Name: super-resolution imaging to study co-localization of proteins and synaptic markers in primary neurons
Uploaded: 2020-10-31T15:00:00
Description: Watch this Scientific Journal Video about Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons at JoVE.com

作者感谢爱德华多·米科蒂对手稿的建设性批评。这项研究得到了B brightFocus A2019296F、Fondo di Beneficenza - Gruppo Intesa Sanpaolo （LC）、Fdazione 区域每拉赖斯卡生物医学组织 （Care4NeuroRare CP_20/2018） （CN） 和玛丽·斯科多夫斯卡-居里创新培训网络 （JK） 的支持。

1. 主要文化
<ol><li>培养小鼠海马原神经元在室盖玻片（如伊比迪μ-滑动8井或N得不好实验室-Tek室盖玻璃），符合#1.5（0.17毫米）盖玻片厚度的客观要求。</li>
	<li>涂覆室盖玻片，带 100 μL 聚 L-lysine （100 μg/mL）。</li>
	<li>第二天，用无菌磷酸盐缓冲盐水（PBS）清洗两次室盖玻片。</li>
	<li>要获得小鼠原发神经元，请将海马从P1-P4幼崽23中分离。</li>
	<li>将解剖的海马放在10 mL的解剖?...

<ol>
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阐明突触的结构和组成对于理解调节记忆和认知的生理和病理过程至关重要。虽然在正常状态下，突触是记忆的积木，它们也是复杂的神经系统疾病（如阿尔茨海默氏症32）的根据。此处描述的协议用于研究神经元蛋白与称为 SIM 的超分辨率显微镜技术的共同定位。使用特定的照明模式，SIM 可以达到约 0.1 μm 的分辨率，这适用于研究突触，通常测量在 0.03 和 0.15 μm 之间。对于更?...

自从1897年福斯特和谢林顿在1897年第一次描述突触以来，对突触的理解和看法发生了巨大的变化。从那时起，我们对神经元交流的知识及其背后的分子过程呈指数级增长。很明显，突触可以被认为是一个双隔间系统：一个包含用于释放神经递质的囊泡的突触前隔间，以及一个具有受体3的突触后隔间。在过去的二十年里，这种简单化的观点已经演变成一个复杂的蛋白质网络，这些蛋白质是将发射机结合成信号4所需的蛋白质。
理解的收获部分是由于超分辨率技术，克服了传统光显微镜的衍射极限，以适应突触的尺寸更好5，6，7，8，9，10。6,7,8,9,105由于衍射限制，光学显微镜不能达到分辨率超过200纳米横向11，12。11,为了绕过这个限制，建立了超分辨率技术，使用不同的方法，并达到不同的子衍射极限分辨率：SIM，STED（刺激发射耗竭显微镜），PALM（照片激活定位显微镜）和风暴（随机光学重建显微镜）13，,14。SIM通过将衍射光栅插入激励光束路径15，使基于激光的广场显微镜系统的空间分辨率翻倍。可移动光栅可衍射激光束，形成已知的照明模式，通常是条纹。这种有目的的结构光模式叠加到荧光染料（样品）的未知空间分布上。由两种模式形成的干扰边缘编码，否则无法区分的精细细节与正常的宽场显微镜。最终的超解析图像是通过结合和解码与数学方法相同的样本的多个原始图像通过衍射光栅的平移和旋转获得的。超分辨率图像的分辨率达到100纳米的横向和500纳米的轴向2D-SIM15或100nm在横向和250纳米的轴向为3D-SIM16。16
由于许多神经系统疾病，突触功能障碍在发病和进展17，18中起着主要作用，对突触的新理解更为重要。阿尔茨海默病、唐氏综合症、帕金森病、普里昂病、癫痫、自闭症谱系障碍和易碎X综合征等都与突触组成、形态和功能19、20、21、22,20,21,的异常有关。
最近，我们使用一组SUMO特异性抗体，使用SIM显示SUMO蛋白质的原生海马神经元与突触前和后突触标记突触物理和PSD95在超分辨率水平23的共同定位。这使我们能够确认SSUMO在神经元中的定位的生化和共体显微镜证据。
在这里，我们描述了一个协议，研究蛋白质在小鼠海马原神经元的定位。同时，此协议可适应不同类型的原发神经元培养。

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			<td >Alexa</td>
			<td>Thermo Fisher Scientific</td>
			<td>-</td>
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			<td >Antibody SENP1</td>
			<td >Santa Cruz</td>
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			<td>Antibody</td>
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			<td >B27 Supplement</td>
			<td >Life Technologies</td>
			<td >17504044</td>
			<td>Chemical</td>
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			<td >Bovine serum albumin&#160;</td>
			<td>Merck</td>
			<td>5470</td>
			<td>Chemical</td>
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			<td >CaCl2</td>
			<td >Merck Life Science</td>
			<td >21115</td>
			<td>Chemical</td>
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			<td >Chambered coverslips</td>
			<td>Ibidi</td>
			<td>80826</td>
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			<td>Thermo Fisher Scientific</td>
			<td>-</td>
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			<td >FBS (Hyclone)</td>
			<td>GIBCO</td>
			<td>SH3007002 (CHA1111L)</td>
			<td>Serum</td>
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			<td >FluoSpheres carboxylate-modified microspheres, 0.1 &#956;m, yellow&#8211;green fluorescent</td>
			<td>Thermo Fisher Scientific</td>
			<td>F8803</td>
			<td>Equiment</td>
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			<td >Glucose</td>
			<td >Merck Life Science</td>
			<td >G8769</td>
			<td>Chemical</td>
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			<td >Glutamax</td>
			<td>GIBCO</td>
			<td>35050061</td>
			<td>Chemical</td>
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			<td >HEPES</td>
			<td >Merck Life Science</td>
			<td>H3537</td>
			<td>Chemical</td>
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			<td >L-Cystein&#160;</td>
			<td >Merck Life Science&#160;</td>
			<td>C6852-25g</td>
			<td>Chemical</td>
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			<td >MAP2</td>
			<td>Merck</td>
			<td>AB15452</td>
			<td>Antibody</td>
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super-resolution imaging to study co-localization of proteins and synaptic markers in primary neurons

突触是神经元的功能元素，其缺陷或损失是几种神经退行性和神经性疾病的基础。成像研究被广泛用于研究其在生理和病理条件下的功能和可塑性。由于其尺寸和结构，蛋白质的定位研究需要高分辨率成像技术。在该协议中，我们描述了一个程序，用于研究在初级神经元中使用结构化照明显微镜 （SIM） 在超分辨率级别使用突触标记的目标蛋白质的共同定位。SIM 是一种图案照明技术，可使广场显微镜的空间分辨率翻倍，细节达到 100 nm 左右。该协议指示强大的联合本地化研究所需的控制和设置，并概述正确分析成像数据的统计方法。

我们在这里介绍研究神经元蛋白共同定位的标准工作流程。我们首先校准了显微镜，然后对样品进行了 SIM 分析。为了校准系统，我们使用直径为0.1 μm的荧光微球。在获取珠子的超解析 3D-SIM 图像后，对底层图像数据进行 Fourier 转换，以将其重新转换为空间频率表示。在图2A中，不同的花型显示为超分辨率细节水平的指示。接下来，我们测量了通过计算单个珠的强度轮廓峰值...

Watch this Scientific Journal Video about Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons at JoVE.com

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

该协议演示了如何使用超分辨率显微镜来研究蛋白质在原发神经元培养物中的共定位。

超分辨率成像研究原发神经元中蛋白质和突触标记的共同定位

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. ...

突触是神经元的功能元素，其缺陷或损失是几种神经退行性和神经性疾病的基础。成像研究被广泛用于研究其在生理和病理条件下的功能和可塑性。由于其尺寸和结构，蛋白质的定位研究需要高分辨率成像技术。在该协议中，我们描述了一个程序，用于研究在初级神经元中使用结构化照明显微镜在超分辨率水平上与突触标记目标蛋白质的共同定位。SIM 是一种图案照明技术，可使宽场显微镜的空间分辨率翻倍，细节达到约 100 纳米。该协议指示强大的联合本地化研究所需的控制和设置，并概述正确分析成像数据的统计方法。但是，允许超分辨率分析的关键是采集过程中使用的试剂，如与目标衍射指数兼容的室盖玻片和安装解决方案。要获得小鼠海马原发神经元，请将海马从P1分离至P4幼崽。板细胞每井7万个细胞，每井体积200微升。等到海马原发神经元在电镀后12至14天完全成熟，进行共定位研究。将每井PBS 200微升中4%的甲状甲醛PFA加入到神经元中，以快速修复它们。去除溶液，用1%牛血清白蛋白BSA在PBS中孵育样品，每井200微升，在室温下一小时，被动地用不相关的蛋白质覆盖板的所有自由结合表面进行分析。与0.2%Triton X-100的相同缓冲液相比，基于BSA的阻尼缓冲液比0.2%Triton X-100的缓冲液更有效地降低了抗体背景。加入原抗体。作为阴性控制，不要向其中一口油井添加任何原抗体。使用 SIM 兼容的安装剂（例如 ProLong 玻璃抗fade安装剂）安装电池。用盖玻璃盖住和保护细胞，例如直径为八毫米的圆形盖玻璃。也可以使用方形的。将室盖玻片存放在室温下，在获取图像前至少等待 48 小时。钻石玻璃在超分辨率收购前至少需要两天的固化时间。使用两种策略来保证抗体特异性。第一种是使用至少两种不同的抗体，针对同一基质。第二种策略是抗体中和，通过与纯化的蛋白质靶点孵育，或用于提高抗体。我们经常使用尼康制造的 N-SIM 超分辨率显微镜进行分析。以下说明旨在独立于使用显微镜获取 SIM 图像。还必须最大限度地提高显微镜的性能，以便对仪器进行精确校准，包括校正环、激光对焦和光栅块对焦。在采集 SIM 图像之前，系统需要使用特定子分辨率尺寸的荧光珠进行适当的校准。一个例子是四普克微球。这些珠子染色有不同的荧光染料，允许用一个样品校准不同的激光器。在水浴中，声波约1.8倍10到第8荧光微球10分钟。在双蒸馏水中稀释荧光微球的500分之一。再声一次，再用10分钟。将稀释的珠子的15微升移液放入室盖玻片的井中。让溶液在室温下干燥五分钟。添加 10 微升的安装解决方案，例如ProLong 玻璃，在上面放置一个 8 毫米的盖玻片。等待至少 48 小时，以便进行适当的固化。打开显微镜和激光。让系统预热，以达到所有显微镜组件的热平衡，SIM超分辨率显微镜系统至少需要三个小时。选择 100 倍目标。通过将激光器与衍射光栅块的中心对齐来开始校准。在 N-SIM 系统中，一个千分尺旋钮和专用摄像机允许光束向目标中心。插入显微镜的室盖玻片进行观察。通过调整目标校正盖，将系统设置为室体盖玻片厚度。NIS软件是N-SIM超分辨率显微镜系统的专有软件，具有调节颜色校正的自动功能。调整每个通道的光栅块焦点，以确保样品上聚焦的结构化图案照明。该软件为此任务提供自动功能。获取多色微球的原始 3D SIM 图像。计算每个独立波长的超解析图像的 Fourier 变换。如果转换的图像无法获得正确的花样图案，则重新启动校准，因为尚未实现超分辨率。在超解析图像中，选择单个微球并计算每个通道的强度轮廓，以测量实现的分辨率。它现在应该接近100纳米横向。通过覆盖微球的多通道采集来执行通道注册。目标是横向和轴向地混合所有通道信号。这将消除不同通道错位导致的色差，并帮助共同本地化分析。通过使用 SIMcheck 的功能照明相位步骤和照明模式焦点（一套用于开源应用程序 ImageJ 的插件）确认校准质量。在共和或宽场模式下使用 40 倍目标开始分析样本。这允许导航示例，保持良好的细节和较大的视野。使用 MAP2 抗体信号检测表示神经元过程的区域。在共合模式下获取样品的图像，以确定染色的质量。切换到目标 100X。将机油涂抹到 100 倍目标上。获取宽场或共和图像，稍后将用于评估超解析图像的质量。切换到 3D SIM 模式。使用对话窗口设置采集参数，选择可用的最高位深度设置以最大化颜色信息。通常，16 位是标准选择。此外，为了提高信噪比，请选择低频值进行采集，例如一兆赫。使用直方图窗口，设置激光功率，获得信号的线性响应，避免信息丢失，限制图像中的饱和像素。N-SIM 系统使用 Andor iXon3 摄像机。当工作在 16 位时，选择 16，000 的目标强度，以确保摄像机的线性响应。或者，选择 30，000 到 45，000 之间的范围，以最大化收购的动态范围。在成像样品时将激光功率设置为 0.1% 到 50%，曝光时间设置为 50 毫秒到 2 秒之间。超过 50% 的激光功率可能导致使用的荧光团快速光漂白。开始以 3D SIM 模式获取图像。使用 SIMcheck，ImageJ 的一套免费插件，评估原始图像的采集质量。如果 SIMcheck 未检测到任何伪影或质量问题，请从完整的技术复制中获取至少 10 张图像，以便进行统计分析。采集的图像是需要处理的原始数据，以获取用于进一步分析的重建 SIM 图像。在协议中，我们建议使用突触物理和PSD-95作为突触前和后突触标记。但是，可以使用其他标记。大量的文献支持使用其他蛋白质，如德布林和低音作为突触标记。3D SIM 采集的图像是原始图像，需要处理才能获取重建的超解析图像。原始图像的重建不正确会导致伪影，影响样本的分析。因此，应高度重视正确选择重建参数。使用显微镜重建分析软件处理原始图像，以获得超解析图像。或者，使用免费可用的 ImageJ 插件 fairSIM 来重建原始图像。使用显微镜重建软件或 ImageJ 插件 SIMcheck 计算超解析图像的 Fourier 变换。对于每个通道，一个好的重建图像应返回一个花一样的图像。如果重建的图像无法重新创建花状形状，请从原始图像重新启动，然后通过修改重建参数（如线性滤波、apodization 和零阶抑制）进行重建。在 NIS 软件中，使用预览来监视更改参数如何影响最终解析的图像、修改参数的照明调制对比度、高分辨率噪声抑制和对焦抑制。使用基于 ImageJ 的插件 NanoJ-SQUIRREL 分析重建的图像，以公正地检测伪影，以评估超解析图像的质量。在分析共同本地化时，我们使用了两种方法。第一种是基于配置文件分析的可视化方法，它显示联合本地化的单个事件，并确定每个通道的贡献。作为研究突触标记和感兴趣的蛋白质之间的共同定位的第一步，采取超解析的图像，并分析单个位置以确定信号重叠。识别超解析图像上的单个位点。获取荧光信号的强度剖面到感兴趣的位点。如果剖面分析建议单位点共定位，则可以通过计算 Pearson 和 Mander 的系数对整个图像进行更一般性的分析。使用JACoP，一个图像J插件，确定两个参数的共同本地化，皮尔逊和曼德的。在校准了系统并评估了重建图像的质量之后，我们接下来开始分析与 MAP2、神经元标记、PSD-95、后突触标记和我们的目标蛋白质 SUMO1 有抗体染染的主要神经元培养物。我们首先分析了以40倍目标执行四通道共体显微镜的样品。在选择表示神经元过程的区域时，我们切换到了 100 倍的目标。我们获取了同一区域的共和图像和 SIM 图像，以评估 NanoJ-SQUIRREL 的重建质量，并执行共同定位分析。阐明突触的结构和组成对于理解调节记忆和认知的生理和病理过程至关重要。虽然在正常状态下突触是记忆的构建基块，但它们也是复杂神经系统疾病的基础，例如痴呆症最常见的疾病之一。此处描述的协议允许使用称为结构化照明显微镜的超分辨率显微镜技术研究突触的蛋白质组成。

Research

JoVE Journal

Neuroscience

Laser Capture Microdissection of Drosophila Peripheral Neurons

Laser Capture Microdissection of Drosophila Peripheral Neurons

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

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

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

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 ...

Super-resolution Imaging of Neuronal Dense-core Vesicles

神经元的密集核心囊泡的超分辨率成像

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

初级量子神经元轴突运输的实时成像点标记的BDNF

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

海马神经元中突触囊泡吞的测量

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

Overexpressing 突触前蛋白培养神经元突触囊泡再生的光学检测

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...