用于研究髓磷脂中央微管蛋白依赖性缺陷的无标记非线性光学器件
1Centro de Investigaciones en Óptica A.C. (CIO), 2Division of Sciences and Engineering, Department of Chemical, Electronic, and Biomedical Engineering,Universidad de Guanajuato, 3Institute of Physiology,Benemérita Universidad Autónoma de Puebla, 4Dirección General de Desarrollo Internacional,Benemérita Universidad Autónoma de Puebla
Summary
在本文中，我们提出了一种协议，通过简单，创新的二次谐波产生显微镜方法检测微管负载的少突胶质细胞在微管疾病模型中。
Abstract
大脑中细胞骨架成分的令人满意的可视化具有挑战性。微管、微丝和中间丝网络在所有神经组织中的普遍分布，以及荧光蛋白融合策略结果的可变性以及它们对作为发色载体的抗体和药物的动态研究的有限适用性，使得经典的光学方法不如其他蛋白质有效。当需要研究微管蛋白时，由于分子的非中心对称组织，二次谐波的无标记产生是非常合适的选择。当与显微镜结合时，该技术可以定性地描述生物样品中平行微管束的体积分布，具有处理未固定和未透化的新鲜组织的额外优势。这项工作描述了如何使用商业的二次谐波生成显微镜设置对微管蛋白进行成像，以突出显示少突胶质细胞富含微管蛋白的结构中的微管，如髓鞘功能减退伴基底神经节萎缩和小脑（H-ABC）微管蛋白病，一种最近描述的髓鞘疾病。
Introduction
组织和器官制剂中细胞骨架结构的光学成像并非易事。细胞骨架丝无处不在，因此，如果对上皮样品中的α-微管蛋白或β-肌动蛋白或潜在的角蛋白进行通用染色，则信号可能会在整个样品中均匀分布。为了将染色限制在更有意义的细胞成分亚群中，可以使用靶向表达1的转基因小鼠或计划使用亚型特异性抗体。虽然后者很少在市场上（而且很少存在2，3，4），但转基因动物模型可能是可用的。但是，它需要由实验室购买并妥善安置，以及该过程所涉及的所有费用。某些抗体或化学物质，例如荧光团偶联药物，如鬼笔环肽或紫杉醇，可能部分或完全与活细胞或组织的使用不相容，因此将其适用性限制在仅研究固定样品。
在微管蛋白的情况下，必须考虑另一个方面，即聚合物对固定的敏感性。众所周知，用甲醛进行常规化学固定不足以最佳地保持微管的完整性5。此外，最近的一份报告证实，甲醛交联诱导微管超微结构的细微变化，类似于某些药物或生理分子（如GTP6）结合时发生的情况。
因此，在未染色、未固定的样品中直接观察微管通常是可取的。为了实现这一目标，一种技术解决方案是二次谐波产生（SHG）显微镜7，它基于平行微管束作为干扰团并在用强烈的脉冲红外激光正确照射时发射倍频光的能力。虽然胶原蛋白和肌球蛋白可以产生更强，更稳定的二次谐波信号，这是已知唯一能够倍频的另外两种生物材料，但迄今为止，来自微管蛋白的信号主要用于研究有丝分裂纺锤体重排8，9，10和轴突微管形态11，12，13。
在这项工作中，我们介绍了SHG显微镜作为诊断工具的新用途，以区分受微管蛋白β4A（TUBB4A）微管蛋白病影响的中枢神经系统（CNS）组织与健康对应物14。在这种主要由神经性微管蛋白亚型引起的一些突变中发生的突变，如导致基底神经节和小脑（H-ABC）髓鞘减退和萎缩的突变，诱导少突胶质细胞中的微管过度充盈15，16;反过来，细胞骨架改变与髓鞘形成等下游效应有关，运动和感觉通路严重受损16，17，18，19。这项工作中使用的taiep小鼠模型在少突胶质细胞中显示出异常的微管含量，并概括了H-ABC患者的大多数感觉运动症状17。该协议解释了如何将结构成像为胼胝体和小脑，它们通常是高度髓鞘的，并且在人类患者以及taiep rat19中受到严重影响，以突出健康和突变组织之间SH信号的差异。
Protocol
所描述的所有程序均符合墨西哥政府《关于健康研究的一般卫生法条例》（NOM-062-ZOO-1999）第七章中批准的法律和法规，并符合《美国国立卫生研究院实验动物护理和使用指南》的建议，并经瓜纳华托大学和贝内梅里塔自治大学生物伦理学研究机构委员会批准 普埃布拉。 1. 显微镜设置 打开显微镜系统。 打开脉冲激光器，以确保在样品提取和制备后…
Representative Results
由于生物组织中存在的异构载体数量非常有限，因此使用该方法获得的图像具有固有的低背景水平，这是该方法的显着优势之一。 当对胼胝体的纤维进行成像时，可以在太极脑中一致地找到纤维状的短结构和圆形元素（图3B），而对照脑的胼胝体在整个大脑区域显示出更加异质和各向同性的信号（图3A）。差分信号的起源具?…
Discussion
SHG显微镜是一组非线性光学技术的一部分，其中包括双光子激发显微镜，三次谐波产生显微镜和相干反斯托克斯拉曼散射显微镜，这些技术有助于将传统光学显微镜的应用范围扩展到生命科学20。
具体来说，SHG显微镜的主要优点和缺点与相同的条件有关：信号发生器是非中心对称的21。这种特殊的建筑条件通常存在于无机和有机晶体领域，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家科学与技术委员会（CONACYT）通过以下赠款的支持：infraestructura 226450给副总裁-CIO，infraestructura 255277给副总裁，FORDECYT-PRONACES/194171/2020给V.H.。我们感谢CIO的Juvenal Hernández Guevara在视频制作方面的支持。
Materials
| 405/10 nm BrightLine(R) single-band bandpass filter | Semrock | FF01-405/10-25 | 32 mm diameter, with housing ring |
| Black Nylon, Polyurethane-Coated Fabric | Thorlabs | BK5 | 5' x 9' (1.5 m x 2.7 m) x 0.005" (0.12 mm) Thick |
| Blades for vibratome | any commercial; e.g. Wilkinson Sword | Classic stainless steel double edge razor blades | |
| Cell culture dishes, 35 mm | any commercial; e.g. Falcon | 351008 | |
| Confocal microscope | Zeiss | LSM710NLO AxioObserver Z1 | Inverted microscope, objective used is LCI Plan-Neofluar 25x/0.8 NA |
| Cooler | any commercial | Any insulated, polystyrene box could work, to mantain the sample at about 37 °C | |
| Corn stach | e.g. Maizena | From the supermarket | |
| Coverslips #1.5 | any commercial | Rectangular | |
| Cyanoacrylate glue | e.g. Loctite | To glue the brain to the masking tape | |
| Fine forceps | fine science tools | 11412-11 | To manipulate tissue sections by handling from the meninges |
| Fine scissors | fine science tools | 14370-22 | To cut the skin |
| Fine scissors curved tip | fine science tools | 14061-09 | To cut along the midline |
| Formaldehyde 37% | Sigma-Aldrich | 252549 | To dilute 1:10 in PBS |
| Friedman Rongeur | fine science tools | 16000-14 | To cut the bone |
| Gel packs | any commercial | Prewarmed to 37 °C, to help mantaining the temperature inside the cooler | |
| Glass Pasteur pipette, modified | any commercial | To transfer the tissue section | |
| Hanks′ Balanced Salt solution (HBSS) | Gibco | 14025-076 | Could be prepared from powders |
| Kelly hemostats | fine science tools | 13018-14 | To separate the bone |
| Masking tape | any commercial | To protect th surface of the specimen plate | |
| NDD module, type C | Zeiss | 000000-1410-101 | To detect the signal, reducing light loss. Housing the 000000-1935-163 filter set with the SP485 |
| Offset bone nippers | fine science tools | 16101-10 | To cut the bone |
| Phosphate buffered saline (PBS) | Gibco | 10010-031 | Could be prepared from powders or tabs |
| Pulsed laser | Coherent | Chameleon Vision II | 680–1080 nm tunable laser |
| Scalpel | any commercial | Straight blade with sharp point | |
| Standard pattern forceps | fine science tools | 11000-18 | |
| Vannas spring scissors | fine science tools | 15018-10 | To cut meninges that remain joined to both the slice obtained from vibratome cutting and the section glued to the specimen plate. |
| Vibratome | any commercial; e.g. Leica | VT1200 |
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Cite This Article
Piazza, V., Alata, M., Hernandez, V. H., Eguibar, J. R., Cortes, C. Label-Free Non-Linear Optics for the Study of Tubulin-Dependent Defects in Central Myelin. J. Vis. Exp. (193), e63449, doi:10.3791/63449 (2023).