原代大鼠海马神经元由康涅狄格大学（美国康涅狄格州斯托尔斯）生物医学工程系的George Lykotrafitis博士和Shiju Gu提供。Abberior STED仪器位于开放研究资源和设备中心的先进光学显微镜设施中，由NIH资助S10OD023618授予Christopher O'Connell。这项研究由美国国立卫生研究院资助R01AG065879授予Nathan N. Alder。

使用 STED 显微镜揭示神经元中的线粒体超微结构

作者没有什么可透露的。

该协议介绍了使用人神经母细胞瘤细胞系SH-SY5Y和原代大鼠海马神经元与新型IMM靶向PKMO染料进行活细胞STED成像。由于PKMO的新颖性，目前很少有使用这种染料进行STED活体成像的出版物。使用这些细胞类型进行STED成像带来了挑战，特别是因为神经元细胞的线粒体较窄。该协议的一个局限性是使用的PKMO染料，因为它可能对细胞有毒。不同的细胞和细胞系对染料的反应不同，因此可能需要调整染料浓度和孵育时间，以在不伤害细胞的情况下优化强信号的结果。建议的解决方案是降低浓度并增加染色时间63;然而，这可能会导致染色较差，而不会增加细胞活力。
与PKMO类似，商业染料Live Orange mito（材料表）也表现出一些细胞毒性。该染料用于多种培养细胞，但无法在 RA 分化的 SH-SY5Y 细胞中成功表现出与未分化对应物相同的参数（我们未发表的观察结果）。然而，可以针对该探针和所选细胞类型优化合适的染色方案。使用这种染料时，检测器设门时间为1-1.05至7-7.05 ns， 表1 中的所有其他参数保持不变。通常，用 200-250 nM 活橙线粒体染色细胞 45 分钟可产生与 PKMO 结果相当的结果。较短时间的较高浓度染色或相同时间或稍长的较低浓度染色会产生不同的结果，并且可能有利于其他细胞类型或生长条件。
由于轴突和树突投射的性质以及成像时的线粒体分布，原代大鼠海马神经元成像与永生化细胞不同。协议这一部分的一个困难是，接种密度决定了原代培养物是否能够粘附和健康生长，并且在更高的密度下，投影往往会过度生长 DIV 10。因此，从这些原代神经元成像的线粒体可能来自细胞体，而不是投射;然而，从较低的起始细胞密度成功生长会在后期生长时产生更好的成像结果。关键是要确保低背景和失焦光，以便为STED提供最佳对比度。为了解决对细胞群的担忧，在补充 B27 的神经元生长培养基中培养原代海马细胞可防止神经胶质细胞的生长，并且来源报告说 <5% 的细胞是星形胶质细胞，并且生长培养基中缺乏 NbActiv1 补充剂可将培养物中的星形胶质细胞数量减少到 <2%87。对于培养的SH-SY5Y细胞和原代大鼠海马神经元，用于生长的PDL涂层会导致图像中的背景雾度。使用（表1）中报告的设置可以实现足够的信噪比，并且反卷积消除了观察到的大部分背景。
除了这里介绍的成像外，还可以在成像之前或期间对细胞进行治疗或施加压力。例如，添加叔丁基过氧化氢 （tBHP） 会诱导氧化应激，并且可以监测添加后线粒体随时间的变化。添加带有荧光标签的淀粉样蛋白-β （Aβ） 可以监测该肽与线粒体的关系以及线粒体结构随时间变化的分布。线粒体健康与AD密切相关，并被广泛支持在Aβ毒性中发挥作用43,71,72。值得注意的是，SH-SY5Y 细胞的分化状态会影响 Aβ 蛋白前体 （AβPP） 定位85，应仔细构建使用 AβPP 的实验。
作为如何调整该方案的一个例子，显示荧光变体Aβ（1-42）-HiLyte 647可以在成像前15分钟添加到PKMO染色的细胞中（补充图1）。成像参数相似（补充表2），主要区别在于对较窄的线粒体进行成像时需要更小的针孔。使用 STED 对 Aβ-HiLyte647 进行成像需要较少的总激发 （6%-8%） 和 STED 耗尽 （10%-12%） 激光功率和较少的累积 （6）。检测器门控也从 0.1 ns 扩展到 10 ns。虽然不需要Aβ的STED分辨率，但原始STED的整体信噪比和Aβ粒径优于共聚焦图像，也可以进行后续的反卷积。当与PKMO染色的原始STED或去卷积STED图像合并时，收集STED图像和Aβ的原始STED z堆栈投影似乎特别有用（补充图1B，C）。两个通道都是在单帧步长中收集的。在应力处理或其他添加后，可以获得与 图 2 中列出的类似和 图 5 中所示的瞬态定位的测量值（如适用）和嵴架构差异。
此处未报道但已被其他人报道的线粒体活细胞 STED 中双标记的其他可能方法包括使用 SNAP 标记蛋白93、Halo 标记蛋白，以及使用具有通用靶标的其他细胞渗透性染料，例如 mtDNA63。值得注意的是，SNAP 和 Halo 标记的标记策略会影响成像时产生的荧光信号强度和寿命94。此外，虽然该协议提供了可应用于分割线粒体的几个分析示例，但软件包可以对这些图像执行许多其他分析。

线粒体是内共生起源的真核细胞器，负责调节几个关键的细胞过程，包括中间代谢和 ATP 产生、离子稳态、脂质生物合成和程序性细胞死亡（细胞凋亡）。这些细胞器拓扑复杂，包含建立多个亚区室的双膜系统1（图1A）。线粒体外膜 （OMM） 与胞质溶胶相互作用并建立直接的细胞器间接触 2,3。线粒体内膜 （IMM） 是一种能量守恒膜，可维持主要以电膜电位 （ΔΨm） 形式存储的离子梯度，以驱动 ATP 合成和其他需要能量的过程 4,5。IMM 进一步细分为紧密贴合 OMM 的内边界膜 （IBM） 和由嵴膜 （CM） 结合的称为嵴的突出结构。该膜从嵴内空间 （ICS） 和膜间空间 （IMS） 描绘了最内层的基质隔室。
线粒体具有基于连续和平衡的裂变和融合过程的动态形态，该过程由动力蛋白超家族6 的机械酶控制。融合允许增加连接和网状网络的形成，而裂变导致线粒体碎片化，并能够通过线粒体自噬去除受损的线粒体7。线粒体形态因组织类型8 和发育阶段9 而异，并受到调节以允许细胞适应包括能量需求10,11 和压力源12 在内的因素。线粒体的标准形态特征，例如网络形成的程度（互连与碎片化）、周长、面积、体积、长度（纵横比）、圆度和分支程度，可以通过标准光学显微镜测量和量化，因为这些特征的大小大于光的衍射极限 （~200 nm）13。
Cristae 结构定义了线粒体的内部结构（图 1B）。嵴形态的多样性可大致分为扁平（层状或盘状）或管状囊泡状14。所有嵴都通过称为嵴结 （CJ） 的管状或槽状结构附着在 IBM 上，这些结构可用于将 IMS 与 ICS 和 IBM 与 CM15 分开。嵴形态受 IMM 的关键蛋白质复合物调控，包括 （1） 位于 CJ 并稳定 IMM-OMM 触点 16 的线粒体接触位点和嵴组织系统 （MICOS），（2） 调节嵴重塑的视萎缩 1 （OPA1） GTP 酶17,18,19，以及 （3） 在嵴尖端 （CT） 形成稳定寡聚体组装体的 F1FO ATP 合酶 20，21.此外，IMM 富含非双层磷脂磷脂酰乙醇胺和心磷脂，可稳定高度弯曲的 IMM22。Cristae 也是动态的，在各种条件下表现出形态变化，例如不同的代谢状态 23,24、不同的呼吸基质 25、饥饿和氧化应激 26,27、细胞凋亡 28,29 和衰老30.最近，研究表明，嵴可以在几秒钟的时间尺度上经历重大的重塑事件，这突显了它们的动态性质31.嵴的几个特征可以量化，包括单个嵴内结构的尺寸（例如，CJ宽度，嵴长度和宽度）以及将单个嵴与其他结构相关的参数（例如，嵴内间距和嵴相对于OMM的入射角）32。这些可量化的嵴参数显示出与功能的直接相关性。例如，线粒体 ATP 产生的程度与嵴的丰度呈正相关，量化为嵴密度或归一化为另一个特征的嵴数（例如，每个 OMM 面积的嵴）33,34,35。由于IMM形态由纳米级特征定义，因此它包括线粒体超微结构，这需要成像技术提供大于光衍射极限的分辨率。如下所述，此类技术包括电子显微镜和超分辨率显微镜（nanoscopy）。
中枢神经系统（CNS）的神经和神经胶质细胞特别依赖线粒体功能。平均而言，大脑仅占总重量的 2%，但利用了 25% 的葡萄糖，占身体耗氧量的 20%，使其容易受到能量代谢的损害36.进行性神经退行性疾病 （ND），包括阿尔茨海默病 （AD）、肌萎缩侧索硬化症 （ALS）、亨廷顿舞蹈症 （HD）、多发性硬化症 （MS） 和帕金森病 （PD），是迄今为止研究最广泛的病理学，研究工作范围从了解这些疾病的分子基础到寻求潜在的治疗预防和干预措施。ND 与氧化应激增加有关，部分源于线粒体电子传递链 （ETC） 37 产生的活性氧 （ROS），以及线粒体钙处理 38 和线粒体脂质代谢39 的改变。这些生理改变伴随着与 AD 40、41、42、43、44、ALS 45,46、HD 47、48,49、MS 50 和 PD51,52,53 相关的线粒体形态缺陷.这些结构和功能缺陷可以通过复杂的因果关系耦合。例如，鉴于嵴形态稳定了 OXPHOS 酶54，线粒体 ROS 不仅由 ETC 产生，而且还会破坏 ETC 所在的基础设施，促进前馈 ROS 循环，从而增强对氧化损伤的敏感性。此外，嵴紊乱已被证明会触发线粒体 DNA （mtDNA） 释放和与自身免疫、代谢和年龄相关疾病相关的炎症通路等过程55。因此，线粒体结构的分析是全面了解ND及其分子基础的关键。
观察嵴的流行方法，包括透射电子显微镜、电子断层扫描和冷冻电子断层扫描 （cryo-ET） 以及 X 射线断层扫描，特别是冷冻软 X 射线断层扫描，已经揭示了重要的发现，并适用于各种样品类型 56、57、58、59、60.尽管最近在更好地观察细胞器超微结构方面取得了进展，但这些方法仍然需要样品固定，因此无法直接捕获嵴的实时动态。超分辨荧光显微镜，特别是结构照明显微镜（SIM）、随机光学重建显微镜（STORM）、光活化定位显微镜（PALM）、膨胀显微镜（ExM）和受激发射耗尽（STED）显微镜等形式，已成为观察结构的流行方法，这些结构需要低于衍射极限的分辨率，这限制了经典的光学显微镜方法。当ExM与另一种超分辨率技术结合使用时，结果令人印象深刻，但样品必须固定并在凝胶61中染色。相比之下，SIM、PALM/STORM 和 STED 都已成功用于活体样品，通常对 IMM 进行染色的新型和有前途的染料为线粒体嵴动力学的实时成像提供了一种新颖而简单的方法 62,63,64,65,66。用于STED成像的活体染料的最新进展改善了染料的亮度和光稳定性，并且这些染料以比其前身更高的特异性靶向IMM。这些发展允许通过超分辨率成像收集长期延时和 z 堆栈实验，为更好地分析线粒体超微结构和动力学的活细胞打开了大门。
本文提供了使用 STED63 用 PKmito Orange （PKMO） 染料染色的未分化和分化的 SH-SY5Y 细胞的活细胞成像方案。SH-SY5Y 细胞系是从亲本细胞系 SK-N-SH 的三个亚克隆衍生物，由转移性神经母细胞瘤67、68、69、70 的骨髓活检产生。该细胞系是ND研究中常用的体外模型，特别是AD、HD和PD等疾病，其中线粒体功能障碍与10,43,71,72,73密切相关。通过操纵培养基将 SH-SY5Y 细胞分化为具有神经元样表型的细胞的能力已被证明是神经科学研究的合适模型，而无需依赖原代神经元细胞10,74。在该方案中，将视黄酸（RA）加入细胞培养基中以诱导SH-SY5Y细胞的分化。RA 是一种维生素 A 衍生物，已被证明可以调节细胞周期并促进调节神经元分化的转录因子的表达75。还提供了从大鼠海马中分离的神经元的培养和活细胞成像的方案。海马体已被证明受到线粒体变性的影响，并与皮层一起在衰老和ND 76,77,78,79,80中起重要作用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >0.05% Trypsin-EDTA</td>
			<td >Gibco</td>
			<td >25300054</td>
			<td ></td>
		</tr>
		<tr >
			<td >0.4% Trypan blue</td>
			<td >Invitrogen</td>
			<td >T10282</td>
			<td ></td>
		</tr>
		<tr >
			<td >0.5% Trypsin-EDTA, no phenol red</td>
			<td >Gibco</td>
			<td >15400054</td>
			<td ></td>
		</tr>
		<tr >
			<td >100 X antibiotic-antimycotic</td>
			<td >Gibco</td>
			<td >15240062</td>
			<td ></td>
		</tr>
		<tr >
			<td >100 X/1.40 UPlanSApo oil immersion lens</td>
			<td >Olympus</td>
			<td ></td>
			<td >Equipped in Olympus IX83 microscope for STED setup described in Section 4</td>
		</tr>
		<tr >
			<td >All-trans-retinoic acid</td>
			<td >Sigma</td>
			<td >R2625</td>
			<td ></td>
		</tr>
		<tr >
			<td >Amyloid-&#946; (1-42, HiLyte Fluor647, 0.1 mg)</td>
			<td >AnaSpec</td>
			<td >AS-64161</td>
			<td >Other fluorescent conjugates available</td>
		</tr>
		<tr >
			<td >B27 supplement (50 X), serum free</td>
			<td >Gibco</td>
			<td >17504044</td>
			<td ></td>
		</tr>
		<tr >
			<td >Cell Counter (Countess II FL)</td>
			<td >Life Technologies</td>
			<td >AMQAF1000</td>
			<td ></td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Eppendorf</td>
			<td >5804-R</td>
			<td ></td>
		</tr>
		<tr >
			<td >Counter slides</td>
			<td >Invitrogen</td>
			<td >C10283</td>
			<td ></td>
		</tr>
		<tr >
			<td >Conical tubes (15 mL)</td>
			<td >Thermo Fisher Scientific</td>
			<td >339650</td>
			<td ></td>
		</tr>
		<tr >
			<td >Cuvettes (Quartz Cells)</td>
			<td >Starna Cells, Inc.</td>
			<td >9-Q-10</td>
			<td >Used with Spectrometer as described in Section 1.3</td>
		</tr>
		<tr >
			<td >DMEM (high glucose with sodium pyruvate)</td>
			<td >Gibco</td>
			<td >11995073</td>
			<td >Used for SH-SY5Y cell materials as described in Section 1</td>
		</tr>
		<tr >
			<td >DMEM (high glucose no sodium pyruvate)</td>
			<td >Gibco</td>
			<td >11965092</td>
			<td >Used for primary cell materials as described in Section 2</td>
		</tr>
		<tr >
			<td >DMEM (phenol red-free)</td>
			<td >Gibco</td>
			<td >31053028</td>
			<td >Used for imaging as described in Section 3</td>
		</tr>
		<tr >
			<td >DMSO</td>
			<td >Sigma</td>
			<td >D8418</td>
			<td ></td>
		</tr>
		<tr >
			<td >DNAase I from bovine pancreas</td>
			<td >Sigma</td>
			<td >DN25</td>
			<td >Used for primary cell materials as described in Section 2.2.1 and 2.2.2</td>
		</tr>
		<tr >
			<td >DPBS (no calcium, no magnesium)</td>
			<td >Gibco</td>
			<td >14190144</td>
			<td ></td>
		</tr>
		<tr >
			<td >E18 Rat Hippocampus</td>
			<td >Transnetyx Tissue</td>
			<td >SDEHP</td>
			<td ></td>
		</tr>
		<tr >
			<td >Ethanol (200 proof)</td>
			<td >Fisher Bioreagents</td>
			<td >BP28184</td>
			<td ></td>
		</tr>
		<tr >
			<td >Fetal bovine serum (FBS), not heat-inactivated</td>
			<td >Gibco</td>
			<td >26140079</td>
			<td >For cultured cells, in Section 1</td>
		</tr>
		<tr >
			<td >Fetal bovine serum (heat inactivated)</td>
			<td >Gibco</td>
			<td >10082147</td>
			<td >For primary cell culture, Section 2</td>
		</tr>
		<tr >
			<td >Filter sterilization unit (0.1 &#181;m, 500 mL)</td>
			<td >Thermo Fisher Scientific</td>
			<td >5660010</td>
			<td ></td>
		</tr>
		<tr >
			<td >FIJI (Is Just ImageJ) and Trainable Weka Segmentation (TWS) plug-in</td>
			<td >--</td>
			<td >--</td>
			<td >Free, open-source image analysis software that includes plug-ins including Trainable Weka Segmentation described in Section 5; TWS plug-in from ref. 90 of the main text</td>
		</tr>
		<tr >
			<td >GlutaMAX supplement (100 X)</td>
			<td >Gibco</td>
			<td >35050061</td>
			<td >Glutamine supplement used for primary cell materials described in Section 2.1.2</td>
		</tr>
		<tr >
			<td >Hausser Scientific bright-Line and Hy-Lite Counting Chambers</td>
			<td >Hausser Scientific</td>
			<td >267110</td>
			<td ></td>
		</tr>
		<tr >
			<td >HBSS (no calcium, no magnesium)</td>
			<td >Gibco</td>
			<td >14170120</td>
			<td >Used for primary cell materials described in Section 2.2.1 and 2.2.2</td>
		</tr>
		<tr >
			<td >HEPES</td>
			<td >Gibco</td>
			<td>15630080</td>
			<td ></td>
		</tr>
		<tr >
			<td >Huygens Professional deconvolution software (V. 20.10)</td>
			<td >Scientific Volume Imaging (SVI)</td>
			<td >--</td>
			<td >The deconvolution software used in this protocol and described in Section 5</td>
		</tr>
		<tr >
			<td >IX83 inverted microscope with Continuous Autofocus</td>
			<td >Olympus</td>
			<td >--</td>
			<td >This paper uses a STED Infinity Line system built around an Olympus IX83 inverted microscope, described in Section 4</td>
		</tr>
		<tr >
			<td >Lightbox software (V. 16.3.16118)</td>
			<td >Abberior</td>
			<td >--</td>
			<td >Vendor software used for STED image acquisition, described in Section 4</td>
		</tr>
		<tr >
			<td >Live Orange Mito dye</td>
			<td >Abberior</td>
			<td >LVORANGE-0146-30NMOL</td>
			<td >Live cell imaging IMM-targeting dye described in Discussion</td>
		</tr>
		<tr >
			<td >Neurobasal media</td>
			<td >Gibco</td>
			<td >21103049</td>
			<td >Used for primary cell materials referred to in Section 2.1.2</td>
		</tr>
		<tr >
			<td >Nunc Lab-Tek II 2-well chambered coverglass</td>
			<td >Nunc</td>
			<td >155379</td>
			<td >Can purchase a variety of chambers but make sure the coverglass is #1.5</td>
		</tr>
		<tr >
			<td >Pasteur Pipets (Fisherbrand)</td>
			<td >Thermo Fisher Scientific</td>
			<td >22183632</td>
			<td ></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin (10,000 U/mL)</td>
			<td >Gibco</td>
			<td >15140122</td>
			<td ></td>
		</tr>
		<tr >
			<td >PKmito Orange dye</td>
			<td >Spirochrome</td>
			<td >SC053</td>
			<td ></td>
		</tr>
		<tr >
			<td >Poly-D-lysine</td>
			<td >Gibco</td>
			<td >A3890401</td>
			<td ></td>
		</tr>
		<tr >
			<td >SH-SY5Y Cell line</td>
			<td >ATCC</td>
			<td >CRL2266</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sodium pyruvate (100 mM)</td>
			<td >Gibco</td>
			<td >11360070</td>
			<td >Used for primary cell materials described in Section 2</td>
		</tr>
		<tr >
			<td >Spectrometer (GENESYS 180 UV-Vis)</td>
			<td >Thermo Fisher Scientific</td>
			<td >840309000</td>
			<td ></td>
		</tr>
		<tr >
			<td >STED Expert Line microscope</td>
			<td >Abberior</td>
			<td >--</td>
			<td >STED setup can be customized, but at time of purchase instrument was considered Abberior&#8217;s Expert Line; brief description of setup is available in Section 4 of protocol</td>
		</tr>
		<tr >
			<td >T25 flask (TC-treated, filter cap)</td>
			<td >Thermo Fisher Scientific</td>
			<td >156367</td>
			<td >Other culture vessels and sizes available</td>
		</tr>
	</tbody>
</table>

using live cell sted imaging to visualize mitochondrial inner membrane ultrastructure in neuronal cell models

线粒体在细胞中起着许多重要作用，包括能量产生、Ca2+ 稳态的调节、脂质生物合成和活性氧 （ROS） 的产生。这些线粒体介导的过程在神经元中发挥特殊作用，协调有氧代谢以满足这些细胞的高能量需求，调节 Ca2+ 信号传导，为轴突生长和再生提供脂质，并为神经元发育和功能调整 ROS 的产生。因此，线粒体功能障碍是神经退行性疾病的核心驱动因素。线粒体结构和功能是密不可分的。形态复杂的内膜具有称为嵴的结构内褶皱，拥有许多执行线粒体特征过程的分子系统。内膜的结构特征是超微结构，因此太小，无法通过传统的衍射极限分辨显微镜进行可视化。因此，关于线粒体超微结构的大多数见解都来自固定样品的电子显微镜。然而，超分辨率荧光显微镜的新兴技术现在提供低至数十纳米的分辨率，从而可以可视化活细胞中的超微结构特征。因此，超分辨率成像提供了前所未有的能力，可以直接对线粒体结构、纳米级蛋白质分布和嵴动力学的精细细节进行成像，从而提供将线粒体与人类健康和疾病联系起来的基本新见解。该协议介绍了使用受激发射耗竭（STED）超分辨率显微镜来可视化活的人神经母细胞瘤细胞和原代大鼠神经元的线粒体超微结构。该程序分为五个部分：（1） SH-SY5Y 细胞系的生长和分化，（2） 原代大鼠海马神经元的分离、铺板和生长，（3） 用于活 STED 成像的细胞染色程序，（4） 使用 STED 显微镜进行活细胞 STED 实验的程序作为参考，以及 （5） 使用示例进行分割和图像处理的指导，以测量和量化内膜的形态特征。

Mitochondria are eukaryotic organelles of endosymbiotic origin that are responsible for regulating several key cellular processes, including intermediary metabolism and ATP production, ion homeostasis, lipid biosynthesis, and programmed cell death (apoptosis). These organelles are topologically complex, containing a double membrane system that establishes multiple subcompartments1 (Figure 1A). The outer mitochondrial membrane (OMM) interfaces with the cytosol and establishes direct inter-organelle contacts2,3. The inner mitochondrial membrane (IMM) is an energy-conserving membrane that maintains ion gradients stored primarily as an electric membrane potential (&#916;&#936;m) to drive ATP synthesis and other energy-requiring processes4,5. The IMM is further subdivided into the inner boundary membrane (IBM), which is closely appressed to the OMM, and protruding structures called cristae that are bound by the cristae membrane (CM). This membrane delineates the innermost matrix compartment from the intracristal space (ICS) and the intermembrane space (IMS).
Mitochondria have a dynamic morphology based on continuous and balanced processes of fission and fusion that are governed by mechanoenzymes of the dynamin superfamily6. Fusion allows for increased connectivity and formation of reticular networks, whereas fission leads to mitochondrial fragmentation and enables the removal of damaged mitochondria by mitophagy7. Mitochondrial morphology varies by tissue type8 and developmental stage9 and is regulated to allow cells to adapt to factors including energetic needs10,11 and stressors12. Standard morphometric features of mitochondria, such as the extent of network formation (interconnected vs. fragmented), perimeter, area, volume, length (aspect ratio), roundness, and degree of branching, can be measured and quantified by standard optical microscopy because the sizes of these features are greater than the diffraction limit of light (~200 nm)13.
Cristae architecture defines the internal structure of mitochondria (Figure 1B). The diversity of cristae morphologies can be broadly categorized as flat (lamellar or discoidal) or tubular-vesicular14. All cristae attach at the IBM through tubular or slot-like structures termed cristae junctions (CJs) that can serve to compartmentalize the IMS from the ICS and the IBM from the CM15. Cristae morphology is regulated by key protein complexes of the IMM, including (1) the mitochondrial contact site and cristae organizing system (MICOS) that resides at CJs and stabilizes IMM-OMM contacts16, (2) the optic atrophy 1 (OPA1) GTPase that regulates cristae remodeling17,18,19, and (3) F1FO ATP synthase that forms stabilizing oligomeric assemblies at cristae tips (CTs)20,21. In addition, the IMM is enriched in nonbilayer phospholipids phosphatidylethanolamine and cardiolipin that stabilize the highly curved IMM22. Cristae are also dynamic, demonstrating morphological changes under various conditions, such as different metabolic states23,24, with different respiratory substrates25, under starvation and oxidative stress26,27, with apoptosis28,29, and with aging30. Recently it has been shown that cristae could undergo major remodeling events on a timescale of seconds, underscoring their dynamic nature31. Several features of cristae can be quantified, including dimensions of structures within individual cristae (e.g., CJ width, crista length, and width) and parameters that relate individual crista to other structures (e.g., intra-cristae spacing and cristae incident angle relative to the OMM)32. These quantifiable cristae parameters show a direct correlation with function. For instance, the extent of mitochondrial ATP production is positively related to the abundance of cristae, quantified as cristae density or cristae number normalized to another feature (e.g., cristae per OMM area)33,34,35. Because IMM morphology is defined by nanoscale features, it comprises mitochondrial ultrastructure, which requires imaging techniques that provide resolution greater than the light diffraction limit. As described below, such techniques include electron microscopy and super-resolution microscopy (nanoscopy).
The neural and glial cells of the central nervous system (CNS) are particularly reliant on mitochondrial function. On average, the brain constitutes only 2% of the total body weight, but utilizes 25% of the total body glucose and accounts for 20% of body oxygen consumption, making it vulnerable to impairments in energy metabolism36. Progressive neurodegenerative diseases (NDs), including Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), Huntington&#39;s disease (HD), multiple sclerosis (MS), and Parkinson&#39;s disease (PD), are some of the most extensively studied pathologies to date, with research efforts ranging from understanding the molecular underpinnings of these diseases to seeking potential therapeutic prevention and interventions. NDs are associated with increased oxidative stress originating in part from reactive oxygen species (ROS) generated by the mitochondrial electron transport chain (ETC)37, as well as altered mitochondrial calcium handling38 and mitochondrial lipid metabolism39. These physiological alterations are accompanied by noted defects in mitochondrial morphology that are associated with AD40,41,42,43,44, ALS45,46, HD47,48,49, MS50, and PD51,52,53. These structural and functional defects can be coupled by complex cause-effect relationships. For example, given that cristae morphology stabilizes OXPHOS enzymes54, mitochondrial ROS are not only generated by the ETC, but they also act to damage the infrastructure in which the ETC resides, promoting a feed-forward ROS cycle that enhances susceptibility to oxidative damage. Furthermore, cristae disorganization has been shown to trigger processes such as mitochondrial DNA (mtDNA) release and inflammatory pathways connected to autoimmune, metabolic, and age-related disorders55. Therefore,&#160;analysis of&#160;mitochondrial structure is key to a full understanding of NDs and their molecular underpinnings.
Popular methods of viewing cristae, including transmission electron microscopy, electron tomography and cryo-electron tomography (cryo-ET), and X-ray tomography, in particular cryo-soft X-ray tomography, have revealed important findings and work with a variety of sample types56,57,58,59,60. Despite recent advancements toward better observation of organellar ultrastructure, these methods still come with the caveat of requiring sample fixation and, therefore, cannot capture real-time dynamics of cristae directly. Super-resolution fluorescence microscopy, particularly in the forms of structured illumination microscopy (SIM), stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), expansion microscopy (ExM), and stimulated emission depletion (STED) microscopy, have become popular ways of viewing structures requiring resolution below the diffraction limit that constrains classical methods of optical microscopy. When ExM is used in conjunction with another super-resolution technique, the results are impressive, but the sample must be fixed and stained in a gel61. By comparison, SIM, PALM/STORM, and STED have all been successfully used with live samples, and new and promising dyes that generally stain the IMM provide a novel and easy approach for live imaging of mitochondria cristae dynamics62,63,64,65,66. Recent advancements in live dyes for STED imaging have improved dye brightness and photostability, and these dyes target the IMM at a higher degree of specificity than their predecessors. These developments allow the collection of long-term timelapse and z-stack experiments with super-resolution imaging, opening the door to better live cell analysis of mitochondrial ultrastructure and dynamics.
Herein, protocols for live cell imaging of undifferentiated and differentiated SH-SY5Y cells stained with the PKmito Orange (PKMO) dye using STED63 are provided. The SH-SY5Y cell line is a thrice subcloned derivative from the parental cell line, SK-N-SH, generated from a bone marrow biopsy of metastatic neuroblastoma67,68,69,70. This cell line is a commonly used in vitro model in ND research, particularly with diseases such as AD, HD, and PD, in which mitochondrial dysfunction is heavily implicated10,43,71,72,73. The ability to differentiate SH-SY5Y cells into cells with a neuron-like phenotype through manipulating culture media has proven a suitable model for neuroscience research without relying on primary neuronal cells10,74. In this protocol, retinoic acid (RA) was added to the cell culture medium to induce the differentiation of SH-SY5Y cells. RA is a vitamin A derivative and has been shown to regulate the cell cycle and promote the expression of transcription factors that regulate neuronal differentiation75. A protocol for culturing and live cell imaging of neurons isolated from the rat hippocampus is also provided. The hippocampus has been shown to be affected by mitochondrial degeneration and, along with the cortex, plays an important role in aging and ND76,77,78,79,80.

作者聚焦：解码线粒体衰老 

使用活细胞STED成像可视化神经元细胞模型中的线粒体内膜超微结构

Our research program focuses on structural and functional aspects of mitochondria, particularly as they relate to aging related dysfunction. To this end, we use a broad array of biophysical, biochemical, and structural techniques to explore the basic mechanisms of mitochondrial aging and to develop therapeutic interventions to preserve and restore mitochondrial health. Mitochondrial function and structure are inextricably related.Standard light microscopy techniques are suitable for measuring large scale mitochondrial features like basic morphology and network structures. Analyzing mitochondrial ultra structure or features that require higher resolution than afforded by traditional optical microscopy is typically done using electron microscopy or tomography on fixed samples. Super resolution microscopy is a fluorescence based imaging technique that measures features beyond the diffraction limit.For mitochondria, this includes measurements of nano scale protein distribution and cristae architecture. The stead imaging protocol described here allows researchers to measure the detailed structure of the inner membrane in living cells. Live cell imaging allows us to observe and quantitatively analyze complex features of cristae under physiologically relevant conditions without the need for sample fixation.This will give researchers novel insights into the dynamic changes that occur in ultra structural features and their temporal responses to stressors and pharmacological compounds.

该协议描述了培养细胞和原代细胞的细胞生长条件，重点是活细胞STED成像和线粒体嵴的后续分析。使用ImageJ对未分化的SH-SY5Y（图3A）和RA分化的SH-SY5Y（图3B）细胞的线粒体进行的投影可以收集为具有传统共聚焦和STED的z-stacks，然后可以对原始STED图像进行反卷积。也可以进行延时成像，然后进行去卷积（图3C，D）。使用原代大鼠海马神经元的成像参数略有不同（表1），共聚焦和原始STED图像可以作为z堆栈获得，并且可以对原始STED图像进行反卷积（图4A）。也可以对原代神经元的线粒体进行延时成像（图4B）。一般来说，延时图像应该能够显示线粒体动态事件。
当用于分割的样本的原始 STED 和解卷积 STED z 堆栈投影看起来一致时，将执行定量测量。TWS插件使用去卷积的STED图像进行分割以制作概率掩码，然后用于创建嵴的二进制掩码以获得尺寸和形状参数（图5A）。此模板中的区域保存在 ROI 管理器中，如果需要，可以应用于原始 STED 图像以测量相对强度的差异。去卷积的STED投影也可用于确定给定区域的嵴周期性和密度（图5B）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65561/65561fig01.jpg"> 图1：线粒体形态。 线粒体有一个定义不同亚区室 （A） 的双膜系统。Cristae 是具有明确特征的内膜的内褶 （B）。缩写：OMM，线粒体外膜;ICS，嵴内间隙;IMS，膜间空间;CM， 嵴膜;IBM，内边界膜;IMM，线粒体内膜;CT，嵴尖;CJ， 嵴交界处。 <a href="https://www.jove.com/files/ftp_upload/65561/65561fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65561/65561fig02.jpg"> 图 2：工作流程示意图。 SH-SY5Y细胞或原代大鼠海马神经元在PDL涂层的盖玻片上生长。SH-SY5Y细胞平行生长，在六天内保持未分化或RA分化。从海马切片中分离出 7 天后，在 PDL 涂层的盖玻片上生长原代大鼠海马神经元。一旦准备好成像，用PKMO染色细胞并用STED成像。然后对原始STED图像进行反卷积处理，并在FIJI中处理解卷积图像，以获得尺寸和形状测量值，例如嵴密度、面积、周长、圆度和纵横比。 <a href="https://www.jove.com/files/ftp_upload/65561/65561fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65561/65561fig03.jpg"> 图 3：SH-SY5Y 细胞中线粒体的成像。 显示了具有PKMO染色的非分化（A）和RA分化（B）SH-SY5Y细胞的线粒体的代表性共聚焦（左）、原始STED（中）和惠更斯去卷积STED图像z堆栈投影（右）。显示了 RA 分化的 SH-SY5Y 细胞的 30 秒间隔和 5 次迭代的延时摄影 （C），其中选定的区域（白框）在 （D） 上使用这些区域的缩放图像进行扩展，而无需插值。比例尺： A，B，250 nm; C，D， 1 μm. <a href="https://www.jove.com/files/ftp_upload/65561/65561fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65561/65561fig04.jpg"> 图 4：原代大鼠海马神经元中线粒体的成像。 显示了来自原代大鼠海马神经元的线粒体的代表性共聚焦（左）、原始 STED（中）和惠更斯去卷积 STED（右）图像 z 堆栈投影 （A）。显示了这些神经元中线粒体间隔 25 秒和 5 次迭代的延时 （B）。比例尺： A，250 nm; B， 1 μm. <a href="https://www.jove.com/files/ftp_upload/65561/65561fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65561/65561fig05.jpg"> 图 5：在 ImageJ 中处理去卷积的 STED 图像。 显示了 Trainable Weka Segmentation 插件测量嵴大小和形状的代表性用法 （A）。从左到右，显示了以下图像：去卷积的STED图像，基于TWS插件分割的概率图，使用概率图作为输入的FIJI阈值掩码，带有ROI轮廓的掩码，以及覆盖在原始去卷积STED图像上的ROI。与这些物体相对应的面积、周长、圆度和纵横比测量值可在 补充表1中找到。图中显示了使用去卷积STED图像测量峰峰值距离的线图，作为嵴密度的读数（B）。比例尺：0.5 μm。 <a href="https://www.jove.com/files/ftp_upload/65561/65561fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td>
			<td>像素尺寸 （nm）</td>
			<td>停留时间 （μs）</td>
			<td>线路符合</td>
			<td>STED 采集期间的 561 nm 激发 （%）</td>
			<td>775 nm STED 耗尽功率 （%）</td>
			<td>步长 （nm）</td>
			<td>针孔 （AU）</td>
			<td>延时摄影间隔（秒）</td>
			<td>延时迭代</td>
		</tr><tr><td>无差分 SH-SY5Y</td>
			<td>20</td>
			<td>4</td>
			<td>10</td>
			<td>15</td>
			<td>20</td>
			<td>200</td>
			<td>1.0</td>
			<td>30</td>
			<td>5</td>
		</tr><tr><td>RA-Differe-ntiated SH-SY5Y</td>
			<td>20-25</td>
			<td>4</td>
			<td>10-12</td>
			<td>15</td>
			<td>20-22</td>
			<td>150-200</td>
			<td>1.0</td>
			<td>30</td>
			<td>5</td>
		</tr><tr><td>原代神经元</td>
			<td>20-25</td>
			<td>4</td>
			<td>10</td>
			<td>10</td>
			<td>25</td>
			<td>200</td>
			<td>0.7</td>
			<td>30</td>
			<td>5</td>
		</tr><tr><td colspan="10">注意：像素大小可能因成像要求和对图像进行反卷积的意图而异。反卷积需要适当的采样。没有反卷积的原始 STED 图像的像素大小可以达到 30 nm。</td>
		</tr></tbody></table>表 1：STED 采集参数摘要。 显示用于每种细胞类型、未分化 SH-SY5Y、RA 分化的 SH-SY5Y 和原代大鼠海马神经元的 2D STED 成像的设置。对于所有延时摄影，根据 ROI 大小以不同的间隔进行了 5 次迭代。
补充图1：添加淀粉样蛋白-β（Aβ）的SH-SY5Y细胞成像。 显示了具有PKMO染色（顶部）和Aβ-HiLyte647（底部）的RA分化的SH-SY5Y细胞的代表性共聚焦（左）、原始STED（中）和去卷积STED（右）图像（A）。显示了原始 PKMO STED（绿色）与原始 Aβ STED（品红色）（B）或去卷积 PKMO STED（绿色）与去卷积 Aβ STED（品红色）（C） 的合并 z 堆栈投影。比例尺：0.5μm。 <a href="https://www.jove.com/files/ftp_upload/65561/Suppl%20Fig.pdf" target="_blank">请点击这里下载此文件。</a>
补充表1：分段嵴的大小和形状测量。 显示了面积 （μm2）、周长 （μm）、圆度和纵横比的大小和形状测量值，对应于 图 5A 中从分段线粒体中概述的物体。 <a href="https://www.jove.com/files/ftp_upload/65561/65561_Supplementary%20Table%201.xlsx" target="_blank">请点击这里下载此文件。</a>
补充表2：淀粉样蛋白β样品的采集参数摘要。 显示用于未分化和RA分化的SH-SY5Y细胞中PKMO和Aβ-HiLyte647的2D STED成像的设置。Aβ-HiLyte647 的共聚焦可以单独使用，因为没有特定的结构可以解析;此处显示了较小粒径的 Aβ-HiLyte647 的 STED 图像。 <a href="https://www.jove.com/files/ftp_upload/65561/65561_Supplementary%20Table%202.xlsx" target="_blank">请点击这里下载此文件。</a>
补充文件 1：淀粉样蛋白β治疗方案。<a href="https://www.jove.com/files/ftp_upload/65561/65561_Supplementary%20File%201.docx" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Decoding Mitochondrial Aging at JoVE.com

该协议提供了培养的SH-SY5Y细胞和原代大鼠海马神经元的繁殖，分化和染色的工作流程，用于使用受激发射耗竭（STED）显微镜进行线粒体超微结构可视化和分析。

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of ...

我们的研究计划侧重于线粒体的结构和功能方面，特别是与衰老相关的功能障碍。为此，我们使用广泛的生物物理、生化和结构技术来探索线粒体衰老的基本机制，并开发治疗干预措施来保持和恢复线粒体健康。线粒体的功能和结构是密不可分的。标准光学显微镜技术适用于测量大规模线粒体特征，如基本形态和网络结构。分析线粒体超结构或特征需要比传统光学显微镜更高分辨率，通常使用电子显微镜或固定样品断层扫描来完成。超分辨率显微镜是一种基于荧光的成像技术，可测量超出衍射极限的特征。对于线粒体，这包括纳米级蛋白质分布和嵴结构的测量。这里描述的稳定成像方案允许研究人员测量活细胞中内膜的详细结构。活细胞成像使我们能够在生理相关条件下观察和定量分析嵴的复杂特征，而无需固定样品。这将使研究人员对超结构特征中发生的动态变化及其对压力源和药理化合物的时间响应提供新的见解。

using-live-cell-sted-imaging-to-visualize-mitochondrial-inner

Research

JoVE Journal

Neuroscience

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

2.3K 次观看.  University of Connecticut. 我们的研究计划侧重于线粒体的结构和功能方面，特别是与衰老相关的功能障碍。为此，我们使用广泛的生物物理、生化和结构技术来探索线粒体衰老的基本机制，并开发治疗干预措施来保持和恢复线粒体健康。线粒体的功能和结构是密不可分的。标准光学显微镜技术适用于测量大规模线粒体特征，如基本形态和网络结构。分析线粒体超结构或特征需要比传统光学显微?...

视频: 作者聚焦：解码线粒体衰老 

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of reactive oxygen species (ROS). These mitochondria-mediated processes take on specialized roles in neurons, coordinating aerobic metabolism to meet the high energy demands of these cells, modulating Ca2+ signaling, providing lipids for axon growth and regeneration, and tuning ROS production for neuronal development and function. Mitochondrial dysfunction is therefore a central driver in neurodegenerative diseases. Mitochondrial structure and function are inextricably linked. The morphologically complex inner membrane with structural infolds called cristae harbors many molecular systems that perform the signature processes of the mitochondrion. The architectural features of the inner membrane are ultrastructural and therefore, too small to be visualized by traditional diffraction-limited resolved microscopy. Thus, most insights on mitochondrial ultrastructure have come from electron microscopy on fixed samples. However, emerging technologies in super-resolution fluorescence microscopy now provide resolution down to tens of nanometers, allowing visualization of ultrastructural features in live cells. Super-resolution imaging therefore offers an unprecedented ability to directly image fine details of mitochondrial structure, nanoscale protein distributions, and cristae dynamics, providing fundamental new insights that link mitochondria to human health and disease. This protocol presents the use of stimulated emission depletion (STED) super-resolution microscopy to visualize the mitochondrial ultrastructure of live human neuroblastoma cells and primary rat neurons. This procedure is organized into five sections: (1) growth and differentiation of the SH-SY5Y cell line, (2) isolation, plating, and growth of primary rat hippocampal neurons, (3) procedures for staining cells for live STED imaging, (4) procedures for live cell STED experiments using a STED microscope&#160;for reference, and (5) guidance for segmentation and image processing using examples to measure and quantify morphological features of the inner membrane.

This protocol presents a workflow for the propagation, differentiation, and staining of cultured SH-SY5Y cells and primary rat hippocampal neurons for mitochondrial ultrastructure visualization and analysis using stimulated emission depletion (STED) microscopy.

科研

神经科学

Propagation, Differentiation, and Preparation of SH-SY5Y Cells for Live Cell Imaging

To begin, rapidly thaw a frozen stock of SH-SY5Y cells in FBS. Supplemented with 10%DMSO and dilute tenfold with pre-warmed media. Then centrifuge the cells and remove the supernatant.Re-suspend the cell pellet in five milliliters of pre-warmed media and seed cells in a T25 flask. To prepare cover slips coated with PDL apply 600 microliters of 50 micrograms per milliliter PDL solution to each well of sterile chambered cover slips. The volume of PDL added to each well varies based on well size.Incubate the cover slips set room temperature for one hour. After incubation, remove the PDL solution and rinse the cover slip thrice with 1.8 milliliters of distilled water. Allow the coated chamber to air dry for two hours.Once the SH-SY5Y cells reach 80 to 90%confluency, dissociate them by adding trypsin solution. Neutralize the trypsin by adding a pre-warmed DMEM medium. Centrifuge the cell suspension and re-suspend the pellet in DMEM.Count the cells on an automated cell counter. Next, seed the cells at a density of 1.5 times 10 to the fourth cells per square centimeter onto the PDL coated chambered cover glass. On day one and day three, replace the medium with DMEM supplemented with either five or 2%FBS respectively, 1%antibiotic antimycotic, and either 10 micromolar RA or 95%ethanol of the same additive volume to serve as the vehicle control for differentiation.Prepare a PKMO stock in pre-warmed Phenyl red free DMEM supplemented with two or 10%FBS dependent on differentiation state, 1%antibiotic antimycotic, and 20 millimolar hepes. On day six, rinse the cells twice with imaging media and incubate cells with the PKMO solution at 37 degrees celsius and 5%carbon dioxide for 30 minutes. After staining, rinse the cells thrice with pre-warmed imaging media.For the final wash, incubate the cells for 30 minutes at 37 degrees celsius, and 5%carbon dioxide. After incubation, replace the final wash with fresh pre-warmed imaging media containing 20 millimolar hepes.

用于活细胞成像的SH-SY5Y细胞的增殖、分化和制备

Imaging SH-SY5Y Cells by STED Microscopy and Subsequent Analysis of Mitochondrial Ultrastructure

Open the LightBox software for image acquisition. To select the laser and filter sets, use parameters for an orange dye by selecting the dye used in the staining from the dye list. Using an overview, click on live to focus the cells.Once the cells are in focus, adjust the confocal acquisition settings. Next, select the rectangular ROI button and create a region of interest around a mitochondria of interest by clicking and dragging to shape the region. To adjust detector gating for stimulated emission depletion or stead acquisition, next to the general menu, select the gating menu or click and hold to add the menu to the view.For stead acquisition, set the excitation laser to 15 to 20%and the stead depletion laser to 20 to 25%with 10 line accumulations. Use a pixel dwell time of four microseconds and a pixel size of 20 to 25 nanometers. Perform time lapse by selecting the time dropdown menu, then setting the number of iterations to five and a time interval of 25 or 30 seconds.A Z-stack can be taken using the volume option and adjusting the desired Z volume range and step size. Open stead image deconvolution software to deconvolute raw stead images with the software algorithm. After ensuring microscopic parameters are correct, select the express button and set the deconvolution type too fast, standard, aggressive, or conservative for varying degrees of deconvolution power.Execute the deconvolution. Save the images in ICS2 format. In image J, click on file then open to open the ICS2 files from the deconvolution software.Select plugins, then segmentation followed by trainable Weka segmentation to open the deconvoluted stead images in the trainable Weka segmentation plugin. In the segmentation settings, select the Gaussian blur, membrane projections and sobel filter features. Label one class as cristae and the other as background.Next, draw a line over the structure to assign to either class. Then select the add button on the right hand side for either cristae or background. Then select the train classifier button on the left hand side to generate a map based on the information provided to the plugin.Click the save classifier button to save the classifier settings for future segmentation. Use the cristae probability map to threshold the image in image J to generate a binary mask, and then go to analyze, then analyze particles. Finally, draw a multi-point line, adjust the line thickness to several pixels wide and spline the line to fit the mitochondria.In this study, imaging of mitochondria in undifferentiated, retinoic acid differentiated SH-SY5Y cells with time-lapse imaging as shown. The deconvoluted stead images can be used to determine cristae periodicity in a given area and cristae size and shape measurements.