这项工作得到了基础科学研究所 (IBS-R015-D1) 和由教育部 (2017R1A6A1A03015642) 资助的韩国国家研究基金会 (NRF) 的基础科学研究项目的支持。

所有手术程序均经成均大学机构动物护理和使用委员会 (IACUC) 批准。
1. 样品准备
注: 所有外科器械在动物搬运前都要用高压釜。在一个专门用于外科手术的房间里进行所有外科手术。手术室所有人员都应佩戴无菌的手术袍和手套。
<ol><li>
组织固定
	<ol>
<li>在 PBS 中准备两个10毫升的注射器, 每个灌满磷酸盐缓冲盐水 (pbs) 和4% 多聚甲醛 (粉煤灰)。</li>
		<li>麻醉7-12 周大的老鼠, (C57BL/6J 或任何性别的鼠标线) 通过管理混合的 zoletil 和甲苯噻嗪 (1:1, 20 毫克/千克) 或替代适当的麻醉机制 (例如, 混合80毫克/千克氯胺酮和10毫克/千克甲苯噻嗪) 腹腔注射。</li>
		<li>一旦鼠标到达麻醉的手术平面 (使用脚趾捏-反应方法), 暴露心脏通过切断皮肤和肋骨笼用剪刀。</li>
		<li>用小剪刀剪右心房, 用针 (灌注率 = 4 毫升/分, 每种溶液的容积 = 10 毫升), 通过左心室依次排出血液并灌注 PBS 和粉煤灰溶液。 注意: 如果过程成功完成, 鼠标就会变硬。使用眼膏或绝育是没有必要的, 因为程序是终端。可跳过固定程序。然而, 建议这一步骤尽量减少结构变形, 包括机械组织损伤和缺血性轴突肿胀。有关组织固定的进一步细节, 请参阅本条, g.j.等。10
</li>
	</ol>
</li>
	<li>
脑切片的制备
	<ol>
<li>用手术剪刀把老鼠斩首腹胸和腹部。</li>
		<li>使用适当的小剪刀取出头皮和骨膜, 直到完全暴露颅骨。</li>
		<li>用小剪刀把前额骨和颅骨沿矢状缝线切开, 小心不要损伤大脑。</li>
		<li>用细钳轻轻地取出颅骨和硬脑膜。</li>
		<li>用柔软的塑料勺子提取大脑, 注意不要损伤组织。</li>
		<li>冲洗和浸泡在4% 粉煤灰溶液中提取的大脑在4摄氏度30分钟。</li>
		<li>用 vibratome 将固定的大脑切片成 100-150 µm 节。 注: 有关组织准备的进一步细节, 请参阅本文的塞格夫等。11. 对于后续的程序, 应将组织切片成比间隔厚度更薄的切片。</li>
	</ol>
</li>
	<li>
脑切片的安装
	<ol>
<li>为每个组织切片准备1张玻璃滑梯和2平方盖眼镜 (22 x 22 x 0.17 毫米)。</li>
		<li>使用玻璃切割机将方形罩玻璃切成一半 (两个矩形件)。</li>
		<li>在幻灯片玻璃上使用超级胶水将两个半盖眼镜连接起来。 注意: 两个半杯之间的空间应该比组织切片的大小稍大一些。</li>
		<li>用刷子或吸管收割脑切片, 并将纸巾放在隔板之间的滑动玻璃上。注意不要折叠组织。</li>
		<li>在组织表面分配 100 ul 的 PBS。</li>
		<li>将另一方形盖子玻璃放在组织的顶部。在这一阶段避免气泡的加入。</li>
		<li>使用指甲油 (或合适的粘合剂) 在盖子玻璃周围密封, 以防止 PBS 蒸发, 并在随后的成像过程中灰尘污染。 注意: 实验者可能在这个阶段暂停。</li>
	</ol>
</li>
</ol>2. 校准
<ol><li>在成像前至少在显微镜上切换1小时, 以允许激光源的热稳定 (见材料表)。然后, 打开用于光谱扫描的软件 (参见材料表), 然后单击 GUI 顶部的 "获取" 按钮 (图 3a).
</li>
	<li>打开白光激光器和光电倍增管 (PMT) 的软件快门 (图 3a)。</li>
	<li>在获取模式下的下拉列表中选择xyΛ模式, 然后将激光输入模式自动从恒定百分比更改为恒功率。取消选中自动 SP 移动的复选框。然后, 将输入激光器的光谱窗口设置为 470–670 nm 和光谱步长到 4 nm 的λ激发 Lambda 扫描设置(图 3b)。</li>
	<li>通过双击或移动用于光谱范围的调整条 (图 3c), 将 PMT 的频谱范围设置为450-690。 注: 此光谱范围应包括输入源的完整带宽。</li>
	<li>选择一个水浸泡物镜, 适当的高数值孔径 (NA > 0.7), 并给予任何价值的 PMT 和激光功率, 以激活AOBS 配置和实时扫描按钮。通过检查AOBS 配置中的反射来切换光学路径 (图 3d)。</li>
	<li>在显微镜阶段安装参考镜 (见材料表)。镜面应面对物镜。如果不容易把镜子放在显微镜台上, 把镜子粘在平板上 (如滑动玻璃)。</li>
	<li>控制显微镜阶段, 使焦平面与镜面表面对齐。</li>
	<li>调整 PMT 增益和激光功率考虑探测器的动态范围, 然后将激光输入模式从恒定的百分比转换为恒功率。 注: 通常情况下, PMT 增益为 500 (V), 相对激光功率为 0.1%, 用于 570 nm。</li>
	<li>在伪色模式下确认, 以检查波长范围内没有饱和度。如果观测到饱和度, 则降低激光功率 (图 3a)。</li>
	<li>运行Lambda 扫描获取。</li>
	<li>从舞台上移除镜像, 并在没有样本的情况下重复相同的捕获, 以获取暗参照 (即暗偏移)。</li>
	<li>以Multistacked TIF格式保存数据。</li>
</ol>3. SpeRe 图像采集
<ol><li>将已安装的组织置于显微镜阶段。要大致将组织与物镜的焦平面对齐, 可通过眼片使用广域荧光模式。</li>
	<li>使用实时扫描, 控制显微镜阶段, 使焦平面与组织中感兴趣的区域对齐。为避免来自盖板滑动的背景噪音, 请从玻璃组织界面中选择至少15微米深度的目标区域。</li>
	<li>使用步骤2.1–2.10 中描述的相同过程获取目标区域的光谱图像堆栈。</li>
	<li>以Multistacked TIF格式保存组织的数据和暗偏移量。 注意: 实验者可能在这个阶段暂停。</li>
</ol>4. 图像处理与分析
<ol><li>在 ImageJ 中打开参考镜和脑组织的光谱数据 (multistacked TIF)。</li>
	<li>为已打开的图像栈选择 ROIs (感兴趣的区域), 即参考镜的中心区域和用于脑组织的轴突纤维的片段。</li>
	<li>获取所选 ROIs 的原始光谱, 方法是运行图像→堆栈→在 ImageJ 菜单上绘制 Z 轴轮廓。</li>
	<li>打开暗偏移数据, 一个为参考镜和其他为脑组织采取。</li>
	<li>通过运行图像来获取暗偏移的光谱→堆栈→在 ImageJ 菜单上绘制 Z 轴轮廓。</li>
	<li>使用复制和粘贴选项保存所有获得的光谱。 注: 对于 SpeRe 成像, 建议使用中心成像场最小化离轴光学畸变。轴突纤维可能是结构上的异构沿其长度。因此, 建议在小轴突段 (通常 < 5 µm) 中选择 ROI, 以最小化部分体积工件。</li>
</ol>5. 基线校正和 SpeRe 信号分析
<ol><li>从参考镜和脑组织的光谱中减去偏移光谱。</li>
	<li>通过划分光谱的最大强度来正常化每个频谱。</li>
	<li>将轴突的归一化谱除以参考镜的归一化光谱, 并从归一谱中减去 DC 偏移量。</li>
	<li>通过将获得的频谱拟合为正弦曲线来测量波数频率 (参见材料表), 然后通过对等的方法将获得的波数转换为周期性。</li>
	<li>使用图 4c中的等式将波数周期性转换为轴突直径。</li>
</ol>

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作者宣布了相互竞争的金融利益: j. 和蔡是本文所描述的专利未决技术的发明者。

SpeRe 是一种新的基于光谱干涉的无标签成像模式, 它首次提供了实时髓轴突中的纳米尺度信息。在当前的采集协议中, 轴突直径的空间分辨率为 10 nm。此外, SpeRe 利用数量级较低的光剂量比其他超分辨率镜;因此, 它是免费的毒性和漂白。SpeRe 将为研究髓轴突的纳米级动力学提供一个新的途径。
本文所描述的协议是基于声光学和点探测器 (即PMT) 介导的光谱扫描。该方法有利于获取全视场的光谱图像。然而, 时间分辨率受频谱扫描的限制, 这通常是 > 十五年代在我们当前的配置。作为一种替代方法, 可以将高速光谱仪结合在一起, 以便对一个小的视野9进行实时观测。该系统可以简单地从传统的共焦系统, 通过光谱仪切换 PMT 和添加白光激光器。对于采集软件, 振镜位置需要与光谱仪的运行同步。在单点测量的情况下, 时间分辨率是由分光镜的帧速率决定的, 可用于最近超快 spectroscopes 的 > 10 赫。这个亚毫秒的时间分辨率应该足够观察髓轴突在体内的大部分生理动力学。SpeRe 是基于光反射的;因此, 检测到的光与输入光的波长相同。相反, 荧光涉及光谱偏移 (即斯托克斯移位);因此, 检测到的光与输入光的光谱是可分离的。此功能有助于同时获取相同标本上的 SpeRe 和荧光 (如图 4所示)。荧光吸收输入光会影响 SpeRe 测量, 但典型荧光染色的吸收率微乎其微低 (< 1%)。
注意, SpeRe 有一个有限的角检测范围-只有几乎平行于成像平面的轴突可以被检测到。SpeRe 的这个角限制在 13.5° 9左右。为了获得特定的兴趣取向, 样品可能会倾斜。如斑马鱼胚胎等透明标本, 可以通过旋转标本进行完全容积的采集。另外一个实际的限制是, 安装在样品上的盖玻片产生强烈的背反射, 导致高背景信号的产生, 如图 5所示;建议避免这种肤浅的区域。基于可见光的 SpeRe 在脑组织中有20µm 的 1/e 衰减长度。获得可靠光谱信息的典型组织穿透深度仅限于100µm. 如果使用较长的输入源, 则可能更深层次的成像。最后, 通过与衍射有限荧光显微镜的比较, 证明了 SpeRe 的有效性。这显然不足以评估纳米精度 (图 4)。最近的技术, 如相关的光电子显微术和扩张显微镜, 将提供一种方法来验证 SpeRe 在纳米级的制度13,14。

在哺乳动物神经系统中, 髓鞘通过将轴突纤维包裹成多层膜鞘来提供快速的神经传导和轴突完整性。其多层结构由由等离子膜 (5 纳米)、细胞质 (~ 3 nm) 和胞外空间 (~ 7 nm)1、2组成的交替纳米薄膜组成。光学显微术, 包括最近的超分辨率显微镜, 不适合观察纳米髓鞘动力学, 由于其分辨率不足, 由于光学衍射3,4,5。尽管电子显微镜可以提供髓鞘纳米结构的细微细节, 但由于具有化学固定和 ultrasectioning6、7的高侵入性样品制剂, 它与生物系统不相容..直到最近, 还没有一种技术适用于髓轴突的纳米级动态观测。
Schain等人此前曾报道, 髓轴突呈现彩色光反射率8。通过对反射光的光谱分析, 我们设计了一种新的成像方式, 用于髓轴突的纳米成像, 称为光谱反射 (SpeRe)9。SpeRe 是基于在髓鞘多层结构中发生的薄膜干扰 (图 1)。通过对各种轴突的光学模拟, 揭示了反射谱是波数的周期性函数, 其周期性 (<img alt="Equation 1" src="/files/ftp_upload/57965/57965eq1.jpg">) 与轴突直径 (d) 成反比。这种简单的关系<img alt="Equation 2" src="/files/ftp_upload/57965/57965eq2.jpg">() 提供了 SpeRe 数据的轴突直径的简便量化。利用这一点, 我们发现在我们的前报告中, 在轻度外伤性颅脑损伤下的流行轴突肿胀。
SpeRe 系统以共焦显微镜为基础, 由专门的激光源和过滤器组成 (图 2)。输入源是白光激光器, 提供了红外区域可见的宽带频谱输出。对于光谱扫描, 系统配备两个声光器件: 一种声光可调谐滤波器 (AOTF), 用于从输入宽带源和声光光束分配器 (AOBS) 中传送选定的波长, 以指导选定的反射波长到探测器。高光谱共焦显微镜软件 (见材料表) 提供了一个可定制的光谱扫描选项, 以顺序获取各种输入波长的反射图像。此外, 色差能严重干扰光谱测量;因此, 建议使用复消色差物镜。
注意, 白光激光器产生不均匀的光谱输出, 光学元件也影响光谱剖面。因此, 需要对获得的光谱进行校准, 以便随后进行定量分析。受保护的银镜通常用作参考, 在整个可见区域上提供几乎恒定的反射率 (> 97%)。获得的光谱然后除以参考光谱从镜子。
光谱扫描的光谱步长决定了采集速度;因此, 它需要进行优化。由于一个较大的轴突具有较高的频谱周期, 它需要更精细的光谱取样。例如, 一个直径为10µm 的轴突, 是最大的生理轴突之一, 其光谱周期为 ~ 8 nm。应用采样标准, 采用 4 nm 的光谱采样间隔, 覆盖了小鼠神经组织的所有生理轴突。这种方法通常需要几秒钟的全光谱扫描, 因此不适合在体内应用, 其中生理运动 (如呼吸和心跳) 干扰稳定的光谱习得。我们以前通过检测一个自定义的垂直显微镜来解决这个问题, 它设计为每个点使用阵列光谱仪 (采集速度≈每像素30毫秒) 获得全部频谱。
在本报告中, 我们描述了一个关于固定脑切片的 SpeRe 成像的详细协议, 可以在商业高光谱显微镜下进行 (见材料表)。因此, 该协议可以由没有光学仪器专业知识的实验者完成。我们还讨论了 SpeRe 数据的获取和分析的潜在问题和疑难解答。

<table>
	<tbody>
		<tr>
			<td>Glass cutter</td>
			<td>-</td>
			<td>-</td>
			<td>Can be purchased in a local convenience store or online stores.</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>-</td>
			<td>-</td>
			<td>Can be purchased in a local convenience store or online stores.</td>
		</tr>
		<tr>
			<td>Apochromat objective 40&#215;, NA 1.1</td>
			<td>Leica Microsystems</td>
			<td>15506357</td>
			<td>Water-immersion type</td>
		</tr>
		<tr>
			<td>Fluoromyelin Green</td>
			<td>Thermo Fisher</td>
			<td>F34651</td>
			<td>Alternatively, Fluoromyelin Red (F34652) can be used.</td>
		</tr>
		<tr>
			<td>Leica SP8 TCS microscope</td>
			<td>Leica Microsystems</td>
			<td>SP8</td>
			<td>Refer to the &#34;Configuration of microscope&#34; in Introduction Section for details.</td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>Leica Microsystems</td>
			<td>LAS-X</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>MathWorks</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Mirror</td>
			<td>Thorlabs</td>
			<td>PF10-03-P01</td>
			<td>Coated with protected silver.</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Life technologies</td>
			<td>14190-136</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Biosolution</td>
			<td>BP031a</td>
			<td>4% v/v in PBS</td>
		</tr>
		<tr>
			<td>Cover slip</td>
			<td>Thermo Fisher</td>
			<td>3306</td>
			<td>Thickness: #1 (0.13 to 0.17 mm)</td>
		</tr>
		<tr>
			<td>Slide glass</td>
			<td>Muto Pure Chemicals</td>
			<td>5116-20F</td>
			<td>Thickness: ~1 mm</td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>Henkel</td>
			<td>Loctite 406</td>
			<td>Use a dispensing equipment to avoid skin or eye contact.</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Brainetree Scientific</td>
			<td>BS-8000 DUAL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica Biosystems</td>
			<td>VT1200S</td>
			<td>-</td>
		</tr>
		<tr>
			<td>White-light laser</td>
			<td>NKT photonics</td>
			<td>EXB-6</td>
			<td>EXB-6 was discontinued and replaced by EXU-6.</td>
		</tr>
	</tbody>
</table>

spectral reflectometric microscopy on myelinated axons in situ

在哺乳动物神经系统中, 髓鞘通过将轴突纤维包裹成多层螺旋来提供电绝缘。在其高度组织的亚细胞体系结构的启发下, 我们最近开发了一种新的成像方式, 命名为光谱反射 (SpeRe), 它能够实现前所未有的无标签的现场髓轴突的纳米级成像。其基本原理是通过对多层亚细胞结构的反射谱进行分析, 获得纳米信息。在本文中, 我们描述了一个详细的分步协议, 以执行一个基本的 SpeRe 成像的神经组织使用一个商业共焦显微系统, 配备了白光激光器和可调谐滤波器。本文介绍了样品制备、光谱数据采集、图像处理等程序, 以获取纳米信息。

根据该协议, 用外源染色、髓鞘靶向荧光 (见材料表) 制备了固定脑切片。用商业高光谱共聚焦显微镜结合共焦荧光成像 (图 4a) 对脑切片进行 SpeRe 成像。对于 SpeRe, 输入光学强度设置为5µW/µm2 , 像素停留时间为1µs。这种光剂量比常规荧光共聚焦显微镜12级低。它需要 ~ 十七年代为光谱扫描470-670 毫微米在间隔4毫微米 (51 张图片)。
SpeRe 信号沿髓轴突的中心进行局部定位, 这是由光学反射的几何所预期的。从轴突段的反射谱, 得到了波数周期性, 随后转换为轴突直径 (图 4b, c)。SpeRe 测量的直径与基于荧光的测量结果吻合良好 (图 4d)。残余小误差可能来源于基于荧光法的衍射有限分辨率或 SpeRe 的不完全几何模型。
SpeRe 成像是基于光学反射的, 因此基于二氧化硅的盖玻片可以引入显著的背景噪声。在我们的光学设置中, 当盖玻片的成像深度小于5µm 时, 背景噪声相当可观, 但在成像深度大于15µm 时避免了 (图 5)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57965/57965fig1.jpg"> 图 1: SpeRe 原则.髓轴突有多层薄膜结构。入射光在界面上部分地反射, 如菲涅尔定律所描述。这些反射光波相互干扰;因此, 由此产生的反射光对纳米信息进行编码。光谱反射 (SpeRe) 通过对髓轴突反射光的解码获得纳米信息。这一数字已从跆拳道, J等。9<a href="/files/ftp_upload/57965/57965fig1large.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/57965/57965fig2.jpg"> 图 2: SpeRe 系统的布局.SpeRe 系统是基于共焦反射显微镜。对于光谱扫描, 在励磁路径上需要另外三个组件: (1) 超连续谱激光器, (2) 声光可调谐滤波器 (AOTF) 和 (3) 声光光束分配器 (AOBS)。宽带激光源是通过 AOTF 进行光谱滤波, 以传输带宽窄的选定波长。AOBS 函数作为光束分流器为选定的波长引导励磁光到样品。然后光线通过振镜扫描仪和物镜在样品上发生事件。样品中的反射光是 descanned, 由共焦针孔空间过滤, 并由光电倍增管 (PMT) 收集而成。<a href="/files/ftp_upload/57965/57965fig2large.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/57965/57965fig3.jpg"> 图 3: 软件设置.(a) 图形用户界面 (GUI)。在主窗口中, 主要有五个子面板: 成像设置、激光安装、光学设置、探测器设置和图像查看器。(b) 要选择图像模式的下拉菜单。对于 SpeRe, xyλ被选中。(c) 调整探测器的光谱窗口的小组。(d) 设立声光分束器 (AOBS) 的小组。选取 "反射" 来引导反射光到探测器。<a href="/files/ftp_upload/57965/57965fig3large.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/57965/57965fig4.jpg"> 图 4: SpeRe 在脑组织中.(a) SpeRe 的小鼠脑组织的图像, 染色的是髓鞘的荧光 counterstaining (fluoromyelin)。(b) 从 (a) 的白色虚线框中获得的代表性反射光谱。(c) 模拟参考曲线来估计轴突直径的频谱周期性。蓝色星号是从 (b) 获取的数据点。(d) (a) 盒装区域的荧光强度横向剖面。用荧光信号估计的髓鞘外径为760纳米, 与 SpeRe 测量结果吻合较好。从荧光测量的衍射误差中, 也能得到残余误差。比例条, 5 µm.<a href="/files/ftp_upload/57965/57965fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57965/57965fig5.jpg"> 图 5: 盖子滑动的作用.(a、 b)在同一侧位的脑组织的反射图像从组织-盖玻片界面的不同深度获得: 在3µm (a) 和15µm (b)。比例条, 10 µm.<a href="/files/ftp_upload/57965/57965fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Spectral Reflectometric Microscopy on Myelinated Axons In Situ at JoVE.com

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

髓轴突原位光谱 Reflectometric 显微术

在这里, 我们提出了一个分步协议的成像髓轴突在固定脑切片使用无标签的纳米尺度成像技术的基础上的光谱反射。

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

spectral-reflectometric-microscopy-on-myelinated-axons-in-situ

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

7.3K 次观看.  Sungkyunkwan University. 在这里, 我们提出了一个分步协议的成像髓轴突在固定脑切片使用无标签的纳米尺度成像技术的基础上的光谱反射。

视频: Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

在果蝇幼虫分部轴突在体内的突触小泡的可视化

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

科研

神经科学

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

实时成像背根轴突后根切断术

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

光谱共焦成像的荧光标记的尼古丁受体敲在慢性尼古丁政府小鼠

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

分析小鼠沿着轴突生长的雪旺氏细胞发展

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脑轴突的激光Nanosurgery<em&gt;在体内</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

尼古丁诱导的钙信号和神经递质的释放了沿着腹侧海马轴突实时成像

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

快速<em&gt;原位</em使用寡核苷酸探针对大鼠多聚甲醛为前缀的大脑5-羟色胺综合征&gt;杂交

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

The electrophysiological technique of intracellular recording is demonstrated and used to determine spectral sensitivities of single photoreceptor cells in the compound eye of a butterfly.

光感受器细胞光谱灵敏度在从昆虫模型决定<em&gt;在体内</em&gt;细胞内记录

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...