Two-Beam Phase Correlation Spectroscopy: A label-free holographic method to quantify particle flow in (bio)fluids

Figure 1 Principle of 2B-ΦCS. (a) Sketch of the microfluidic channel illuminated by a perpendicular light beam. The sample flowing through the microchannel is monitored by phase imaging. (b)-(d) The workflow of 2B-ΦCS. (e) ACFs and CCF curves calculated from the phase-time traces.

Figure 2 2B-ΦCS measurement in density and size of rat RBCs. (a) The workflow of in-vitro 2B-ΦCS measurement on rat blood. (b) Phase images of flowing RBCs at six different times (specified in the insets). (c, d) Histograms of (c) density and (d) diameter measurements.

Figure 3 In-vivo 2B-ΦCS measurement of blood flow velocities in an artery and a vein of a zebrafish embryo. (a) The schematic DHM setup for 2B-ΦCS measurement on blood cell flow in zebrafish vessels; (b) The autocorrelation and cross-correlation curves calculated from two phase-time trajectories extracted from vascular images; (c) Statistical chart of blood flow velocity in arteries and veins, respectively.

Characterizing the transport of nano- and microscopic particles (e.g., flow of blood cells in vessels and active movement of vesicles along microtubules in cells) is important for understanding many biological processes. A variety of techniques have been proposed to quantify the dynamics of particle flow, including particle image velocimetry (PIV) and Doppler optical coherence tomography (DOCT). PIV, which involves continuous imaging of particles to analyze the speed and direction of the flow pattern, is limited to sparse tracer particles. DOCT utilizes low coherence interference and Doppler frequency shift analysis to detect the blood flow in blood vessels, and currently, the time resolution of ODT is mainly limited by the necessity to record hundreds of holograms for each swept wavelength. Fluorescence correlation spectroscopy (FCS), especially the dual-focus FCS proposed in recent years, has also proven to be a valuable tool for the quantitative assessment of particle flow. In dual-focus FCS, the fluorescence signals from the two foci are registered as a function of time; then, time autocorrelation analysis of the intensity yields quantitative information about diffusion and flow. However, both FCS and dual-focus FCS require either intrinsically fluorescent particles, or particles labeled with fluorescent moieties. The unavoidable photobleaching sets limits to its application and calls for techniques not relying on fluorescence.

The refractive index (RI) is an intrinsic optical parameter related to the electrical permittivity of the material. The RI of biological particles, subcellular organelles or cells differ from their (usually aqueous-based) surrounding biofluid and, therefore, the RI can provide the necessary contrast against the background. Once fluorescence contrast is replaced by phase contrast, we can quantitatively access the dynamics of flowing microparticles using the concept of dual-focus FCS but in a label-free manner. Centering on the above ideas, Prof. Peng Gao's research group from Xidian University and Prof. G Ulrich Nienhaus's research group proposed and demonstrated two-beam phase correlation spectroscopy (2B-ΦCS) as a label-free approach for the quantification of particle flow, such as in-vivo blood flow or particle flow through a standard microfluidic chip.

In 2B-ΦCS, digital holography microscopy (or other quantitative phase microscopy approaches) was used to perform quantitative phase imaging for flowing particles continuously. Then, the phase fluctuation time traces are acquired from the DHM holograms, and correlation analysis yields information on concentration and velocity of flowing particles. 2B-ΦCS overcomes photobleaching and phototoxicity issues inherent in fluorescence-based approaches, and thus can be widely applied in many fields including biomedicine. The authors utilized 2B-ΦCS to realize the in vitro measurement of rat blood cell density and in vivo measurement of zebrafish blood flow velocity. The relevant research results were published in Photonics Research, Volume 11, No. 5, 2023(Lan Yu, Yu Wang, Yang Wang, Kequn Zhuo, Min Liu, G. Ulrich Nienhaus, Peng Gao. Two-beam phase correlation spectroscopy: a label-free holographic method to quantify particle flow in biofluids[J]. Photonics Research, 2023, 11(5): 757).

The workflow of 2B-ΦCS for the quantification of particle flow: First, acquiring the holograms of flowing particles continuously by digital holography microscopy (DHM). Second, acquiring the phase fluctuation time traces in two selected circular areas in the phase images. Third, the correlation curves from phase fluctuation time traces are calculated, and the further correlation analysis yields information on the concentration and velocity of flowing particles. After comparing the correlation analysis using the intensity image sequence and phase image sequence, it is confirmed that the contrast provided by the phase of the flowing particles can provide the correlation curves with significantly-enhanced amplitude and signal-to-noise ratio (SNR).

2B-ΦCS was applied to measure the density of rat blood cells in vitro. Fresh rat blood from a rat was extracted and diluted with PBS at a volume ratio of 1:100. Once the diluted blood was pumped in, and flowed in a microfluidic channel, 280 holograms were taken. 2B-ΦCS analysis reveals the density of the rat blood cells is 4.7 ± 1.9×106 µL-1 (mean ± s.d.), and the average diameter of rat blood cells was 4.6 ± 0.4 µm (mean ± s.d.), as shown in Figure 2.

In addition, 2B-ΦCS was utilized to monitor blood flow in live zebrafish embryos. 6000 holograms of the veins and arteries of zebrafish embryos were recorded continuously using DHM. The phase image of blood vessels can be reconstructed from the holograms. An exemplary phase image reconstructed from the hologram series (Figure 3) shows RBCs in a vein (posterior cardinal vein, PCV) and an artery (dorsal aorta, DA) in phase contrast. The correlation analysis of 2B-ΦCS on 15 zebrafish embryos reveals that the blood flow velocities arteries (red square) and veins (gray square) are 290 ± 110 μm s-1 and 120 ± 50 μm s-1, respectively.

The authors highlighted their finding: 2B-ΦCS utilizes the phase difference of particles against the surrounding media as the substitute for extraneous fluorescence labels. The correlation analysis on the phase images can quantitatively access the flow velocity, diameter, and concentration of flowing particles in a label-free manner. The potential of 2B-ΦCS will be further enlarged when being combined with deep learning, for instance, to measure particles of different sizes simultaneously. The authors also envisage that this technology will be widely applied in many fields, including blood inspection and water quality monitoring.


图1 双光束相位相关光谱(2B-ΦCS)原理。(a)利用数字全息显微系统连续采集流场中微粒的相位图像序列;(b-d)提取相位图像序列中两个圆形观察区域内随时间变化的相位信号;(e)根据两个相位轨迹计算的自相关和互相关曲线

图2 2B-ΦCS对大鼠血细胞密度与直径的测量。(a) 测量流程图;(b)流动血细胞在六个不同时刻的相位图像;(c,d)血细胞密度和直径的测量结果直方图

图3 2B-ΦCS对斑马鱼胚胎血液流速的体内测量。(a)使用DHM装置采集斑马鱼胚胎的血管部位相位图像;(b)从血管图像中提取的两个相位-时间轨迹计算出的自相关和互相关曲线;(c)动脉和静脉的血液流速统计图




基于以上思路,西安电子科技大学郜鹏教授课题组和德国KIT的G. Ulrich Nienhaus教授课题组提出并验证了用于流动中的微粒检测的双光束相位相关光谱方法(2B-ΦCS)。在2B-ΦCS中,利用数字全息显微(也可以是其他定量相位显微方法)对连续流动的微粒进行定量相位成像。之后,通过对相位图像序列中两个相邻圆域内随时间变化的相位曲线进行自相关和互相关分析,在无需标记的前提下获得了微粒的流速、直径、浓度等信息。文中,作者利用2B-ΦCS实现了大鼠血细胞密度的体外测量和斑马鱼血液流速的在体测量。相关研究成果发表于Photonics Research 2023年第5期


应用1:使用2B-ΦCS对大鼠血细胞的密度进行体外测量。结合微流控通道,对稀释100倍的大鼠血液进行测量。280组测量结果的分布直方图显示大鼠的血细胞密度为4.7 ± 1.9 × 106 µL–1 (平均值±标准差)、血细胞平均直径为4.6 ± 0.4 µm (平均值±标准差),如图2所示。

应用2:利用2B-ΦCS对斑马鱼胚胎血管内血流进行了动态测量。利用DHM连续记录了斑马鱼的静脉和动脉血管的6000帧全息图。从DHM的全息图中可以再现出血管的相位图像,在相位图像序列中分别选取两个圆形区域以覆盖斑马鱼的动脉和静脉血管。之后,对两个圆域内的相位-时间曲线进行相关分析,可以获得动脉和静脉中血细胞的流速。通过对15条斑马鱼进行测量,统计得到动脉和静脉的血液流速分别为290 ± 110 μm s–1 和120 ± 50 μm s–1,如图3所示。