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近红外光在生物样品传输时会受到运动散射体的强烈散射,这种散射会引起近红外相干散射光场的相位改变,探测器可以探测这种光斑波动。相位变化受生物样品特性的影响,因此,相位的变化率可以反映生物样品内部散射颗粒的动态特性,通常用吸收系数μa和约化散射系数μs′对其进行表征[1]。
组织表面下的运动信息由位于 r处t时刻的散射光电场 E(r, t)表征,运动散射体的均方位移通常用布朗运动模型和生物组织中的随机流动模型描述[14-16]。因此,生物样品中散射光场的自相关函数可由与近红外扩散方程类似的相关谱扩散方程给出[17-18]:
$ \begin{array}{c} G_1(\boldsymbol{\rho}, \tau)= \\ \frac{3 S \mu_{\mathrm{s}}{ }^{\prime}}{4 \pi} \frac{\exp \left(-\sqrt{3 \mu_{\mathrm{a}} \mu_{\mathrm{s}}{ }^{\prime}+6 k_0{ }^2 \mu_{\mathrm{s}}{ }^{\prime} D_{\mathrm{B}} \tau} \cdot \boldsymbol{\rho}\right)}{\boldsymbol{\rho}} \end{array} $
(1) 式中:$ G_1(\boldsymbol{\rho}, \tau)=\left\langle\boldsymbol{E}^*(\boldsymbol{\rho}, t) \boldsymbol{E}(\boldsymbol{\rho}, t+\tau)\right\rangle$表示光场自相关;E(ρ, t+τ)为位于 ρ处t+τ时刻的光场强度;E*(ρ, t)为位于ρ处t时刻的光场强度复共轭;S为各向同性光源的总发射功率;DB表示散射体的有效布朗扩散系数,反映强散射介质的动态特性;τ为相关时间延迟,当时间延迟增大时,光场相关强度减小;ρ为光源与光纤探测器间的距离;k0为介质中光的波数。
通过测量样本表面的散射光强随时间的变化可以获得散射粒子的运动信息[19],光强的自相关函数为:
$ G_2(\tau)=I(t) I(t+\tau) $
(2) 式中:I(t)为t时刻的光强,表示在积分时间tint内的平均值;I(t+τ)为在t+τ时刻的光强[3]。
在实验中,光强通常比电场强度更容易获取,散射光电场归一化自相关函数g1(τ)通常由测量的光强归一化自相关函数g2(τ)给出[20],因此实际测量的是光强归一化自相关函数,即:
$ g_2(\tau)=\frac{G_2(\tau)}{I(t)^2}=\frac{I(t) I(t+\tau)}{I(t)^2} $
(3) g2(τ)能直观反映近红外光在生物组织内部的吸收和散射情况,从而反向拟合出生物组织的吸收系数μa、散射系数μs′以及散射体的运动速度等特性参数。
近红外扩散相关光谱测量过程的示意图如图 1所示。光强归一化自相关函数g2(τ)曲线的斜率表征了生物样品的动态特性[2],衰减斜率更大的曲线对应的组织中细胞运动速度更快,因此,通过计算散射光强的归一化自相关函数可以反映生物样品一定的内部特性。
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DCS检测系统数据采集与数据分析的操作系统为Windows 10(专业版64位),运行内存为16.0 GB,计算机处理器为Intel(R) Core(TM) i7-10875H CPU@2.30 GHz 2.30 GHz。
软件系统核心算法是对探测器检测到的动态激光散斑光强进行归一化自相关运算,从而获得归一化自相关函数g2(τ)。单光子探测器将探测到的光脉冲信号转换为高速TTL电脉冲信号,系统数据采样和计数原理如图 3所示。
上位机程序使用频率为fs的采样时钟去读取离散的脉冲信号,得到采样数据S(i),S(i)表示在第i·Δt时刻计数器积累的光子数,其中单位时间间隔Δt=1/fs,Nint表示积分时间tint内采样的总数,因此,Nint=tint·fs。则在第i·t的单位时间间隔内收到的光子数为s(i)=S(i+1)-S(i)。因此光强的归一化自相关函数表示为:
$ g_2\left(n=\tau f_{\mathrm{s}}\right)=\frac{\langle s(i) s(i+n)\rangle}{\langle s(i)\rangle\langle s(i+n)\rangle} $
(4) 此时,延时变量τ=n/fs,n为采样增量, 〈〉表示在(Nint-n)点范围内的平均值。光强的归一化自相关函数的公式可展开为:
$ g_2\left(\tau=n / f_{\mathrm{s}}\right)=\frac{\sum\limits_{i=1}^{N_{\text {int }}-n} s(i) s(i+n)}{\sum\limits_{i=1}^{N_{\text {int }}-n} s(i) \sum\limits_{i=n+1}^{N_{\text {int }}} s(i)}\left(N_{\text {int }}-n\right) $
(5) 采集数据时需要考虑存储空间的分配,例如设置积分时间tint=1 s,采样频率fs=1 MHz,则每次采样总数Nint为1×106个点。计数器的数据位宽是32位,4 bytes,因此,每次采样的数据至少要分配4 Mbytes的存储空间。硬件系统采集到的脉冲数值保存在采集卡内存中,上位机程序调用数据采集卡(data acquisition,DAQ)采集模块,使用低频时钟采集计数器的缓存区数据,获得采样数据S(i),DAQ采样时钟由外部信号源提供,频率fs为1 MHz~20 MHz,采样频率越高,单位时间内的采样数据量越大,但运算时间也会越长。软件的高速采样提高了采集量和测量时间分辨率,大多数DCS对样品的测量是缓慢的,高时间分辨率测量将改善对运动粒子的识别。除此之外还能够使用较少的探测器实现高空间分辨率成像,可以将成像帧率降低到秒甚至更少,从而实现动态成像。程序结构由while循环和JKI有限状态机组成[21],可以实现对动态激光散射光斑光强的长期闭环跟踪和控制,获得稳定的采样数据,解决了硬件自相关器不容易实现连续长期监测的问题,还可以利用软件自相关器实现同时监测通道的持续监测,拓展了软件自相关器在长期持续监测方面的应用。
近红外扩散相关光谱系统软件界面如图 4所示。程序模块主要包括参数设置与计数采样、相关算法和实时监测3个模块。参数设置与计数采样模块如图 4a所示,可以实现计数采集和工作参数的控制、APD开关控制、数据读取的方式选择和计数数据的分析,也可以实现软件退出及数据保存;DCS检测系统相关算法模块如图 4b所示,可以实现光子数采集,光功率显示,动态激光散斑光强的归一化自相关运算并作图显示;如图 4c所示,可以实现数据计算的实时监测。
近红外扩散相关光谱系统的研制
Design and experiment of a near-infrared diffusion correlation spectrum system
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摘要: 近红外扩散相关光谱(DCS)是一种先进的无创检测组织深层血流流速变化的动态光散射技术。为了解决光子相关器价格昂贵、灵活性差、应用局限、计算效率低的问题,采用将光子相关器软件化的方法,设计了一种近红外扩散相关谱检测系统。该系统以单纵模长相干激光器作为光源,采用单光子探测器探测散射光信号,用数字信号采集模块采集高速数字信号,并对数字脉冲信号进行计数,基于软件完成了对动态散射光斑光强归一化自相关的计算。结果表明,该系统计算出生物样品波动漫反射信号的时间平均强度自相关函数符合理论,验证了系统检测功能的可靠性;在客观评价指标方面,可成功区分不同的生物样品,并且能够分别体积分数差为1%的同种生物样品。该系统可以实时监测生物样品整体的光学特性和动态特性,具有体积小、成本低、灵活性好、可拓展性强的特点,为轻量化DCS检测系统的发展提供了一种新的解决方案。Abstract: Near-infrared diffusion correlation spectroscopy (DCS) is a relatively new method widely used for blood flow detection because deep biological tissues can be penetrated by near-infrared light, which has a low absorption coefficient and strong penetrating ability. The DCS technique differed from traditional biological sample detection techniques in that it used the time-averaged intensity autocorrelation function of fluctuating diffuse reflectance signals to detect blood flow within tissues, which could achieve non-destructive and continuous quantitative detection of biological samples. In response to the problems of low computational efficiency, high system price, cumbersome operation and application limitations of the current DCS detection system by hardware technology for scattered spot attenuation calculation, a near-infrared diffusion correlation spectrum detection system based on software was proposed and designed. Considering the light source requirement for correlation, a single longitudinal mode laser was used as the light source for this system, a single photon detector was used to detect the scattered light signal, and the signal was transmitted by the digital signal acquisition module, and the calculation of normalized autocorrelation for dynamic scattered light spot intensity was performed by the software. The experimental results show that the time-averaged intensity autocorrelation function of the fluctuating diffuse reflectance signals obtained from the detection of biological samples by the system is in accordance with the theory, which verifies the reliability of the detection function of the system; in terms of objective evaluation indexes, the system successfully discriminates between different biological samples and is able to separate biological samples of the same species with a difference in volume fraction of 1%. The system can monitor the overall optical and dynamic characteristics of biological samples in real-time and is characterized by small size, low cost, good flexibility, and strong expandability, which provides a new solution for the development of a lightweight DCS detection system.
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