作者承认来自 NSERC 发现赠款 (#05573) 和加拿大创新基金会 (#222084) 的研究经费用于资助活体微 CT. w 病房是加拿大骨骼和肌肉发育方面的研究椅。

这项研究得到了布洛克大学动物保育委员会的批准, 并按照加拿大动物保育理事会制定的指导原则21进行。
1. 使用异氟醚气体进行麻醉
<ol><li>预填充丙烯酸玻璃孵化室与高品质的 O2在一个连续的流速约1-2 升/分钟从一个麻醉机 (补充图 1)。</li>
	<li>先将大鼠转移到孵化室尾部, 然后关闭孵化室盖, 形成密闭密封。</li>
	<li>开始填补孵化室与兽医级的异氟烷在3-4% 伏/v 溶解在 O2在连续流速为1-2 升/分钟 (补充图 1)。 注意: 废弃的麻醉气体会对处理程序产生不利影响。一个清道夫系统 (即, 木炭过滤器或排气直接进入油烟罩) 必须始终到位。</li>
	<li>当老鼠不再能够站立, 将大鼠转移到面罩或鼻锥接受1-3% 的异氟醚在 O2中溶解, 流速为1-2 升/分. 大鼠主要通过鼻子呼吸, 所以, 只要鼻子被面罩或鼻子覆盖锥, 将有足够的麻醉分娩。</li>
	<li>将眼部润滑应用于眼部细腻的细胞膜上, 以保护它们免受任何逃逸的异氟醚气体。 注意: 确保眼部润滑无抗生素, 因为这可能影响干预的结果。</li>
	<li>测量眼睑 (眼睛闪烁反应, 轻柔刺激眼睑开口) 和踏板 (对捏的反应的后肢撤退);随着麻醉深度的增加, 在踏板反射之前眼睑反射将缺席 (补充图 2)。</li>
	<li>当达到适当的麻醉水平和鼠已经失去了眼睑和踏板反射, 维持在 0.5-2% 的异氟醚溶解在 O2在流量为1-2 升/分钟的大鼠。</li>
	<li>持续监测鼠的呼吸率整个过程中, 保持恒定的视觉上的鼠直接与内部监测系统或通过直播视频饲料 (补充图 3)。</li>
</ol>2. 大鼠后肢的定位和抑制
<ol><li>将老鼠放在碳纤维扫描仪床上的仰卧位上 (补充图 4)。</li>
	<li>将右脚放在可锻的泡沫管中, 脚趾伸出管子的末端。用牙蜡把脚紧紧地抱在泡沫内, 然后把管子紧紧地关上。确保保持脚的管子的直径足够牢固地装入塑料管。</li>
	<li>将塑料管滑入 X 射线扫描仪床 (补充图 5)。</li>
	<li>延长鼠后肢, 直到它绷紧。不要 over-extend 腿对老鼠造成任何伤害 (补充图 5), 因为这可能会导致肢体由于呼吸的剧烈运动而引起非自愿的移动。</li>
	<li>拉左腿 (non-scanned 后肢) 与尾巴出扫描视野和朝向躯干, 远离要扫描的扩展腿. </li>
	<li>用掩蔽胶带固定左腿 (non-scanned 后肢) 和位置的尾部。不要使用任何更多或更少的粘性 (即, 管道胶带或画家的磁带), 因为这些材料会伤害鼠时, 他们被删除 (胶带) 或不提供足够强大的持有 (画家的磁带) (补充图 6)。</li>
	<li>将老鼠的身体固定在臀部、肩部和头部, 并用掩蔽胶带。将面罩或鼻锥固定在鼠身上 (补充图 6)。 注: 涂抹胶带的粘合面, 以消除其粘附在鼠毛上的能力。不要涂抹遮蔽胶带的两端, 这样它就可以牢固地固定在扫描床上。</li>
	<li>将大鼠裹在兽医包中以限制热量损耗 (补充图 6)。 注意: 在全身麻醉下, 大鼠由于其大的表面重量比19,20而迅速失去热量。</li>
	<li>通过对大鼠 (直接或通过视频直播) 保持恒定的视觉效果, 在整个手术过程中持续监测大鼠的呼吸速度。 注意: 这里设置了5分钟, 扫描采集是依赖于采集设置, 恢复时间为60分钟。</li>
	<li>继续获取µCT 图像。 注意: 扫描采集的精确规范是针对每种扫描仪类型、软件系统和具体的研究问题, 但是, 在文献中存在着几种方法学出版物1,2,9。</li>
</ol>3. 麻醉恢复
<ol><li>在体内µCT 扫描完成后, 停止对鼠的异氟醚的流动, 但保持 O2的1-2 升/分钟流量。</li>
	<li>当老鼠恢复马达控制 (1-2 分钟), 把它从呼吸器中取出, 并允许它单独在一个 general-purpose 加热垫上放置在低热的笼子里。已知大鼠在全身麻醉下的体温在1° c 以下时会降低体温19。不要让老鼠无人看管, 直到它恢复了足够的意识来维持胸骨卧床。 注意: 从我们的研究小组的轶事证据报告说, 立即从异氟醚麻醉恢复后, 大鼠开始吃, 所以重要的是他们的食物和水可供他们在恢复期间。虽然我们已经观察到这种行为, 重复全身麻醉不会引起食物摄入量或体重的显著增加1,9。</li>
</ol>

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作者没有什么可透露的。

该协议为观众提供了第一个详细的指导方针, 正确的麻醉, 放置和约束的大鼠在在体内µCT 扫描的后肢肢体。这些指南使在体内µCT 扫描系统的用户能够获得高分辨率和高质量的胫骨图像, 可用于量化3维骨微结构。必要的关键步骤, 以确保适当的定位和克制, 包括适当的麻醉的大鼠, 以及延长后肢远离所有其他关键结构, 直到它是绷紧, 但不是在一个不自然的立场。为了获得最佳的影像学结果, 必须将大鼠麻醉到完全放松的程度, 并失去眼睑和踏板的反射。此外, 扫描腿应延长, 整个脚和踝应克制在泡沫。上述方法, 以实现扫描腿的最佳定位, 将确保: 1) 研究中的大鼠后肢在同一方向上始终如一地定向, 从而允许 x 射线束穿过每条腿的同一区域, 因为它旋转在样品附近;2) 不会发生后肢的自发和非自愿运动, 从而最大限度地减少运动工件干扰所采集图像质量的可能性;3) 防止物体的障碍物 (即、尾部), 从而最大限度地减少部分体积效应的可能性, 从而产生不精确的 BMD 和 TMD 测量。这些指南可能会被修改为每一个单独的大鼠, 因为异氟醚的代谢和定位的速度取决于应变和体型大小22。最常见的在体内扫描机器是专为小动物模型 (即, 小鼠, 大鼠, 兔, 豚鼠) 和将有可互换的动物阶段, 允许扫描不同的动物大小。因此, 他们可以容纳广泛的身体重量。
虽然在体内µCT 扫描允许老鼠重新定位和扫描如果从初始扫描获得的图像质量较差, 重复扫描将使大鼠暴露于额外剂量的辐射和异氟醚麻醉 一段长时间。每月重复照射 600 mGy 的大鼠胫骨四月以上对骨微结构没有造成不良影响, 与对侧后肢的1相比, 但这并不能确定两次扫描重复的安全性立即继承。所述技术的进一步限制包括需要延长后肢绷紧的力量, 使其保持静止, 这可能会导致骨骼结构的某些变化。虽然在扫描过程中后肢的限制的严重性取决于每个研究目标, 以前的研究从我们的实验室每月重复在体内µCT 成像的一个后肢导致了不同的皮质微结构参数, 偏心, 相对于对侧后肢, 没有经过重复的扩展, 稳定和扫描1。偏心是一个测量的椭圆形形状的皮质骨和变化的反应, 改变 load-bearing。因此, 当使用这种定位和限制后肢的方法重复在体内µCT 成像时, 应考虑在评估和解释对 load-bearing 微结构参数的变化。
虽然已为骨组织的成像和分析提供了上述的指导方针, 但在对后肢的软组织进行成像时, 必须对该协议作轻微的调整。具体而言, 必须考虑到后肢从躯干延伸和克制的方式, 因为目前的程序畸形在扫描期间将软组织 (肌肉、脂肪组织) 定位为异常定位。因此, 当外推这个模型用于成像软组织的后肢, 应作出一些调整的限制技术, 以减少或消除改变的组织定位的相互关系。
此外, 这些指导方针是根据我们研究小组的经验专门编写的, 但是, 它们可以被修改以适应其他商业上可用的在体内µCT 扫描仪。其他建议的方法来定位和限制后肢可能是由制造商提供的在体内µCT 扫描系统。最商业化的在体内µCT 单位列出聚丙烯, 扩大聚苯乙烯, 和塑料管与牙科蜡举行一个突出的脚作为可接受的材料和方法, 以抑制扫描腿。然而, 该协议提供了更有控制和一致的定位和约束的扫描腿, 并始终如一地产生高质量的图像。本方法所提出的准则要求对大鼠进行麻醉所需的专用设备, 如汽化器、管子、口罩、感应室和氧气。虽然与注射麻醉剂相比, 该设备的成本略高, 但它使研究人员能够迅速和精确地诱导麻醉在特定深度的意识, 这提供了比替代的优势方法.
使用本方法视频中概述的指南, 研究人员利用高分辨率的在体内µCT 技术来调查他们对兴趣的干预将能够正确和始终如一地定位和抑制大鼠后肢的高高质量的 x 射线成像。这将提供一个连续的领域内的在体内µCT 图像获取, 并作为一个步骤, 以优化的一致性和准确性的研究, 并使比较跨研究的文献。同样, 这些协议和方法可以扩展, 以用于其他啮齿动物物种, 包括小鼠, 虽然一些变化将需要2,10。例如, 在泡沫管脚的克制可以包括脚踝, 以尽量减少腿部移动的可能性, 在扫描。此外, 充分的脚将适合于泡沫持有人。因此, 脚趾不伸出的结束的持有人, 因为他们做时, 保护一只老鼠的脚。此外, 鼠标的身体不需要同样的约束与磁带的老鼠。在扫描过程中, 小的鼻锥可以用来维持小鼠的麻醉。如果没有更小的鼻锥, 你可以在可用的鼻锥上固定一个丁腈手套, 并在手套上做一个小切口, 提供一个空间, 使它能适合老鼠的鼻子提供麻醉, 同时保持鼻子周围的密封。
虽然胫骨近端是研究大鼠骨微结构改变的主要部位, 但对其他骨骼部位 (如股骨和腰椎) 的正确和一致定位的指导原则应进行调查, 并建立文学中的一致性。然而, 在进行未来的研究涉及腰椎成像, 必须考虑到脊柱成像提供辐射接触周围的器官和组织。

使用在体内微计算机断层扫描 (µCT) 是一个强大的工具, 包括非破坏性成像的内部结构在高分辨率使用啮齿类动物模型。非破坏性的性质,在体内µCT 允许重复成像相同的啮齿动物随着时间的推移。这一特性不仅减少了实验设计中所需的啮齿动物总数, 从而减少了学科的变异, 而且也使研究人员能够理解对干预的长期反应。随着使用重复的在体内µCT, 实验鼠和大鼠已阐明发展变化的骨骼微结构和骨密度 (BMD) 在整个生命周期1,2,3 ,4,5,6,7,8以及对干预的骨骼健康响应, 如节食9,10,卵巢切除7,11和药理剂8,12,13。骨密度和骨微结构的具体骨骼部位, 即胫骨近端, 股骨和腰椎, 是指整体骨骼健康和维持骨折的风险, 所以在量化反应的主要措施干预.
在体内µCT 图像采集涉及 two-dimensional x 射线投射在多个角度获取, x 射线源和探测器围绕被调查的动物旋转, 14,15。结果图像的质量取决于许多因素, 包括但不限于: 选定的采集参数 (即、空间分辨率、X 射线电压、安培量、旋转步骤、应用滤镜、曝光时间)、µCT 的限制扫描仪 (即, scanner-based 工件, 如环形工件或灰尘, 造成裸奔或部分体积的影响) 和适当的定位和约束的动物。这些因素中的前两个可以由用户在某种程度上纵 , 这取决于特定的扫描机、研究目标以及优化扫描仪功能所需的校正或获取的图像的处理。后者的这些因素, 正确定位的啮齿目动物在扫描之前, 可以实现, 无论是 scanner-based 的限制或获取参数, 被选中, 以达到一个特定的研究目标。虽然许多出版物涉及活体成像已经发表在文献14,15,16,17, 经典的手稿风格是这样详细的 "如何" 信息不能包括在内。因此, 本文和视频指南的目的是填补这一空白。在这里, 我们的目的是指导用户的在体内µCT 扫描仪如何麻醉鼠, 并定位和抑制后肢, 以产生高质量的图像, 可以分析, 以更准确地量化的结果骨微结构。
为了量化最精确的骨密度和骨骼微结构值, 防止 x 射线束通过后肢以外的物体的障碍物是必不可少的。当 x 射线通过不同厚度和密度的物体和组织时, 一些 x 射线被它们通过的材料吸收 (即衰减)。由于样品的质量密度受其厚度和周围组织的存在和厚度的影响, 因此必须以同样的方式扫描用于确定 BMD 的校准幻影。因此, 如果 x 射线光束是通过对象 (即、尾部) 穿过感兴趣区域之前或之后, 这些对象将吸收一些 x 射线能量, 并会干扰所获得的传输图像。此外, 这些扫描将很难模拟时, 扫描的幽灵, 必须密切类似样本扫描。因此, 这些衰减差异导致骨密度测量的不精确性。因此, 为了方便和准确, 最好限制 x 射线源、感兴趣区域和 x 射线探测器之间的障碍物数量。
骨结构的纵向评估从干预的临床前模型涉及重复麻醉的动物, 以限制他们的运动在扫描协议。为了制服接受µCT 扫描的动物, 有几种全身麻醉方法, 包括注射和吸入麻醉1,2,4,5,6,12. 不同于吸入麻醉剂, 如异氟醚, 重复全麻使用可注射麻醉剂导致体重减轻, 手术耐受性和其他生理参数的显著变化, 特别是大鼠和豚鼠, 建议严重禁忌症重复使用18,19,20。虽然异氟醚是高度挥发性和允许快速诱导和恢复, 注射麻醉剂产生不同程度的麻醉和时间在麻醉下取决于应变, 性别, 身体组成, 禁食状态, 和昼夜循环动物.注射麻醉药也对其使用造成额外的障碍, 因为它们受到国家管理机构的高度管制。吸入麻醉然而, 涉及直接分娩进入呼吸系统;这种方法允许更快的诱导和恢复时间, 更好地控制麻醉的长度和深度19,20。吸入麻醉方法的局限性包括其对专用汽化设备的要求以及感应、维护和恢复过程中心率和血压的一些变化18,19。

<table>
	<tbody>
		<tr>
			<td>Isoflurane</td>
			<td>Fresenius Kabi Animal Health</td>
			<td>108737</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer</td>
			<td>Dispomed</td>
			<td>990-1091-3SINEWA</td>
			<td></td>
		</tr>
		<tr>
			<td>Scavengers/Charcoal Filters</td>
			<td>Dispomed</td>
			<td>985-1005-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-CT Scanner</td>
			<td>Bruker microCT</td>
			<td>SkyScan 1176</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental wax</td>
			<td>Kerr Dental Laboratory</td>
			<td>623</td>
			<td></td>
		</tr>
		<tr>
			<td>Foam (Backer Rod)</td>
			<td>Rona</td>
			<td>CF12086</td>
			<td>1&#8221;x10&#8217;</td>
		</tr>
		<tr>
			<td>Plastic tube</td>
			<td>Bruker microCT</td>
			<td>SP-3010</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon-fiber bed</td>
			<td>Bruker microCT</td>
			<td>SP-3002</td>
			<td></td>
		</tr>
		<tr>
			<td>Vet Wrap/Bandage</td>
			<td>Dura-Tech</td>
			<td>17473</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic Gel</td>
			<td>OptixCare</td>
			<td>006CLC-4256</td>
			<td>Antibiotic-free</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Sunbeam</td>
			<td>000731-500-000</td>
			<td></td>
		</tr>
	</tbody>
</table>

proper positioning and restraint of a rat hind limb for focused high resolution imaging of bone micro-architecture using in vivo micro-computed tomography

使用在体内微计算机断层扫描 (µCT) 是一个强有力的工具, 涉及在高分辨率的活体动物模型的内部结构的无损成像。这允许反复成像相同的啮齿动物随着时间的推移。这一特性不仅减少了实验设计中所需的啮齿类动物总数, 从而减少了学科的变异, 同时也使研究人员可以评估对干预的纵向或 life-long 反应。为了获得高质量的图像, 可以进行处理和分析, 以便更准确地量化骨微结构的结果,在体内µCT 扫描仪的用户必须正确麻醉鼠, 并定位和抑制后肢。要做到这一点, 就必须把老鼠麻醉到完全放松的程度, 而踏板反射就会丢失。这些指南可能会被修改为每一个单独的大鼠, 因为异氟醚的代谢速率可以根据应变和身体大小而变化。适当的技术为在体内µCT 图像采集能够准确和一致的测量骨微结构内和跨研究。

这种麻醉方法的大鼠, 并定位和约束的后肢为在体内µCT 成像有助于获得高质量的图像适合分析胫骨微结构。对大鼠后肢的正确定位包括腿部被完全伸展, 整个脚和踝部被抑制在泡沫中 (图 1A), 从而获得足够质量的图像来分析小梁和皮质微结构 (图 1B)。后肢的位置和限制不足 (图 1C) 可能会导致带有移动工件的图像 (图 1D), 而未完全从视图扫描字段 (图 1E) 中删除的尾部将干扰 X 射线通过扫描样本 (图 1F) 进行衰减, 并改变 BMD 和组织矿物密度 (TMD) 测量值。这些放置错误中的任何一个都将导致质量较差的扫描, 而不应进一步分析。获得质量较差的图像将改变细梁网和后肢皮层结构的量化, 并产生不适当或不确定的数据14。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56346/56346fig1.jpg"> 图1。有代表性的大鼠后肢的位置图像和胫骨近端的相应获得的图像。 (A) 大鼠后肢的适当位置, 踝关节完全克制在泡沫中, 腿延长和尾部拉离胫骨提供足够的图像质量 (B) 胫骨及其小梁和皮质的横断面微建筑。(C) 不正确放置大鼠后肢, 腿未完全伸展, 且未完全抑制在泡沫中的踝关节可能导致 (D) 运动工件, 在横断面上被视为裸奔。(E) 干扰视场的物体, 如未从胫骨抽出的尾巴 (F) 会干扰胫骨的 x 射线衰减, 并可能导致骨密度和 TMD 测量的改变, 尽管视觉上并不明显。在面板的左下角F显示在视野中的部分尾巴, 这会干扰随后通过胫骨的 x 射线束。左侧面板中的红色虚线表示右侧面板上显示的剖面线。<a href="//cloudflare.jove.com/files/ftp_upload/56346/56346fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充图1。异氟醚麻醉装置.异氟醚麻醉装置设置为在 O2中以连续流速1-2 升/分钟的速度在全麻中溶解, 以提供3-4% 的异氟醚。<a href="http://cloudflare.jove.com/files/ftp_upload/56346/Suppl_Fig_1.jpg">请单击此处下载此图.</a>
补充图2。确保麻醉深度。通过用面罩或鼻锥捏住接受连续吸入麻醉剂的老鼠的脚趾来测量踏板的反射。当腿部略微延长时, 疼痛反应更明显。非常强的捏或钳子或夹子的用途可能导致组织损伤, 因此不应该使用。<a href="http://cloudflare.jove.com/files/ftp_upload/56346/Suppl_Fig_2.jpg">请单击此处下载此图.</a>
补充图3。屏幕捕获的实时反馈生理监控摄像头的看法.<a href="http://cloudflare.jove.com/files/ftp_upload/56346/Suppl_Fig_3.jpg">请单击此处下载此图.</a>
补充图4。在碳纤维扫描仪床上放置仰卧位的大鼠.<a href="http://cloudflare.jove.com/files/ftp_upload/56346/Suppl_Fig_4.jpg">请单击此处下载此图.</a>
补充图5。右脚的大鼠约束在一个可塑性泡沫管.右脚的大鼠是克制在一个可塑性泡沫管与脚趾延伸出的管 (这里没有图)。泡沫管被限制在塑料支架 (请参阅特定材料/设备表以获取更详细的信息)。<a href="http://cloudflare.jove.com/files/ftp_upload/56346/Suppl_Fig_5.jpg">请单击此处下载此图.</a>
补充图6。大鼠的位置与右腿伸直直.尾巴和左脚被胶带从右腿 (朝向躯干), 臀部是固定的, 鼠躯干被包裹在兽医包装 (蓝色), 以限制热量损失。<a href="http://cloudflare.jove.com/files/ftp_upload/56346/Suppl_Fig_6.jpg">请单击此处下载此图.</a>

Watch this Scientific Journal Video about Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography at 

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

应用活体显微计算机断层成像技术对骨微结构高分辨成像大鼠后肢的定位与抑制

本文指导用户的在体内微计算机断层扫描 (µCT) 扫描仪如何麻醉, 正确定位和抑制后肢的小鼠在高分辨率成像胫骨的最小运动。结果是高质量的图像, 可以处理, 以准确地量化骨微结构。

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

proper-positioning-restraint-rat-hind-limb-for-focused-high

Research

JoVE Journal

Bioengineering

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

9.8K 次观看.  Brock University. 本文指导用户的在体内微计算机断层扫描 (µCT) 扫描仪如何麻醉, 正确定位和抑制后肢的小鼠在高分辨率成像胫骨的最小运动。结果是高质量的图像, 可以处理, 以准确地量化骨微结构。

视频: Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

高分辨率光纤内窥镜为原位细胞成像

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

科研

生物工程

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

高分辨率三维成像 EX - VIVO生物样品显微CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

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 ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

高强度的实时监控聚焦超声（HIFU）消融<em&gt;体外</em&gt;使用谐运动成像的聚焦超声犬肝脏（HMIFU）

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

在心肌结构，功能的体内定量评估，灌注与活力使用心脏微型计算机断层扫描

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

使用Tomoauto:一个协议，用于高通量自动化低温电子断层扫描

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality that shows great potential for both preclinical and clinical applications. Conventional optical parametric oscillator-based PAT (OPO-PAT) systems are bulky and expensive and cannot provide high-speed imaging. Recently, pulsed-laser diodes (PLDs) have been successfully demonstrated as an alternative excitation source for PAT. Pulsed-laser diode PAT (PLD-PAT) has been successfully demonstrated for high-speed imaging on photoacoustic phantoms and biological tissues. This work provides a visualized experimental protocol for in vivo brain imaging using PLD-PAT. The protocol includes the compact PLD-PAT system configuration and its description, animal preparation for brain imaging, and a typical experimental procedure for 2D cross-sectional rat brain imaging. The PLD-PAT system is compact and cost-effective and can provide high-speed, high-quality imaging. Brain images collected in vivo at various scan speeds are presented.

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

A compact pulsed-laser diode-based photoacoustic tomography (PLD-PAT) system for high-speed in vivo brain imaging in small animals is demonstrated.

高性能紧凑型光声层析成像系统<em&gt;体内</em&gt;小动物脑成像

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality ...

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.