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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

Published: February 08, 2018
doi:
10.3791/57135
, , , ,
1Kellogg Eye Center, Department of Ophthalmology and Visual Sciences,University of Michigan, 2Department of Biomedical Engineering,University of Michigan, 3Institute of Biomedical Engineering,Chinese Academy of Medical Sciences & Peking Union Medical College, 4Department of Radiology,University of Michigan

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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 光声显微镜 (PAM) 使用由二极管泵浦的固态激光器泵浦的光…

Representative Results

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?…

Discussion

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?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

Keywords: Photoacoustic MicroscopyOptical Coherence TomographyDual-modality ImagingChorioretinal ImagingRabbit EyesNon-invasiveLabel-freeOphthalmic ImagingOxygen SaturationBlood DistributionMelaninOptical Parametric Oscillator LaserBeam SplitterTwo-dimensional GalvanometerTelescopeOphthalmic LensWater Circulating Heating BlanketPupil DilationUltrasonic AmplifierLow Pass FilterAmerican National Standards InstituteSpectral Domain OCT
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Tian, C., Zhang, W., Nguyen, V. P., Wang, X., Paulus, Y. M. Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes. J. Vis. Exp. (132), e57135, doi:10.3791/57135 (2018).
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