameter calculations,simulation designs,and experimental analyses were carried out in two aspects: the fluidic system(flow chamber and fluidic drive)and the optical system(laser beam shaping optical path,scattered light signal,and fluorescence signal detection optical path).In the fluidic system design,theoretical calculation formulas for the sample flow focusing diameter were derived through theoretical deduction based on the basic theory of fluid mechanics and the principle of fluid focusing.The flow chamber structure in the flow cytometer was designed using Solidworks software,and simulation analysis was carried out to simulate the fluid flow changes inside the model when the velocity of the sample and sheath fluids changed.Through orthogonal experiments,the optimal sample flow inlet velocity was found to be 1900mm/s,and the optimal sheath flow inlet velocity was240mm/s.Based on the DPM theory,the flow of particles/cells in the sample stream was simulated,and the ideal particle motion trajectory at the optimal focusing effect was obtained.An experimental setup was built,and experimental data was measured and obtained to verify the focusing effect of the fluidic system designed in this study.In the optical system design,a single-laser four-color flow cytometer optical path system was designed and built.The advantages and disadvantages of laser sources in the optical path,as well as the factors affecting scattered light and fluorescence detection,were theoretically analyzed.A multi-channel spectrophotometer optical path structure was designed,selected,and built for laser beam shaping,scattered light,and fluorescence detection.Scattered light was detected by a photodiode with an area of 1mm×1mm,and fluorescence was detected by four channels of photomultiplier tubes with wavelengths of530 nm,570nm,620 nm,and 675 nm.Experimental validation showed that the optical system had a spot size that met the design requirements after collimation and that the detection optical path could effectively collect signals.Based on the research of the two systems,a single-laser multi-color flow cytometer experimental device was designed and built.The difference in cell counting accuracy between the designed experimental device and a traditional flow cytometer was tested,and the results showed no significant difference in accuracy at low concentrations.This confirmed the similarity in cell counting accuracy between the designed experimental device and a traditional flow cytometer.