Two-beam phase correlation spectroscopy:a label-free holographic method to quantify particle flow in biofluids

We introduce two-beam phase correlation spectroscopy(2B-ΦCS)as a label-free technique to measure the dynamics of flowing particles;e.g.,in vitro or in vivo blood flow.2B-ΦCS combines phase imaging with correlation spectroscopy,using the intrinsic refractive index contrast of particles against the fluid background in correlation analysis.This method starts with the acquisition of a time series of phase images of flowing particles using partially coherent point-diffraction digital holographic microscopy.Then,phase fluctuations from two selected circular regions in the image series are correlated to determine the concentration and flow velocity of the particles by fitting pair correlation curves with a physical model.2B-ΦCS is a facile procedure when using a microfluidic channel,as shown by the measurements on flowing yeast microparticles,polymethyl methacrylate microparticles,and diluted rat blood.In the latter experiment,the concentration and average diameter of rat blood cells were determined to be(4.7±1.9)×10^(6)μL^(-1)and 4.6±0.4μm,respectively.We further analyzed the flow of mainly red blood cells in the tail vessels of live zebrafish embryos.Arterial and venous flow velocities were measured as290±110μm s^(-1)and 120±50μm s^(-1),respectively.We envision that our technique will find applications in imaging transparent organisms and other areas of the life sciences and biomedicine.