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DOPC系统主要利用的是光学反演,经过散射组织后的光束将会逆向穿透组织重新聚焦。然而在生物应用中,光束在散射组织内聚焦更具有实际意义,因此,需要引入引导星与波前整形技术结合来实现散射组织内的聚焦。引导星可以与组织内的散射光作用(比如产生频移或明显的波长变化)而区别于大量的背景光噪声,使得反演后的光束最终可以聚焦到引导星处,从而实现散射组织内的聚焦。这些引导星的性质,如位置和尺寸,对最终散射组织中的光学焦点的质量有着决定性的作用[43]。
侵入性的引导星,如荧光分子[44]、二次谐波纳米粒子[45-46]、磁性粒子[47-48]、基因编码蛋白[49]都已成功与基于光学相位共轭的数字化波前整形系统结合,实现了散射组织内的聚焦。这些引导星通过各种方式产生可以被选择性检测到的散射光来指引光学焦点的位置,具有高对比度、小尺寸等优点,但是对于生物应用的开展却没那么安全。生物体中内源性散射体的变化过程,包括位置变化、吸收变化等,也可以用作引导星[50-51]。虽然这些引导星是非侵入性的,但是它们在生物体内通常成片出现,很难被操控与分离。因此一个可操控的并且非侵入性的引导星对于散射组织内的聚焦有着重大的意义。
2011年,研究者首次提出将聚焦的超声波作为引导星与基于相位共轭的波前整形技术结合,将光聚焦在散射组织的深处[52-54],其工作原理如图4所示。如图4(a)中蓝色标记所示中,当入射光照射到散射组织上时,散射效应使得光的传播方向被随机改变(用蓝色标记)。相比于光,超声受散射组织的影响较弱,因此可以通过超声换能器实现散射组织内的超声聚焦。由于声光相互作用,通过超声焦点的一部分散射光将发生频移(用红色标记)。通过添加不同频率的干涉光束来分离被超声波标记的光子和没有发生频率变化的光子。到达探测面的光场可分为3部分:
图 4 TRUE工作原理;(a) 超声引导星标记一部分通过超声焦点的散射光,使其发生频率偏移;(b) 仅对发生频移的光子相位共轭将实现散射组织内的光学聚焦
Figure 4. Operational principle for TRUE; (a) The ultrasonic guide star tags a portion of scattered light that passes through the ultrasonic focus, causing a small amount of frequency shift; (b) The phase conjugate mirror selectively reflects the frequency-shifted light, realizing optical focusing inside the scattering medium
$$ \left\{ {\begin{array}{*{20}{l}} {{E_R} = {A_R}\cos \left( {{\omega _R}t + {\varphi _R}} \right)} \\ {{E_T} = {A_T}\cos \left( {{\omega _T}t + {\varphi _T}} \right)} \\ {{E_S} = {A_S}\cos \left( {{\omega _S}t + {\varphi _S}} \right)} \end{array}} \right. $$ (1) 式中:ER为参考光;ET为标记光;ES为未被标记的样品光。实验中利用声光调制器(一般工作频率为40~60 MHz)将ER移至与ET相近的频率(用深红色标记),则三种光会发生外差干涉形成拍频。但由于ES与另外两种光频率相差太大,其产生的拍频远大于相机的帧率,只能被探测到一个稳定的散斑背景。因此最终被记录的场强为
$$ \begin{split} I(t) =& {\left| {{E_R} + {E_T} + {E_S}} \right|^2} = A_R^2 + A_T^2 + A_S^2 + \\ & 2{A_R}{A_T}\cos \left[ {\left( {{\omega _T} - {\omega _R}} \right)t + {\varphi _T} - {\varphi _R}} \right] \end{split} $$ (2) 由于φR可认为在空间上均匀,标记光的相位就可以利用多步相移提取。在图4(b)中,只对图中红色标记的频移光子进行光学相位共轭,它们将遵循时间反演原理沿着原先的光路返回到声学焦点,实现散射组织内的聚焦。以声学焦点作为引导星,结合基于相位共轭的波前整形技术的聚焦方案被称为Time-reversed ultrasonically encoded optical focusing,简称为TRUE。
与物理的侵入性引导星相比,聚焦的超声波具有非侵入性和易自由操控等优点,更适合应用到生物组织中,然而其较长的波长也使得所形成的光学焦点尺寸较大。为了解决这一问题,将TRUE进行数次迭代能够有效地减小光学焦点尺寸、提升焦点亮度[55-56],也可以用来实现快速成像扫描[57]。尽管理论上迭代的TRUE可以收敛到单个散斑的大小[58],但目前实验上仅仅观察到了2~3倍分辨率的提升和约20倍PBR的提高[55-56]。2013年,Judkewitz等人提出对光学散斑的涨落进行特异性编码,再通过数千次的光场测量提取单个光学散斑所对应的光场,在散射组织内耗时数小时实现了具有单个散斑尺寸(约5 μm)的光学聚焦[59]。2015年,Ruan等人还提出利用聚焦的超声击破血管中的微气泡以提高信号对比度,能够大幅减小光学焦点的尺寸、提升光学焦点的亮度[60]。研发尺寸更小、对比度更高、易自由操控的非侵入性引导星能让有效信号的光场测量更加准确,对于波前整形技术未来在生物光子学的应用具有重要的作用。
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由于生物本身呼吸,心跳和血流等生理运动,生物组织实际上是一个动态散射介质,因此在活体组织内聚焦具有更大的挑战。其中大部分的动态散射过程都是由流动的红细胞造成的,其余的生理活动对于动态过程的贡献较小[35]。针对各类活体应用场景,光学散斑的相关时间应控制在数十微秒到数毫秒之间。由于波前整形的原理要求整个系统的操作时间短于散斑的相关时间,提升TRUE系统的速度对于其在生物活体中的应用至关重要。如图5所示,利用TRUE进行聚焦和DOPC系统相类似,同样包含光场测量与光场调控两个步骤。在光场测量步骤中,干涉光需要附加4个特定的相位0、π/2、π和3π/2或与标记光发生外差干涉,再结合四步相移全息技术来测量标记光的光场。该方法需要记录4幅强度图像并传输到计算机,因此测量速度受到相机速率的限制。通常情况下,对于工作频率为几百赫兹的相机,4幅图的测量与传输时间需要几十毫秒。在完成光场测量与重构后,波前整形技术还需要通过用空间光调制器产生散射光场的相位共轭波面,使得共轭光能够回到原来声学焦点的位置。因此,实现高速的TRUE聚焦需要对光场测量与光场调控2个步骤的速度进行提升。
图 5 TRUE工作流程;(a) 光场测量步骤;(b)光场调制步骤。φ(r)表示由相机测量到的散射光场的相位图
Figure 5. Operational procedures for TRUE; (a) Light field measurement process; (b) Light field modulation process. φ(r), phase map of scattered light, which is captured by the camera
2015年,Wang等人提出了一种单帧测量下的二值化相位提取方案,并结合高速DMD(数字微镜)的高速刷新速率和FPGA的高速并行处理能力,实现了穿透活体小鼠背部聚焦,系统的响应时间约为5.3 ms。但是DMD调制速度的上升(至22 kHz)是以牺牲调制效率得到的(从相位调制的π/4下降到二值化强度调制的1/2π)。另外,由于无法区分被标记的和未被标记的散射光,该方案尚不能直接与超声引导星相结合[61]。2016年,Liu等人提出锁相探测技术非常适用于从大背景中提取小信号的TRUE中,其使用一个具有300×300 pixel的锁相相机完成了0.3 ms内的散射光场测量[62]。与普通CMOS或CCD不同,锁相CMOS的每一像素都有一个高速光电转换器并在内置电路中加入移相器,其利用锁相原理同时输出高频信号的振幅及相位信息,因此可在外差干涉系统中探测高速拍频。该方法中虽然光场测量过程速度极快,但是由于锁相相机的传输速率较低,且其工艺受限导致像素较少(90 000 pixel),并未进行实时光场重构、光场调控、与TRUE焦点的形成[62]。2017年,Hemphill等人提出离轴全息术能够从一帧强度图中提取散射光场信息,并演示了约9 ms的散射光场相位共轭过程,但是该方案需要牺牲像素,计算较为繁琐。而且,该项研究尚不完善,也没有对于其与超声引导星结合后的工作性能进行演示[63]。2017年,Liu等人开发了两帧曝光下的二值化相位测量方案,结合高速的铁电液晶空间光调制器,完整地实现了第一个具有毫秒响应的TRUE聚焦系统[64]。如图6所示,展示出在不同散斑相关时间下散射组织内分别产生的TRUE焦点,可以看到相关时间越短,聚焦性能越差。通过对PBR的数学模型进行数值拟合,得出系统的响应时间约为6 ms。尽管该系统已是目前世界上最快的TRUE系统,但是其系统响应仍然无法跟上活体中的散斑相关过程。
因此,较慢的系统响应速度仍是波前整形技术在生物活体应用开展的主要限制性因素,应分别从光场测量和光场调控方向寻求更具效率的方案,有效提升系统速度。
Wavefront shaping technology based on digital optical phase conjugation (invited)
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摘要: 光在生物组织中传播时,会被微观尺度上不均匀的组织随机散射,这种现象严重制约了光学技术在生物医学中的应用。波前整形技术将散射过程当成一个确定性的过程,通过测量散射效应造成的相位延迟并利用空间光调制器进行逐点补偿,可以实现散射光的操控与重新聚焦。在各类波前整形技术中,基于光学相位共轭的数字化波前整形技术具有可调控自由度高、系统响应速度快等优点,最适宜与生物医学应用相结合,如生物活体成像、操控、治疗等。文中将重点关注基于光学相位共轭的数字化波前整形技术的发展,探讨该技术在应用研发中面临的主要技术瓶颈和挑战,并概述其应用开展情况。Abstract: Optical scattering inherent in biological tissue prohibits optical technologies from being widely used in biomedical applications. Wavefront shaping treats optical scattering as a deterministic process, enabling the control of scattered light by point-by-point phase compensation through spatial light modulators. Among various wavefront shaping technologies, digital optical phase conjugation controls the largest number of degrees of freedom and has the fastest speed, which is suitable for biomedical applications such as live-tissue imaging, manipulation, and therapy. This paper will describe the development of digital optical phase conjugation, discuss the main technological challenges encountered, and speculate on future applications.
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图 4 TRUE工作原理;(a) 超声引导星标记一部分通过超声焦点的散射光,使其发生频率偏移;(b) 仅对发生频移的光子相位共轭将实现散射组织内的光学聚焦
Figure 4. Operational principle for TRUE; (a) The ultrasonic guide star tags a portion of scattered light that passes through the ultrasonic focus, causing a small amount of frequency shift; (b) The phase conjugate mirror selectively reflects the frequency-shifted light, realizing optical focusing inside the scattering medium
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