Design Of The Fluorescence Collection System For Spectral Flow Cytometer

Flow cytometer is a technique for the rapid measurement of biological particles in a single arrangement with uniform flow in the liquid stream.It is widely used in the fields of biological cell cycle and dynamics,apoptosis,and immunology.In the fluorescence multicolor analysis of cells,the traditional flow cytometer uses photomultiplier tube sensor to collect the fluorescence signal that is not full-spectrum and the detection channels are limited,meanwhile it requires complicated fluorescence compensation.The spectral flow cytometer uses light gratings or prisms to separate the fluorescence emitted by the cells through the laser detection zone.The array sensor is used to collect the full fluorescence spectrum,and then the fluorescence full-spectrum is analyzed by data processing algorithm,which makes it more convenient and accurate to realize the multicolor analysis of cells.In this paper,based on the mechanism of flow fluorescence spectroscopy detection,a data acquisition system for fluorescence full-spectrum is designed,using a grating as a spectral dividing element and an array CCD as a photoelectric sensor,which consists of three parts,an optical system,a data acquisition system and a human-computer interaction system.The user can operate the human-computer interaction interface to control data acquisition system to achieve the collection of the fluorescence full-spectrum that is focused on the CCD sensor by the optical system.At the same time,a fluorescence spectrum analysis algorithm based on principal component analysis and multi-curve resolution is proposed.The initial estimate of the pure spectrum is obtained by principal component analysis and evolving factor analysis,and then the pure fluorescence components and component concentrations are obtained by iterative processing of alternating least squares method.The data acquisition system has passed functional tests and wavelength calibration experiments.The experimental results show that the system can perform spectral signal measurements on Ne-Ar standard light sources with a spectral resolution of less than 1 nm.Using the proposed analytical algorithm for fluorescence spectrum to process the auto-fluorescence spectrum of cyanobacteria,the results show that the method can accurately estimate the numbers of pure component in the mixed spectrum and fit the spectrum peaks,thereby accurately estimating the pure component and concentration.This method is not only suitable for the analysis of fluorescence spectrum for spectral flow cytometer,but also can be used for the analysis of other multivariate mixed spectral systems.