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多模态眼前段成像设备的研发及应用

Development and application of multi-modal anterior eye imaging system

来源期刊： 眼科学报 | 2021年1月 第36卷 第1期： 38-45 发布时间： 收稿时间：2023/3/6 11:31:07 阅读量：4051
作者：
段铮昱,
肖鹏,
骆仲舟,
王耿媛,
袁进,
关键词：
多模态光学相干断层成像眼微血流成像眼科成像
multi-modal imaging optical coherence tomography microvascular imaging ophthalmic imaging
DOI：
10.3978/j.issn.1000-4432.2021.01.04
目的：获取眼表图像的综合信息，建立眼表疾病综合诊断和评估。方法：将超高分辨率光学相干断层成像仪(ultra-high resolution optical coherence tomography，UHR-OCT)与基于裂隙灯生物显微镜的微血管成像系统相结合，开发了一种多模态、非接触式的眼科光学成像平台。结果：UHR-OCT模块在组织中实现轴向分辨率约为2 μm 。眼表微血管成像模块在最大放大倍率下横向分辨率约为3.5 μm。通过集成在裂隙灯显微镜成像光学路径的不同模块，多模态成像平台能够执行实时前段OCT结构成像、结膜微血管成像和传统裂隙灯成像功能。利用自主开发的软件，进一步分析结膜血管网络图像和血流图像，获取血管分形维数、血流速度、血管直径等定量形态学和血流动力学参数。结论：通过在健康受试者和角膜炎患者的在体成像测试，表明多模态眼前段成像设备可为眼科临床应用及人工智能提供结构和功能信息数据。
Objective: To obtain the comprehensive information of the anterior eye image, establish complementary information for the diagnosis and evaluation of ocular diseases. Methods: We developed a multi-modal, non-invasive optical imaging platform by combining ultra-high resolution optical coherence tomography (UHR-OCT) with a microvascular imaging system based on slit-lamp biomicroscopy. Results: The uHR-OCT module achieved an axial resolution of approximately 2 μm in tissues. The lateral resolution of the ocular surface microvascular imaging module under maximum magnification was approximately 3.5 μm. By combining the imaging optical paths of different modules, the customized multi-modal eye imaging platform was capable of performing real-time cross-sectional UHR-OCT imaging of the anterior eye, conjunctival vessel network imaging, high-resolution conjunctival blood flow videography, and traditional slit-lamp imaging on a single device. With self-developed software, a conjunctival vessel network image and blood flow videography were further analyzed to acquire quantitative morphological and hemodynamics parameters, including vessel fractal dimensions, blood flow velocity and vessel diameters. Conclusion: The ability of the multi-modal anterior eye imager to provide both structural and functional information for ophthalmic clinical applications can be demonstrated in a healthy human subject and a keratitis patient.
    眼表疾病通俗的是指眼睛表面的疾病，泛指损害累及角膜和结膜眼表正常结构与功能的疾病，例如结膜炎、角膜炎、干眼症都统称为眼表疾病。在眼部结构中，眼表组织与外界直接接触，易发生感染、炎症、创伤等疾病，严重情况会影响人们的健康和生活质量，眼表致盲性疾病是仅次于白内障、青光眼和老年性黄斑变性的世界第4大致盲原因，尤其在发展中国家盛行[1-3]。大多数的眼表疾病存在着多种临床症状。例如角膜炎主要由角膜炎症引起，典型表现为角膜上皮缺损甚至基质浸润，严重的可引起周围结膜血管扩张、变形、充血，甚至导致血管生长异常[4-6]。目前，临床诊断通常采用裂隙灯显微镜、光学相干断层扫描仪(optical coherence tomography，OCT)等多种检查设备，结合荧光染色等成像方式来提供多模式和多维的图像，获取综合信息，进行眼表疾病的诊断和治疗评估[7-8]。
    裂隙灯显微镜是应用最广的眼科常规眼表检查设备[9]。通过向眼睛照射1束狭缝光，利用丁达尔效应的原理，可对角膜、结膜等前段组织的病理变化进行立体评价。然而，传统的裂隙灯显微镜由于其有限的放大倍数和较差的空间分辨率，使其难以对病理结构进行更高精度的评价及准确的生物学参数测量[10-12]。20世纪90年代发展起来的OCT，以其非接触、非侵入性和高分辨率的特性，彻底改变了眼科和视觉科学领域，成为眼科组织断层成像领域的重要诊断工具之一[14-16]。OCT技术在活体人眼角膜、视网膜组织结构成像中已得到广泛应用[17-20]。随着光电技术的不断改进，超高分辨率光学相干断层成像仪(ultra-high resolution optical coherence tomography，UHR-OCT)已经实现了微米级的轴向分辨率[21-22]。UHR-OCT在眼表组织的成像性能可以对泪膜[23]、角膜主要结构成像[24]，以及各层组织厚度测量，如角膜上皮层/上皮细胞，肾小球膜、基质、角膜后弹力层和内皮[25]，这可以帮助临床医生更好地诊断和监测眼部表面病理和疾病，包括角膜炎[25-26]、圆锥形角膜[27]、眼部表面肿瘤[28]等。
    对于眼表疾病，除组织结构的改变外，炎症还会引起眼微血管系统的改变，引起血管扩张、通透性增加、血流增加等一系列与病变严重程度有关的血管功能性变化[5-6]。OCT血管造影等技术已被应用于前段血管成像[29-30]，但只能提供形态学信息，缺乏提供血流速度等定量功能参数的功能。为测量球结膜血管的血流速度和评估其网络的复杂性，本团队开发了功能裂隙灯生物显微镜，实现结膜血管网络成像和高分辨率结膜血流视频成像[10,31]。通过定制的软件，可以对图像和视频进行处理，得到血管直径、复杂程度等形态学参数，以及血流速度等功能参数。上述方法已经在健康受试者以及眼表损伤和炎症等疾病患者的临床研究[31-35]中得到验证，被证明是对球结膜微血管系统进行成像和评估的一种非常简单且成本效益更高的替代方法。
    目前，眼科疾病的诊断和治疗评估通常是基于裂隙灯、OCT等多类传统结构成像设备，结合眼表荧光染色等多种技术，获得的单模态的组织信息，再通过医生进行综合判断。能够同时提供多模态成像和全面诊断信息的一体成像系统逐步成为临床的需求。提高系统分辨率，提供功能成像能力，实现眼科疾病的准确早期诊断和定量分类，是当今眼科影像学及成像设备的发展趋势。多模态一体成像系统不仅可以减少患者从一个检查设备转到另一个检查设备的耗时，提高医疗机构的检查效率，而且可以减少检查设备占据的空间和设备投资。目前，国内外研究人员已经开发了多种多模态系统来结合多种成像模态的优点用于人眼成像[36-45]，但其中大多数集中应用在后段成像[36-40]，用于人眼前段成像较少。另外，有研究证明将OCT系统集成到裂隙灯显微镜中进行前后段成像以提高检查效率[41-42]，OCT和角膜地形图结合的多模态系统已被用于前段生物测量，并应用于圆锥角膜的结构成像[43-44]。
    本研究通过在数码裂隙灯生物显微镜平台上集成自主开发的UHR-OCT和微血管成像系统，开发了一套全新的多模态非接触的眼表多功能成像平台，为眼科临床眼表疾病检查提供结构和功能信息。

1 材料与方法

1.1 成像装置

    多模态眼表成像系统的原理如图1所示。多模态系统集成了裂隙灯显微镜、自主研发的OCT模块和眼微血流成像模块。眼微血流成像系统采用了裂隙灯显微镜的光源和光学系统，数码相机通过裂隙灯固有的相机端口连接到裂隙灯光路。为提高结膜血管网络和血流视频的图像质量，在裂隙灯光源光路中加装滤光片提高红细胞显示强度，无需对裂隙灯进行额外的修改或转接适配器。为获得合适的成像视野，裂隙灯内置光学放大率设为25倍(由放大率表盘设定)，血流成像采用的数码相机放大率为7.5倍(由佳能EOS 60D相机功能设定)，总成像放大率可达约187.5倍。UHR-OCT模块是基于超宽谱光源(MT-850-HP-I，Superlum)的OCT系统。SLD光源发出的光经过隔器、衰减器后，进入分光比为25:75的光纤耦合器，入射光按照比例分入样品臂和参考臂。为匹配偏振态及色散，参考臂和样品臂均装有偏振控制器及相同的光学元件。在样品臂，准直后的光经过二维扫描振镜和聚焦透镜，通过45°安装在在裂隙灯成像镜头前的二向色镜反射入人眼。照射在人眼上的入射光的功率调节到750 μW，低于ANSI(美国眼科标准-眼科仪器的光危害保护)规定的最大允许暴露量[46-47]。进入参考臂的光，经准直后通过中性密度滤波器、聚焦透镜后对准平面反射镜。参考臂的光路长度与样品臂的光路长度匹配。从样品臂上的眼睛和参考臂上的镜子反射的光被反射回光纤耦合器，包含组织信息的光信号随后进入OCT的光谱仪。光谱仪由准直透镜、光栅，图像放大透镜和线阵CCD相机组成。CCD相机信号通过图像采集卡传输进入计算机。

1.2 微血管图像分析

    微血管成像模块获取的结膜血管网络图像和动态血流视频，需进一步应用成像算法处理，以提取定量的血流形态学和血流动力学功能参数。图像处理软件的开发已在之前的文献中叙述[31-32]。对于结膜血管网络图像，首先，通过自适应直方图均衡化来提高图像质量，然后通过减去背景图像来进一步增强图像质量；然后，将增强后的图像转换为二值图像，利用骨架化算法得到网络骨架；最后，对血管骨架图像进行分析，计算分形维数复度，提取反应血管网复杂度的分形维数单分形值(Dbox)和反应血管网密度的多重分形(D0)值，用于评价血管密度和复杂性。对于结膜血流影像，首先采用基于球结膜时空图像的运动校正算法，对眼球运动进行补偿，得到配准图像。在对血管图像进行增强和分割后，利用多帧图像的均值，通过计算血管信号轮廓的半高宽(full width at half maxima，FWHM)来测量血管直径(D，μm)。通过分析红细胞的运动，定量结膜血流动力学参数，直接测出轴向血流速度(Va，mm/s)，以此计算出截面血流速度，截面血流速度乘以血管横截面积(假设血管为圆形截面，由血管直径计算得出)获得血流量(Q，pl/s)[48]。
20230306114411_2434.png
图1 系统结构原理图
Figure 1 Schematic of the eye imaging system

2 结果

    通过在1名健康受试者和1名角膜炎患者进行在体成像实验，初步验证了多模态眼表成像系统的性能及可行性。实验均已获得受试者知情同意，实验程序遵循赫尔辛基宣言原则，按照临床研究设计方案进行(临床批件伦理号：2017KYPJ104)。首先对每位受试者进行正常的裂隙灯检查。然后，依次对角膜中央/角膜损伤区和球结膜区进行OCT成像和微血管成像。最后，对角膜炎患者行裂隙灯荧光染色成像。
    在本研究中，OCT成像以24 000 A-line/s的扫描速率记录图像，获得横向扫描范围为6 mm的OCT断层图像。球结膜血管成像时，首先在数码相机的单图模式下通过设置裂隙灯显微镜的放大倍率为16倍，对应的成像视场约为18×12mm2，图像大小为5 184×3 456像素。然后，微血管成像在佳能60D摄像机的电影剪辑功能下，以60帧/s的速度拍摄球结膜血流视频片段，进行血流动力学分析，裂隙灯显微镜放大率设置为25倍，60D相机长焦效应放大率为7.5倍，成像视场约为1.15×0.86 mm2，图像尺寸为640×480像素。为了测量的准确性，需要从多个区域记录一个受试者球结膜上的5个视频片段，在每个成像位置记录1~2 s。采集到的图像和视频片段，通过自主开发的软件进行处理得到最终的结果。

2.1 健康人

    图2显示了多模态眼表成像系统在健康受试者上获得的实验结果。图2C为健康角膜的UHR-OCT图像。最佳聚焦位置在角膜的中部，因此可以以最高的灵敏度对全角膜进行成像。在UHR-OCT图像放大区域(图2D)，角膜主要层结构如上皮层(EP)、前弹力层(BL)、基质层(ST)、内皮层(ED)清晰可见。图2F为健康受试者结膜血管网络的静态图像。图2G，H显示了使用图像分析软件从图2F处理得到的骨架化网络。计算健康受试者微血管网络的分形维数为：单重分形Dbox=1.56，多重分形D0=1.49(表1)。图2E是从健康受试者的一段结膜血流视频片段中截取的静止结膜血管图像。通过软件计算血流动力学定量结果是：平均血管直径D=17.2 μm，轴向血流速度平均值Va=0.25 mm/s，和血流量平均值Q=59.8 pl/s(表1)。

2.2 角膜炎患者

    图3显示了多模态眼表成像系统对角膜炎患者的实验结果。眼外观裂隙灯成像可见明显的炎性病变。角膜荧光素染色成像突出显示了病变在眼表的位置、大小和边界。UHR-OCT图像显示：病灶在靠近角膜顶端边缘的区域呈高反射结构，角膜病灶清晰可见，侵润角膜上皮层、前弹力层和基质层。计算角膜炎患者微血管网的分形维数为：单分形Dbox=1.65，多重分形D0=1.69(表1)。从角膜炎患者的结膜血流视频片段中截取到的静止的结膜血管图像中，放大区域红色箭头所指显示的是红细胞簇。经图像处理软件计算，角膜炎患者的血流动力学结果为平均血管直径D=23.0 μm，轴向血流速度Va=0.58 mm/s，血流量Q=194 pl/s(表1)。
20230306114552_5449.png
图2 健康受试者多模态成像
Figure 2 Experimental results of the healthy subject
(A，B)健康受试者眼外观裂隙灯成像；(C，D)OCT成像，放大区域显示角膜分层，EP：上皮层，BL：前弹力层，ST：基质层，ED：内皮层；(E)结膜微血管图选定区域的眼微血管动态视频截图；(F)健康受试者结膜微血管图；(G，H)结膜微血管网络骨架化处理结果。
(A, B) Slit-lamp corneal imaging of healthy subjects, the red dashed line indicates the OCT scanning position. (C, D) OCT image of the healthy cornea and the zoom-in area showing clear corneal layer structures. EP: epithelial layer; BL: Bowman’s layer; ST: stroma; ED: endothelial layer. (E) Screenshots of eye microvascular dynamics in the selected area of conjunctival microvascular network. (F) The bulbar conjunctival vessel network image of the healthy subject. (G, H) The segmented vessel network image and the skeletonized vessel network image of the selected region.



图3 角膜炎患者多模态成像
Figure 3 Experimental results of the keratitis patient
(A)角膜炎受试者眼外观裂隙灯成像；(B)角膜炎受试者眼外观裂隙灯角膜荧光素染色成像；(C，D)OCT成像，放大区域清晰显示出角膜病灶位置及浸润深度(E)结膜微血管图选定区域的眼微血管动态视频截图；(F)角膜炎受试者结膜微血管图；(G，H)结膜微血管网络骨架化处理结果。
(A) Appearance image of the keratitis patient’s eye; the red dashed line indicates the OCT scanning position; (B) Fluorescein stain image of the keratitis patient; (C, D) OCT image of the inflamed cornea and the zoom-in area showing the highly reflective, inflamed lesion.(E) Screenshots of eye microvascular dynamics in the selected area of conjunctival microvascular network; (F) The bulbar conjunctival vessel network image of the keratitis patient; (G, H) Results of skeletonization of conjunctival microvascular network.


表1 血流形态学及动力学参数对比
Table 1 Comparison of the quantitative vessel parameters

3 讨论

    本研究首次在传统的裂隙灯显微镜系统上集成了UHR-OCT模块和微血管成像模块，开发了一种能够进行眼前段结构和功能成像的非接触多模态成像平台。成像平台可实现：1)利用UHR-OCT模块实时人眼角膜/结膜高分辨率OCT图像；2)利用微血管成像模块进行结膜血管网络成像和高分辨率结膜血流成像；3)使用裂隙灯显微镜模块进行荧光素染色和裂隙灯成像。针对多模态成像系统获取的血流图像，自主开发了图像分析软件来实现血管参数的定量分析。通过在健康的人体受试者和角膜炎患者进行的在体成像测试，证明了多模态系统的可行性。UHR-OCT模块用于高分辨率角膜结构成像，角膜组织轴向分辨率为约为2 μm。微血管成像模块横向分辨率约为3.5 μm，放大倍数达约为187.5倍，可分辨结膜微血管系统和移动的红细胞簇。在体成像测试已经成功地证明了该系统的可行性，显示了其在眼科临床应用的潜力。通过自主开发的图像分析件，进一步获得定量血流形态学和血流动力学参数，初步认识到健康受试者与角膜炎患者在血管密度、复杂性、血管直径和血流率方面的差异，符合炎症后血管扩张、扭曲和血流增加的临床表现[5-6]。    
    虽然多种多模态的眼科成像系统已被用于人眼成像，但一般只用于眼后段成像。与目前传统的眼科成像方式相比，我们自主研发的多模态眼前成像不仅可以通过UHR-OCT模块提供高于商业设备(轴向分辨率一般为5 μm)的结构信息，而且可以通过微血管成像模块提供球结膜血管的功能信息，具有多模态、多维度的成像功能，有望帮助眼科医生对眼前病变进行更全面的评估、更准确的诊断和定量分类。
    未来的系统改进工作将在UHR-OCT成像速度，实现实时三维成像等方面提升，另外，球结膜血管的形态学和血流动力学参数经验证可以与眼表疾病炎症的状态相关，还需要进行大规模临床研究全面描述这些定量参数之间的关系。此外，由于人工智能技术在眼科图像自动分析及眼科疾病诊断中得广泛应用[49-50]，多模态眼前段成像系统还可以为多模态人工智能应用提供图像数据支持。

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1、于薏,周奕文,杨燕宁.人工智能在眼表疾病中的应用现况[J].国际眼科杂志,2022,22(5):726-730.
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