这项工作得到了国家眼科研究所 4K12EY022299 (YMP) 的慷慨支持, 为视力而战-国际视网膜研究基金会 FFS GIA16002 (YMP), 不受限制的部门支持从研究, 以防止失明, 和密歇根大学眼科和视觉科学系。这项工作利用了国家眼科研究所 P30 EY007003 资助的核心视觉研究中心。

兔子是美国农业部 (USDA) 覆盖的物种。它在生物医学研究中的应用需要遵循严格的规定。所有的兔实验都是按照帕特 (视觉和眼科研究协会) 在眼科和视力研究中使用动物的声明进行的, 这是在大学批准了实验动物协议后进行的。密歇根大学动物使用与护理委员会 (UCUCA) (协议 PRO00006486, PI Yannis)。
1. 系统配置
<ol><li>光声显微镜 (PAM)<ol>
<li>使用由二极管泵浦的固态激光器泵浦的光学参量振荡器 (参量) 作为 PAM 的光源. 选择合适的技术指标, 如脉冲重复率1赫、脉冲持续时间 3-6 ns 和可调谐波长范围 405-2600 nm。</li>
		<li>用两个镜 (M1 和 M2) 反射激光在 570 nm 的发射光束, 然后通过一个半波板衰减器安装在一个机动的旋转阶段, 最后聚焦, 过滤, 并准它由光束准直 (图 1)。优化光束准直器的设计。光束准直器的一个示例配置包括聚焦透镜 L1 (焦距 250 mm)、针孔 (直径50µm) 和准直透镜 L2 (焦距 30 mm)。</li>
		<li>将准直光束除以分光比为 90/10 (反射/透射) 的光束分配器 (BS1)。用光电二极管记录传输部分的脉冲-脉冲激光能量监测。连续偏转反射的部分由镜子 (M3) 和分色镜 (DM) 和光栅扫描它使用二维电流计。振镜是一个共享的组件与频谱域 (SD)-OCT 系统 (描述如下)。</li>
		<li>通过由扫描透镜 (焦距36毫米) 和眼科透镜 (OL, 焦距10毫米) 组成的望远镜传送扫描光束, 最后将其聚焦在兔眼光学的眼底。</li>
		<li>选择一个有适当技术规格的针形超声波换能器, 例如, 中心频率27兆赫, 双向 6 dB 带宽60%。将其与中央视轴上的结膜接触, 捕捉兴奋的光声信号。</li>
		<li>使用超声波放大器 (例如, 增益 57 dB) 放大信号, 通过低通滤波器 (例如, 截止频率 32 MHz) 对其进行过滤, 并以200毫秒/秒的采样速率将高速数字化器数字化。</li>
		<li>在兔眼上方放置一个电能表, 并测量在兔角膜上的激光脉冲能量, 使其低于 ANSI 安全限制160新泽西在 570 nm38。通过同步电子学, 用 Matlab 控制的激光快门来阻挡光束, 避免激光曝光。</li>
		<li>通过数据采集 (数据采集) 板同步激光器、振镜和数字化仪。编程软件进行系统控制和数据采集。</li>
	</ol>
</li>
	<li>光谱域光学相干层析成像 (SD OCT)<ol>
<li>在参考臂 (图 1) 中, 通过在扫描透镜 (SL) 和一块色散补偿玻璃 (DCG) 之后添加眼科透镜 (OL) 来适应基于商用系统的 SD OCT 系统。通过修改, OCT 系统可以实现对兔眼后段的图像处理。</li>
		<li>使用变焦外壳管调整参考臂的长度, 以确保它与样品臂的光学路径长度匹配。使用虹膜来控制反射光的亮度, 以确保其与背散射光强度的匹配, 从而达到最大的图像对比度。</li>
		<li>采用在扫描头上封装的电荷耦合器件 (CCD) 摄像机, 以照明发光二极管 (LED) 作为外部光源, 对兔眼底进行实时可视化。</li>
	</ol>
</li>
</ol>2. 系统对准
<ol><li>初始化电流计的位置, 并通过调整光纤准直器安装和分立方体的螺钉来对准 OCT 系统。 注: 分步程序可在商业获取的 OCT 系统手册中获得, 此处不包括。这一步骤主要是为了确保光纤准直器、参考臂和扫描透镜的正确对准, 以最大限度地提高 OCT 系统的性能。</li>
	<li>调整聚焦透镜焦点周围的针孔的x、 y和z位置, 以便在空间上滤除激光束, 并最大限度地传输激光能量。使用高度测量工具检查针孔前后的激光高度, 以确保它们是相同的。</li>
	<li>调整准直透镜 L2 的倾斜、偏心和z位置以准过滤光束。确保在近场和远场观测时, 光束的大小和高度大致相同。</li>
	<li>通过调整镜面和 DM 的倾斜, 将 PAM 激光光束和 OCT 光束进行共轴结合。在这一步之后, PAM 激光器和 OCT 光应该完全重合, 扫描区在兔眼底是相同的。</li>
	<li>调整 OL 透镜的倾斜和偏心, 使其在光学路径中正确对准。一旦完成, 双模态系统就可以成像了。 注: 你可以使用自动准直法来实现这一点, 即, 检查光线背面反射的 OL 透镜表面, 以确保它回去沿着同样的方式与入射光。</li>
</ol>3. 兔制剂
<ol><li>从动物设施中取一只新西兰白兔, 记录个人信息, 如体重和动物数量。</li>
	<li>山兔平台, 包括身体支持和头部支持的光学表下的成像系统。将水循环加热毯放在车身支撑上, 将循环水的温度设置为38° c, 以帮助保持家兔体温在实验和恢复期间的体温。</li>
	<li>记录手术前的生命体征, 包括总体动物状态、粘膜颜色、心率、呼吸速率和直肠体温。麻醉用氯胺酮 (40 毫克/千克) 和嗪 (5 毫克/千克) 的混合物通过肌肉注射和记录氯胺酮 (附表三控制物质) 的使用情况。通过检查心率、呼吸速率和整体状态来确认麻醉水平。</li>
	<li>放大兔学生使用一滴卡1% 眼科和肾上腺素盐酸2.5% 眼科。</li>
	<li>使用窥镜, 以保持眼睑的方式和应用一滴眼润滑剂滋润角膜。在成像程序之前, 在眼睛中灌输一滴局部丁卡因0.5%。 注: 对于动物可能不适的较长的程序或程序, 在实验前给兔子皮下注射昔以确保动物的舒适性。</li>
</ol>4. SD-OCT 成像
<ol><li>将兔子转移到临床眼底相机的成像平台上, 在 OCT 成像前进行50度眼底、无红色和自发荧光图像。这有助于检查眼睛的光学透明度和识别眼底血管的形态和标志, 如视神经和髓质视网膜血管。</li>
	<li>把兔子转移到 OCT 系统的平台上, 调整它的姿势, 大致定位在 OL 下的一只眼睛。用 LED 灯照亮眼睛。 注: 为方便临床翻译的技术, 兔眼不稳定使用任何其他方法和兔头是刚刚投入头部支持没有任何固定。</li>
	<li>打开 OCT 软件, 首先检查眼底的 CCD 摄像头图像。精细调整头部支持的高度和角度, 如果有必要, 以确保利益区 (roi), 如脉络膜血管和视网膜血管, 是在视野 (视) 的相机。 注意: 如果兔子处于良好的麻醉水平下, 一个连续的成像会话可以持续长达10分钟, 而无需重新调整兔头。</li>
	<li>画一条直线代表 OCT B 扫描兴趣并开始扫描。调整参考臂长度, 以可视化 oct 图像并优化 oct 软件中的色散补偿因子, 以获得最清晰的图像。 注意: 当调整参考臂长度时, 两个镜像的 OCT 图像将一个接一个地出现。正确的图像可以区分的基础上先验知识的眼底解剖。</li>
	<li>设置数据获取参数, 如像素数和平均值, 以及保存图像。</li>
	<li>观察家兔的呼吸速率和心率, 估计麻醉水平和动物舒适度。对于更长的疗程, 第三剂量的补充氯胺酮或吸入异氟醚可以考虑与气管插管, V 凝胶, 或口罩, 以维持在必要的麻醉平面。</li>
	<li>在实验中每2分钟用眼冲洗兔角膜, 以防止角膜表面脱水和角膜浅点上皮角膜。每15分钟监测和记录动物的生命体征。</li>
</ol>5. PAM 成像
<ol><li>戴上合适的激光安全护目镜, 打开参量激光器。</li>
	<li>启动 PAM 控制软件, 将激光波长调到目标团的一个吸收峰值 (例如, 570 nm 用于血红蛋白), 初始化电流计的位置, 并在兔角膜前监测激光能量, 以确保它低于 ANSI 安全限制。</li>
	<li>在三维 (3D) 平移阶段安装超声波换能器, 并将传感器尖端与指向眼底的兔结膜接触。使用一滴眼润滑剂更好地耦合传感器提示和兔结膜。</li>
	<li>打开 LED 照明灯, 通过 Matlab 软件对兔眼底进行可视化。</li>
	<li>设置扫描 ROI (即视网膜血管或脉络膜血管), 包括中心和物理尺寸。打开激光快门, 开始 B 扫描光束。通过大致校准传感器, 你应该能够看到在示波器上检测到的光声信号。如果没有, 稍微调整眼睛的位置, 以扫描不同区域的角膜或切换到另一只眼睛, 并重复上述过程。</li>
	<li>观察示波器上检测到的光声信号, 并微调传感器位置, 使整个 B 扫描信号强度最大化。 注: 由于光束宽度有限, 超声换能器通常有一个小视41。这一步决定了最终 PAM 图像的背景调制。不对称会导致 PAM 图像具有异构背景, 大大降低了图像质量。</li>
	<li>设置数据获取参数。这包括像素数 (e. g、256×256像素)、采样率 (e. g、200毫秒/秒) 和延迟时间。开始数据采集。当完成时, Matlab 软件将自动打开快门以通过激光束, 并关闭快门以阻止激光曝光。 注: 受激光脉冲重复率 (1 kHz) 的限制, 用 256 x 256 像素完成图像的数据采集需要大约1分钟。</li>
	<li>通过最大强度投影 (MIP)13或通过体积渲染38在3D 中处理原始数据并将 PAM 图像可视化为二维 (2D)。</li>
	<li>卸下超声波传感器, 用去离子水冲洗笔尖, 并将其放回存储箱。</li>
	<li>将兔子转移到眼底摄像机, 重新检查眼底。这一步有助于检查是否有任何形态学变化的眼底后, 成像会议。</li>
	<li>在实验中每两分钟用眼冲洗兔角膜, 以防止角膜表面脱水和角膜。每15分钟监测和记录动物的生命体征。 注: PAM、OCT 和眼底成像会议大约需要1小时。</li>
</ol>6. 后成像
<ol><li>眼底再检查后, 如果连接, 请断开 V 型凝胶。用眼冲洗眼睛, 应用芬眼和霉素, 素 B 硫酸盐和地塞米松眼膏, 闭上眼睛。</li>
	<li>将带水循环毯的兔子转移到回收室。把盒子从灯上屏蔽下来, 等到兔子自然苏醒。在此期间, 监测动物的生命体征每15分钟, 并保持记录, 并返回一份副本的动物设施, 保存。</li>
	<li>一旦兔子醒来, 是活跃的, 警觉和正常行走, 把它运回动物设施。如果一个急性实验计划, 安乐的动物使用安乐死解决方案 (e. g., Beuthanasia, 0.22 毫升/千克, 静脉注射在边缘耳静脉) 和处置的胴体。</li>
	<li>关闭软件和激光。清洁光学工作台。</li>
</ol>

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作者没有什么可透露的。

一个完整的和常规的泪膜是必不可少的高质量眼底图像。不规则和恶化的撕裂膜可以显著降低图像质量42。为了保持泪膜的完整性, 防止角膜表面有斑点的角膜, 重要的是用眼非常频繁地润滑角膜, 大约每两分钟。如果对眼睛的不透明度有任何顾虑, 请使用狭缝灯和荧光素条来检查角膜的状况。
较大的动物眼后段成像可能存在一些困难, 包括高频率分量、角膜?...

近几十年来见证了生物医学光声成像领域的爆炸性发展1,2,3,4,5,6,7 ,8。根据光的能量转换成声音, 新出现的微声成像可以可视化的生物样品从细胞器, 单元, 组织, 器官的小动物整个身体, 可以揭示其解剖, 功能, 分子, 遗传,和代谢信息1,2,9,10,11,12。光声成像在一系列生物医学领域中找到了独特的应用, 如细胞生物学13,14, 血管生物学15,16, 神经病学17,18, 肿瘤科19,20,21,22, 皮肤科23, 药理学24, 和血液病25,26。它在眼科的应用, 即光声眼部成像, 已经引起了科学家和临床医生的极大兴趣, 目前正在积极调查中。
与常规使用的眼部成像技术27不同, 如荧光血管造影 (FA) 和哚绿血管造影 (ICGA) (基于荧光对比), 光学相干层析 (OCT) (基于光学散射对比), 其衍生物 OCT 血管造影 (基于红细胞的运动对比), 光声眼成像以光学吸收为对比机制。这是不同于传统的眼科成像技术, 并提供了一个独特的工具, 研究光学吸收特性的眼睛, 这通常是与病理生理状态的眼部组织28。到目前为止, 在光声视觉成像中已经做了大量的出色工作29,30,31,323334 36,37, 但这些研究集中在小动物的眼球后段, 如老鼠和老鼠。开创性的研究充分证明了眼科光声成像的可行性, 但是, 由于大鼠和小鼠眼球尺寸小 (小于 1/3), 临床翻译技术还有很长的路要走。人类。由于超声波在较长距离上传播, 当使用这种技术来成像大眼睛后段时, 信号强度和图像质量会受到很大的影响。
为了达到这个目标, 我们最近报道了使用集成光声显微镜 (PAM) 和光谱域 oct (SD oct)38的活兔无创无标签脉络成像。该系统具有优异的性能, 可根据眼组织的内源吸收和散射对比, 对大动物眼的视网膜和脉络膜进行可视化。家兔的初步结果表明, PAM 可以非区分个别视网膜和脉络膜血管使用激光照射剂量 (〜80新泽西州) 显著低于美国国家标准学会 (ANSI) 安全限制 (160 新泽西州) 在570nm39;OCT 可以清楚地分辨出不同的视网膜层、脉络膜和巩膜。这是第一次演示的大动物后段成像, 使用 PAM, 可能是一个重要的步骤, 以临床翻译的技术考虑到兔眼球大小 (18.1 mm)40是几乎80% 的轴向长度人 (23.9 毫米)。
在这项工作中, 我们提供了一个详细的描述的双模态成像系统和实验协议, 用于无创, 无标签的脉络成像在活兔和演示系统性能通过代表性的视网膜和脉络膜成像结果。

<table><tbody><tr><td>&lt;strong&gt;Dual-modality imaging system&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>OPO laser</td><td>Ekspla (Vilnius, Lithuania)</td><td>NT-242</td><td></td></tr><tr><td>Beam attenuator</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>AHWP10M-600</td><td></td></tr><tr><td>Motorized rotation stage</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>PRM1/MZ8</td><td></td></tr><tr><td>Motorized rotation stage controller</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>TDC001</td><td></td></tr><tr><td>Focusing lens</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>AC254-250-B</td><td></td></tr><tr><td>Pinhole</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>P50S</td><td></td></tr><tr><td>Collimating lens</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>AC127-030-B</td><td></td></tr><tr><td>Photodiode</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>PDA36A&amp;nbsp;</td><td></td></tr><tr><td>Laser shutter</td><td>Vincent Associates Inc. (Toronto, Canada)</td><td>LS6S2T0</td><td></td></tr><tr><td>Laser shutter driver</td><td>Vincent Associates Inc. (Toronto, Canada)</td><td>VCM-D1</td><td></td></tr><tr><td>Dichroic mirror</td><td>Semrock, Inc. (Rochester, NY, USA)</td><td>Di03-R785-t3-25&amp;times;36</td><td></td></tr><tr><td>Scan lens</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>OCT-LK3-BB</td><td></td></tr><tr><td>Ophthalmic lens</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>AC080-010-B-ML</td><td></td></tr><tr><td>Ultrasonic transducer</td><td>Optosonic Inc. (Arcadia, CA, USA)</td><td>Custom</td><td></td></tr><tr><td>Amplifier</td><td>L3 Narda-MITEQ (Hauppauge, NY, USA)</td><td>AU-1647</td><td></td></tr><tr><td>Band-pass filter</td><td>Mini-Circuits (Brooklyn, NY, USA)</td><td>BLP-30+</td><td></td></tr><tr><td>Digitizer</td><td>DynamicSignals LLC (Lockport, IL, USA)</td><td>PX1500-4&amp;nbsp;</td><td></td></tr><tr><td>Synchronization electronics</td><td>National Instruments Corporation (Austin, TX, USA)</td><td>USB-6353</td><td></td></tr><tr><td>OCT module</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>Ganymede-II-HR</td><td></td></tr><tr><td>Dispersion compensation glass</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>LSM03DC</td><td></td></tr><tr><td>Illumination LED light</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>MCWHF2&amp;nbsp;</td><td></td></tr><tr><td>Power meter</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>S121C&amp;nbsp;</td><td></td></tr><tr><td>Power meter interface</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>PM100USB&amp;nbsp;</td><td></td></tr><tr><td>Height measurement tool&amp;nbsp;</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>BHM1</td><td></td></tr><tr><td>Fundus camera</td><td>Topcon Corporation (Tokyo, Japan)&amp;nbsp;</td><td>TRC 50EX</td><td></td></tr><tr><td>Matlab</td><td>MathWorks (Natick, MA, USA)</td><td>2017a</td><td></td></tr><tr><td>Oscilloscope</td><td>Teledyne LeCroy (Chestnut Ridge, NY, USA)</td><td>WaveJet 354T</td><td></td></tr><tr><td>&lt;strong&gt;Animal experiment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Water-circulating blanket</td><td>Stryker Corporation (Kalamazoo, MI, USA)</td><td>TP-700</td><td></td></tr><tr><td>Ketamine hydrochloride injection</td><td>Par pharmaceutical, Inc. (Woodcliff Lake, NJ, USA)</td><td>NDC code 42023-115-10</td><td></td></tr><tr><td>Xylazine hydrochloride</td><td>VetOne (Boise, ID, USA)</td><td>NDC code 13985-704-10</td><td></td></tr><tr><td>Tropicamide ophthalmic</td><td>Akorn Pharmaceuticals Inc. (Lake Forest, IL, USA)</td><td>NDC code 17478-102-12</td><td></td></tr><tr><td>Phenylephrine hydrochloride ophthalmic</td><td>Paragon BioTeck, Inc. (Portland, OR, USA)</td><td>NDC code 42702-102-15</td><td></td></tr><tr><td>Eye lubricant</td><td>Hub Pharmaceuticals LLC (Rancho Cucamonga, CA, USA)</td><td>NDC code 17238-610-15</td><td></td></tr><tr><td>Eyewash</td><td>Altaire Pharmaceuticals, Inc. (Aquebogue, NY, USA)</td><td>NDC code 59390-175-18</td><td></td></tr><tr><td>Tetracaine hydrochloride ophthalmic solution</td><td>Bausch &amp;amp; Lomb, Inc. (Rochester, NY, USA)</td><td>NDC code 24208-920-64</td><td></td></tr><tr><td>Flurbiprofen sodium ophthalmic solution</td><td>Bausch &amp;amp; Lomb, Inc. (Rochester, NY, USA)</td><td>NDC code 24208-314-25</td><td></td></tr><tr><td>Neomycin and Polymyxin B Sulfates and Dexamethasone Ophthalmic Ointment</td><td>Bausch &amp;amp; Lomb, Inc. (Rochester, NY, USA)</td><td>NDC code 24208-795-35</td><td></td></tr><tr><td>Meloxicam injection</td><td>Henry Schein Inc. (Queens, NY, USA)</td><td>NDC code 11695-6925-1</td><td></td></tr></tbody></table>

novel photoacoustic microscopy and optical coherence tomography dual-modality chorioretinal imaging in living rabbit eyes

光声眼部成像是一种新兴的眼科成像技术, 它可以通过将光能转换成声波来非可视化的眼部组织, 目前正在进行密集的调查。然而, 迄今为止报告的大部分工作都是针对小动物 (如大鼠和小鼠) 眼后段的成像, 这对由于眼球尺寸小而对临床人的翻译造成了挑战。这篇手稿描述了一个新的光声显微镜 (PAM) 和光学相干断层扫描 (OCT) 双模系统的后段成像的大动物的眼睛, 如兔。该系统配置, 系统对准, 动物准备, 和双模态实验协议为在体内, 无创, 无标签的脉络成像在兔子是详细的。该方法的有效性通过代表性的实验结果证明, 包括由 PAM 和 OCT 获得的视网膜和脉络膜血管。这篇手稿提供了一个实用的指南, 以再现兔的成像结果和推进大动物的光声眼部成像。

双模态成像系统和实验协议已成功地在作者的实验室使用四新西兰白兔进行了测试。下面展示了一些代表性的结果。
图 1显示了 PAM 和 SD OCT 双模态成像系统的示意图。它由以下几个模块组成: 光声光源、可变激光衰减器、光束准直仪、能量计、扫描头、光电探测和采集模块、OCT 单元和同步电子学。详细?...

Watch this Scientific Journal Video about Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes at JoVE.com

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

这份手稿描述了新的设置和操作程序的光声显微镜和光学相干断层扫描双模系统的无创, 无标签的脉络成像大动物, 如兔。

新型光声显微镜与光学相干层析成像在活兔眼中的双模脉络成像

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into ...

novel-photoacoustic-microscopy-optical-coherence-tomography-dual

Research

JoVE Journal

Engineering

10.8K 次观看.  University of Michigan. 这份手稿描述了新的设置和操作程序的光声显微镜和光学相干断层扫描双模系统的无创, 无标签的脉络成像大动物, 如兔。

视频: Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

集成光声眼底镜检查和谱域光学相干断层扫描

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

科研

生物工程

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

斜扫描激光检眼镜 (奥斯陆) 与光学相干层析成像 (OCT) 的多模态视网膜成像

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

使用光学相干断层扫描对啮齿动物模型中眼部疾病的体内结构评估

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

超高分辨率小鼠光学相干层析成像辅助眼内注射在视网膜基因治疗中的应用

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

医学

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

在体内图像的Cx3cr1gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

神经科学

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

光学相干断层扫描在视网膜病变小鼠模型中的应用

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

生物学

Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding and treatment of ocular diseases. This study introduces an optimized adeno-associated virus (AAV) approach, utilizing specific serotypes and promoters to achieve optimal transfection efficiency in targeted retinal cells, including retinal ganglion cells (RGCs) and M&#252;ller glia. Leveraging the precision of confocal scanning laser ophthalmoscopy (CSLO), this work presents a non-invasive method for in vivo imaging that captures the longitudinal expression of AAV-mediated green fluorescent protein (GFP). This approach eliminates the need for terminal procedures, preserving the continuity of observation and the well-being of the subject. Furthermore, the GFP signal can be traced in AAV-infected RGCs along the visual pathway to the superior colliculus (SC) and lateral geniculate nucleus (LGN), enabling the potential for direct visual pathway mapping. These findings provide a detailed protocol and demonstrate the application of this powerful tool for real-time studies of retinal cell behavior, disease pathogenesis, and the efficacy of gene therapy interventions, offering valuable insights into the living retina and its connections.

This protocol describes the use of adeno-associated virus (AAV) vectors for cell-specific labeling and in vivo imaging using a confocal scanning laser ophthalmoscope (CSLO). This method enables the investigation of different retinal cell types and their contributions to retinal function and disease.

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding ...