资金来源来自波士顿医疗中心的埃文斯医疗基金会提供的资金, 以及来自 NIH 5R01CA183101、CTSI 试点赠款1UL1TR001430、乔斯林试点项目和 CTSI KL2TR001411 的分包合同。

这里描述的所有方法都得到了波士顿医疗中心动物护理和使用委员会 (ACUC) 的批准。
1. 系统设置
<ol><li>奥斯陆系统<ol>
<li>使用超连续谱激光源作为系统激光源。<ol>
<li>用分色镜 (DM1) 将可见光范围 (450-650 nm) 从较高波长范围 (650-2000 nm) 中分离出来。在光束通过偏振光束分配器 (PBS) 后, 用一对色散棱镜展开频谱。</li>
			<li>放置一个狭缝, 以选择激发波长范围 (475-495 nm)。使用反射镜将过滤后的光束反射回棱镜对, 然后将光耦合到单模光纤中 (SMF 1)。</li>
			<li>使用光谱仪来确认单模光纤输出的波长选择。</li>
		</ol>
</li>
		<li>将单模光纤连接到两个级联光纤耦合器, 如图 2所示。第二光纤耦合器中的光纤输出端口之一向奥斯陆系统提供光。</li>
		<li>瞄准首先在奥斯陆系统中进行激光。<ol>
<li>用振镜 (GM1) 偏转激光。通过1:1 望远镜系统将激光器中继到第二个振镜 (GM2), 并通过3:1 望远镜系统进一步中继到眼睛的瞳孔。</li>
			<li>在3:1 望远镜系统内安装一个分色镜 (DM2), 以反映荧光信号。</li>
		</ol>
</li>
		<li>在自定义的鸽子尾滑块上安装3:1 望远镜系统和分色镜 (DM2), 以抵消光轴并创建倾斜扫描照明, 如图 3所示。使用卡尺精确地控制偏移长度根据需要。</li>
		<li>荧光成像光路。<ol>
<li>用分色镜反射荧光, 并继电器到第三振镜, 以消除扫描慢扫描。</li>
			<li>用另外1:1 台望远镜系统将光传给成像物镜。在翻译阶段安装上述光学。 注: 在第三个振镜 (GM3) 下安装两个额外的平移阶段, 以提供在自由度上的冗余, 以优化成像。</li>
		</ol>
</li>
		<li>在具有三自由度 (旋转和两个平移轴) 的舞台上安装最终成像系统。使用平面相机捕捉截面荧光图像。</li>
	</ol>
</li>
	<li>光学相干层析成像系统<ol>
<li>使用相同的超连续谱激光源作为系统激光源。<ol>
<li>用另一种分色镜 (DM3) 将近红外线 (近红外) 范围 (650-900 nm) 与剩余光 (650-2000 nm) 分开。使用长通滤波器进一步限制带宽到 800-900 nm。将光束耦合成单模光纤 (SMF 2)。</li>
		</ol>
</li>
		<li>将单模光纤连接到两个级联光纤耦合器的另一个输入端口, 并与蓝色奥斯陆励磁相结合。将第二光纤耦合器的第二输出端口的光直接定向到 OCT 参考臂, 其中有一个可变中性密度滤波器 (VNDF)、色散补偿板和反射镜。 注: 在第二光纤耦合器上从参考臂和眼睛重组返回的光, 并传送到 OCT 光谱仪收集信号。</li>
	</ol>
</li>
	<li>数据采集<ol>
<li>使用在 LabVIEW 编写的数据采集系统软件, 并从奥克塔7、8、9、10的扫描协议中进行修改。对于每一个 B 扫描, 一个80% 工作周期锯齿与500步是输出的模拟输出板 (AO1), 以控制 x ' 快速扫描镜, GM2。</li>
		<li>仅当镜像处于正向扫描方向时, 在每个步骤中触发行扫描摄像机以获取 OCT 的数据。将线扫描摄像机的曝光时间设置为17µs。</li>
		<li>要获取奥克塔信号, 请在同一 B 扫描位置重复测量5次。</li>
		<li>将 AO 输出速率设置为100赫, OCT 的 A 线速率为50赫。控制 y ' 慢扫描镜, GM1, 由一个倾斜的波形。同步扫描镜像, GM3, 与 GM1, 以消除扫描慢扫描。</li>
		<li>通过另一个模拟输出板 (AO2) 触发平面摄像机, 在每个 y 位置捕获一个荧光图像。裁剪图像大小或 bin 相邻像素以提高所需的速度和灵敏度。</li>
	</ol>
</li>
</ol>2. 系统对齐
<ol><li>调整奥斯陆光源中的狭缝以选择蓝色激发波长。使用光谱仪监测光谱范围约475-490 纳米。</li>
	<li>调整鸽子尾部装入滑块, 将光轴移动5毫米。这将导致在老鼠瞳孔的偏移量为 ~ 1.7 毫米, 导致在视网膜上的斜角。</li>
	<li>用相同的5毫米调整荧光检测光学的平移阶段。</li>
	<li>将最终的荧光成像系统调整为 ~ 30°。</li>
	<li>使用电能表测量光功率。确保蓝色奥斯陆励磁功率为≤0.2 兆瓦和 OCT 激光功率≤0.8 兆瓦, 不会引起视网膜损伤。 注: 根据 ANSI 标准, 最大许可暴露 (MPE) 到视网膜的水平为2兆瓦7,8在可见光范围内。根据 Delori等公式。9、近红外光的 MPE 比可见光高约两倍, 约4兆瓦。</li>
</ol>3.活体动物实验
<ol><li>将12周长的埃文斯大鼠转移到感应室。用4.5% 异氟醚在氧气中麻醉10分钟, 用异氟醚蒸发器的流速为2升/分。<ol>
<li>确认麻醉深度, 这是指在指数捏中缺乏退缩反射所决定的。</li>
	</ol>
</li>
	<li>感应后, 把老鼠放在一个5轴 (x, y, z 平移, 偏航和音高) 持有人。通过加热阶段、循环温水毯或其他适宜的方法在长期试验中提供补充热量。保持麻醉在1.5% 的 isofluorane 与流速为2升/分钟在余下的部分的实验。当不使用具有主动排气的感应腔时, 感应腔应放置在回燃现象或下沉台或在通气管下以清除异氟醚。</li>
	<li>用 1% Tropicamide 眼科溶液扩张瞳孔2分钟。如有必要, 在大鼠眼中应用0.5% 丁卡因眼科溶液进行额外的局部麻醉。在实验中, 每分钟至少用一次商业人工泪让眼睛湿润。</li>
	<li>注射荧光素盐 (10% 瓦特/w) 或 FITC (10% 瓦特/w) 稀释在无菌生理盐水 (0.1-0.3 毫升) 通过尾静脉与1毫升注射器和29G 针。</li>
	<li>打开激光源。放置中性密度过滤器, 在对齐过程中衰减蓝光激发。测量 OCT 光的功率为0.8 兆瓦, 蓝光 < 0.01 兆瓦, 以避免白内障的形成。</li>
	<li>启动振镜扫描和对齐模式。调整眼睛球的高度, 使角膜上固定的激光点。调整大鼠的眼睛位置, 使瞳孔的边缘大致垂直于激光, 并将激光偏移到眼睛的顶端中心约1.5 毫米。</li>
	<li>进一步调整动物持有者, 直到 OCT 图像达到最佳质量。在 x ' 快速扫描方向, 确保横截 B 扫描图像显示为平面。当切换到 y ' 慢扫描方向, 确保横断面 B 扫描图像出现倾斜, 由于倾斜扫描。</li>
	<li>将中性密度滤波器移至蓝光励磁, 并监控摄像机的实时馈源。横断面荧光图像应显示出不同深度的血管。<ol>
<li>调整最终荧光成像系统的聚焦, 达到最佳聚焦。允许微调的眼睛位置在横向平面, 以达到最佳的奥斯陆图像质量。</li>
	</ol>
</li>
	<li>对齐后, 开始获得同时奥克塔和容量荧光素血管造影 (vFA)。</li>
	<li>利用 Matlab 构造奥克塔和奥斯陆的容积图像。这些算法以前是详细描述的10。通过图像分割生成深度分辨的视网膜 vasculatures。</li>
	<li>完成成像后, 关闭激光, 释放动物, 并在眼睛上涂抹一些眼膏, 然后将动物放在康复箱中。</li>
	<li>不要让动物无人看管, 直到它恢复了足够的意识, 以维持胸骨卧床或根据机构政策。</li>
</ol>

<ol>
	<li>Webb, R. H., Hughes, G. W., Delori, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+scanning+laser+ophthalmoscope.">Confocal scanning laser ophthalmoscope.</a> Applied Optics. 26 (8), 1492-1499 (1987).</li><li>Roorda, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+optics+scanning+laser+ophthalmoscopy.">Adaptive optics scanning laser ophthalmoscopy.</a> Optics Express. 10 (9), 405-412 (2002).</li><li>Huang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography.">Optical coherence tomography.</a> Science. 254 (5035), 1178-1181 (1991).</li><li>de Carlo, T. E., Romano, A., Waheed, N. K., Duker, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+optical+coherence+tomography+angiography+(OCTA).">A review of optical coherence tomography angiography (OCTA).</a> International Journal of Retina and Vitreous. 1 (1), 5 (2015).</li><li>Jia, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Optical+Coherence+Tomography+Angiography+of+Choroidal+Neovascularization+in+Age-Related+Macular+Degeneration.">Quantitative Optical Coherence Tomography Angiography of Choroidal Neovascularization in Age-Related Macular Degeneration.</a> Ophthalmology. 121 (7), 1435-1444 (2014).</li><li>Chen, C. -. L., Wang, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+based+angiography+[Invited].">Optical coherence tomography based angiography [Invited].</a> Biomedical Optics Express. 8 (2), 1056-1082 (2017).</li><li>Yi, J., Chen, S. Y., Shu, X., Fawzi, A. A., Zhang, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+retinal+imaging+using+visible-light+optical+coherence+tomography+guided+by+scanning+laser+ophthalmoscopy.">Human retinal imaging using visible-light optical coherence tomography guided by scanning laser ophthalmoscopy.</a> Biomedical Optics Express. 6 (10), 3701-3713 (2015).</li><li>Zhang, X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-band+spectral-domain+optical+coherence+tomography+for+in+vivo+imaging+the+spectral+contrasts+of+the+retinal+nerve+fiber+layer.">Dual-band spectral-domain optical coherence tomography for in vivo imaging the spectral contrasts of the retinal nerve fiber layer.</a> Optics Express. 19 (20), 19653-19667 (2011).</li><li>Delori, F. C., Webb, R. H., Sliney, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+permissible+exposures+for+ocular+safety+(ANSI+2000),+with+emphasis+on+ophthalmic+devices.">Maximum permissible exposures for ocular safety (ANSI 2000), with emphasis on ophthalmic devices.</a> Journal of the Optical Society of America a-Optics Image Science and Vision. 24 (5), 1250-1265 (2007).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volumetric+fluorescence+retinal+imaging+in+vivo+over+a+30-degree+field+of+view+by+oblique+scanning+laser+ophthalmoscopy+(oSLO).">Volumetric fluorescence retinal imaging in vivo over a 30-degree field of view by oblique scanning laser ophthalmoscopy (oSLO).</a> Biomedical Optics Express. 9 (1), 25-40 (2018).</li></ol>

济易持有奥斯陆的未决专利。其他作者声明没有竞争的财政利益。

在这里, 我们已经描述了奥斯陆, 一个在体内容积荧光视网膜成像技术与 FOV 超过了30°。与目前眼科护理成像方法标准的 OCT 相比, 奥斯陆提供了类似的3D 成像能力, 但也允许了 oct 不敏感的荧光对比度。奥斯陆的优势在于它只需要一个光栅扫描, 从而允许 OCT 的无缝组合, 为结构和荧光容积成像提供两种互补技术。
在该协议中, 获得良好图像质量的关键是大鼠眼的清晰度。如果奥斯陆图像被遮蔽, 检查是否有白内障形成。氯胺酮/甲苯噻嗪麻醉、角膜干燥、蓝光照射等几个因素会导致白内障的形成, 这将显著恶化图像质量。预防白内障, 避免连续暴露在蓝光中超过2分钟;至少每分钟涂抹一次人工眼泪, 以防止角膜干燥;并允许眼睛休息至少30秒的成像部分通过阻止光。
我们设想, 奥斯陆可以显著影响荧光成像的临床实践。结果表明, 深度分辨力能有效地消除外源视网膜的信号, 使高对比度的容积法图像下降到单毛细管水平, 这是传统斯洛伐克所无法获得的。显着改善的图像清晰度将允许更灵敏的检测和量化的血-视网膜屏障破坏和视网膜毛细血管渗漏, 标志性黄斑水肿在 DR 和其他脉络膜视网膜血管疾病。
在目前的系统中, CCD 摄像机的速度是每秒20帧, 导致 > 25 秒的采集时间。一个科学的 CMOS 相机将大大提高系统的速度。荧光检测具有与光照相分离的不同光路。在今后的设计中使用相同的光路进行光照和检测, 将简化系统。综述了一种新型的容积式视网膜成像多模态平台, 即斜扫描激光检眼镜 (奥斯陆)。应用奥斯陆和同时光学相干层析成像 (OCT) 对大鼠视网膜的 FOV 角进行体外成像。

眼科和视觉科学从现代光学成像技术中受益很大, 因为视网膜可以很容易地接触到光。荧光视网膜成像是诊断和治疗脉络膜视网膜血管疾病的重要工具, 如糖尿病视网膜病变 (DR) 和年龄相关的黄斑变性 (AMD), 这两者都是导致美国失明的主要原因。
然而, 利用荧光成像技术获得大视场 (FOV)、三维 (3D) 视网膜成像仍具有挑战性。眼底摄影没有深度分辨能力, 也不排斥漫射光。因此, 不同深度的信号混合可以降低图像质量。扫描激光检眼镜 (斯洛伐克) 和共焦斯洛伐克 (cSLO) 可通过共聚焦浇口1降低散射光的影响。然而, 由于其聚焦深度的限制, 斯洛伐克或 cSLO 很难获得3D 人的视网膜图像。自适应光学斯洛伐克 (AOSLO) 可以通过校正人眼所引入的波前畸变来提供高超的分辨率和对比度。然而, AOSLO 仍然需要 z 堆叠的容积成像2。光学相干层析成像 (oct)3和 oct 血管造影 (奥克塔) 系统克服这些限制, 提供三维 (3D) 解剖和血管图像 4,5,6, 但无染料的性质OCT 不能可视化泄漏指示血管功能障碍。
该协议描述了一种新的3D 容量荧光视网膜成像多模态平台, 即斜扫描激光检眼镜 (奥斯陆)。在这个成像系统中, 斜扫描是由鸽子尾滑块产生的, 最后的成像系统以角度对齐, 以检测荧光剖面图像。该系统采用激光扫描方法, 这些技术允许与 OCT 简单地结合, 作为一个互补的体积结构成像方式。目前, 大鼠视网膜的深度分辨率约为25µm, 视野为30°。从根本上说, 奥斯陆允许荧光版的 oct, 并可以同时结合 oct 和奥克塔在一个大 FOV。
在本议定书中, 我们将介绍奥斯陆的设置、对准和构造方法、大鼠视网膜活体成像方法和代表性结果。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Supercontinuum Laser Source</td>
			<td>NKT Photonics</td>
			<td>SuperK EXTREME EXU-OCT6</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichroic Mirror (DM1)</td>
			<td>Thorlabs</td>
			<td>DMLP650R</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichroic Mirror (DM2)</td>
			<td>Chroma</td>
			<td>ZT514/1064rpc</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichroic Mirror (DM3)</td>
			<td>Thorlabs</td>
			<td>DMLP900R</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Mode Fiber (SMF 1)</td>
			<td>Thorlabs</td>
			<td>P3-460B-FC-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Mode Fiber (SMF 2)</td>
			<td>Thorlabs</td>
			<td>P3-780A-FC-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Optic Fiber Coupler</td>
			<td>Thorlabs</td>
			<td>TW850R5A2</td>
			<td></td>
		</tr>
		<tr>
			<td>1:1 Telescope System</td>
			<td>Thorlabs</td>
			<td>AC254-100-A&#215;2</td>
			<td></td>
		</tr>
		<tr>
			<td>3:1 Telescope System</td>
			<td>Thorlabs</td>
			<td>AC254-150-A&#215;2</td>
			<td></td>
		</tr>
		<tr>
			<td>3:1 Telescope System</td>
			<td>Thorlabs</td>
			<td>AC254-50-A&#215;2</td>
			<td></td>
		</tr>
		<tr>
			<td>1:1 Telescope System</td>
			<td>Thorlabs</td>
			<td>AC254-100-A&#215;2</td>
			<td></td>
		</tr>
		<tr>
			<td>Galvo Mirrors (GM1,GM2)</td>
			<td>Thorlabs</td>
			<td>GVS201&#215;2</td>
			<td></td>
		</tr>
		<tr>
			<td>De-sacn Galvo Mirrors (GM3)</td>
			<td>Thorlabs</td>
			<td>GVS011</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective Lens</td>
			<td>Olympus</td>
			<td>UplanSApo 20&#215;/0.75</td>
			<td></td>
		</tr>
		<tr>
			<td>Final imaging system</td>
			<td>Olympus</td>
			<td>UplanFL N 10&#215;/0.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Final imaging system</td>
			<td>Computar</td>
			<td>12-36mm/1:2.8</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>PCO</td>
			<td>Pco.pixelfly usb</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Thorlabs</td>
			<td>FEL0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounted Continuously Variable ND Filter</td>
			<td>Thorlabs</td>
			<td>NDC-50C-4M-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Line Scan Camera</td>
			<td>Thorlabs</td>
			<td>SPL2048-140K</td>
			<td></td>
		</tr>
		<tr>
			<td>Analog Output Board (AO1)</td>
			<td>National Instrument</td>
			<td>PCI-6731</td>
			<td></td>
		</tr>
		<tr>
			<td>Analog Output Board (AO2)</td>
			<td>National Instrument</td>
			<td>PCIe-6351</td>
			<td></td>
		</tr>
		<tr>
			<td>Long pass filter</td>
			<td>Thorlabs</td>
			<td>FEL0800</td>
			<td></td>
		</tr>
	</tbody>
</table>

multimodal volumetric retinal imaging by oblique scanning laser ophthalmoscopy (oslo) and optical coherence tomography (oct)

虽然荧光成像在眼科广泛应用, 但大视野 (FOV) 三维 (3D) 荧光视网膜图像仍然是一个巨大的挑战与最新的视网膜成像方式, 因为他们需要 z 叠加编译容量数据集。新的光学相干层析成像 (oct) 和 oct 血管造影 (奥克塔) 系统克服了这些限制, 提供三维 (3D) 解剖和血管图像, 但 OCT 的无染料性质不能想象泄漏指示血管功能 障碍。该协议描述了一种新的斜扫描激光检眼镜 (奥斯陆) 技术, 提供3D 容量荧光视网膜成像。成像系统的设置产生斜扫描的鸽子尾巴滑块, 并使最终成像系统的角度, 以检测荧光横断面图像。该系统采用激光扫描方法, 因此, 允许简单地将 OCT 作为互补容积结构成像模式。对大鼠视网膜的活体成像进行了演示。荧光素溶液是静脉注射注射, 以产生容量荧光素血管造影 (vFA)。

图 4a显示了大鼠视网膜的横断面 OCT 图像。图 4b-4c显示同一时间获得的奥克塔和奥斯陆 vFA 相同的视网膜横断面图像。奥斯陆使横断面法类似于 OCT B 扫描。与奥克塔相比, 奥斯陆 vFA 横断面图像清楚地识别出神经纤维层 (NFL) 和神经节细胞层 (协鑫) 中的血管, 以及外层网状层 (OPL) 中的毛细血管。图 4d和4g显示了奥克塔和奥斯陆 vFA 图像的表层。与奥克塔不同, 奥斯陆 vFA 图像 (图 4g) 利用荧光发射对比度避免了运动工件 (图 4d中的垂直条纹)。通过比较奥斯陆 vFA (图 4e) 和奥克塔 (图 4h) 在视网膜中间层的图像, 垂直跳水船清楚地显示在奥斯陆 FA 图像, 但不明显在奥克塔。这大概是因为血流速度或血管定向会影响奥克塔信号, 而不是奥斯陆荧光对比度。
图 4f和4i显示了深毛细血管丛层内的图像。在奥斯陆 vFA 蓝色箭头指出的区域比奥克塔在脉管中有更好的对比。奥斯陆的白色箭头指出的静脉的大小比奥克塔的大。总的来说, 奥斯陆 vFA 图像比奥克塔更准确地与实际的血管形态学相似, 因为它不依赖于血流速度或血管方向。视频 1显示了从奥斯陆和奥克塔两个同时获得的容量数据集的en 面飞行。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57814/57814fig1.jpg"> 图1。系统示意图.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57814/57814fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57814/57814fig2.jpg"> 图2。两个级联光纤耦合器的照片, 直接奥斯陆和 OCT 光.光通行证的路线被标记。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57814/57814fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57814/57814fig3.jpg"> 图3。鸽子尾部安装的设置, 用于倾斜照明.(a) 斜光照部分的固体工作模型。(b)放大的视图, 和鸽子尾部安装的单独视图。(d) 拍摄斜照部分的实际设置。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57814/57814fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57814/57814fig4v2.jpg"> 图4。奥斯陆和奥克塔同时获得的大鼠视网膜图像.(a) 《华侨城》 (b) 奥克塔和 (c) 奥斯陆法的横断面图像的例子。面板 (d) 和 (g) 显示从表层的表面 OCT 和奥斯陆 FA 图像。用红色箭头指出了奥克塔图像中的运动伪影。面板 (e) 和 (h) 显示中间层的结果。船只潜入下一层的位置被黄色箭头指出, 奥斯陆法比奥克塔更清楚。面板 (f) 和 (i) 显示的结果, 从深毛细管丛层。奥斯陆的对比比奥克塔在蓝色箭头指出的区域要好。奥斯陆的白色箭头指出的静脉的大小比奥克塔的大。图表中的条形图为200µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57814/57814fig4v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT at JoVE.com

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

在这里, 我们提出了一个协议, 以获得一个大视野 (FOV) 三维 (3D) 荧光和 OCT 视网膜图像的新的成像多式联运平台。我们将介绍系统设置、校准方法和操作协议。在体内成像将被证明, 并有代表性的结果将提供。

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 ...

multimodal-volumetric-retinal-imaging-oblique-scanning-laser

Nanyang Technological University

Research

JoVE Journal

Bioengineering

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

8.4K 次观看.  Boston University School of Medicine. 在这里, 我们提出了一个协议, 以获得一个大视野 (FOV) 三维 (3D) 荧光和 OCT 视网膜图像的新的成像多式联运平台。我们将介绍系统设置、校准方法和操作协议。在体内成像将被证明, 并有代表性的结果将提供。

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

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 ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

宽带超声医疗成像应用，光学检测仪

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Biofunctionalized普鲁士蓝纳米粒子多式联运分子成像应用

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

计算机断层扫描和成骨血管生成耦合光学成像评估头盖骨自体移植和移植物的整合

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

基于光学相干断层扫描的可降解支架组织工程血管发育的无损监测

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

分离的月毛心室肌细胞的光学成像

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

近同步激光扫描在活细胞上的共和和原子力显微镜（康波卡尔）

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

用于血管可视化的双栅格扫描光声小动物成像仪

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...

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 ...