Precision in flow cytometry depends on many factors, the first of which is accurate and stable positioning of the hydrodynamically focused cells. However, no method exists to evaluate the stability of laminar flow and single-cell flow in the flow chamber of the flow cytometer directly because of the small size of the rectangular channel of the flow chamber. In this paper, a method of high-speed particle image velocimetry is proposed to solve this problem. The velocity stability of the particles in the flow chamber is used to evaluate the flow stability of the fluid path of the flow cytometer. The side scattering images of particles are obtained by a high-speed camera. Upon exposure, cells were imaged at random positions in the flow cell, resulting in four different types of the images: blank, inadequate, normal, or overlapped. Normal images were identified utilizing a grey cluster analysis algorithm based on trapezoid whitenization weight functions. A mid-point method is applied to determine the length of the particle track at a fixed exposure time. The variation of the trajectory lengths of the normal images are used to evaluate the stability of the liquid path. Experiments are carried out to verify the feasibility of our method in which different diameter microspheres at different flow rates. The results indicate that the standard deviation and relative standard deviation of the trajectory lengths can be used as the evaluation indices of the liquid path stability of the flow cytometer. © 2016 International Society for Advancement of Cytometry.
Keywords: flow cytometer; grey clustering analysis; high-speed image acquisition; particle velocimetry; stability evaluation.
© 2016 International Society for Advancement of Cytometry.