Epon Post 嵌入相关光和电子显微镜
Summary
我们提出了使用称为 mScarlet 的荧光蛋白进行 Epon 嵌入后相关光学和电子显微镜的详细方案。这种方法可以同时保持荧光和超微结构。该技术适用于各种生物应用。
Abstract
相关光电子显微镜（CLEM）是一种综合显微镜，它结合了荧光显微镜（FM）提供的定位信息和电子显微镜（EM）获取的细胞超微结构的背景。CLEM是荧光和超微结构之间的权衡，通常，超微结构会影响荧光。与其他亲水性包埋树脂（如甲基丙烯酸缩水甘油酯、HM20或K4M）相比，Epon在超微结构保存和切片性能方面更胜一筹。此前，我们已经证明mEosEM可以在四氧化锇固定和Epon包埋中存活。使用mEosEM，我们首次实现了EPON后嵌入CLEM，它同时保持荧光和超微结构。在这里，我们提供了有关 EM 样品制备、FM 成像、EM 成像和图像对齐的分步详细信息。我们还改进了在 EM 成像期间识别 FM 成像的相同细胞的程序，并详细说明了 FM 和 EM 图像之间的配准。我们相信，在传统的电磁设施中，按照这种新协议，可以很容易地实现嵌入相关光和电子显微镜的Epon后。
Introduction
荧光显微镜（FM）可用于获得靶蛋白的定位和分布。然而，靶蛋白周围的背景丢失了，这对于彻底研究靶蛋白至关重要。电子显微镜 （EM） 具有最高的成像分辨率，可提供多个亚细胞细节。然而，EM缺乏靶标标记。通过将FM采集的荧光图像与EM采集的灰度图像精确合并，相关光电子显微镜（CLEM）可以将这两种成像模式1,2,3,4获得的信息结合起来。
CLEM 是荧光和超微结构之间的权衡 1.由于目前荧光蛋白和传统电镜样品制备程序的局限性，特别是使用锇酸（OsO4）和疏水性树脂（如Epon），超微结构总是影响荧光5。OsO4 是 EM 样品制备中不可或缺的试剂，用于提高 EM 图像的对比度。与其他包埋树脂相比，Epon 在超微结构保存和切片性能方面更胜一筹5。然而，在OsO4 和Epon embedding6处理后，没有荧光蛋白可以保留荧光信号。为了克服荧光蛋白的局限性，开发了预包埋 CLEM，其中 FM 成像在 EM 样品制备之前完成 6。然而，预嵌入 CLEM 的缺点是 FM 和 EM 图像之间的配准不精确5.
相反，嵌入后CLEM方法在EM样品制备后进行FM成像，其配准精度可达6-7nm 5,6。为了保留荧光蛋白的荧光，使用极低浓度的OsO4（0.001%）3或高压冷冻（HPF）和冷冻取代（FS）EM制备方法4,7，以牺牲超微结构受损或EM图像的对比度为代价。尽管使用甲基丙烯酸缩水甘油酯作为包埋树脂5，但mEos4b的发展极大地促进了包埋后CLEM的进展。随着 mEosEM 的发展，该 mEosEM 可以在 OsO4 染色和 Epon 包埋中存活下来，首次实现了 Epon 后包埋超分辨率 CLEM，同时保持了荧光和超微结构6.在 mEosEM 之后，开发了几种可以在 OsO4 染色和 Epon 包埋中存活的荧光蛋白 8,9,10,11。这极大地促进了CLEM的发展。
Epon 嵌入后 CLEM 有三个关键方面。首先是荧光蛋白，在EM样品制备后应保持荧光信号。根据我们的经验，mScarlet优于其他报道的荧光蛋白。第二个是如何在EM成像中找到通过FM成像成像的相同细胞。为了解决这个问题，我们改进了这一步的程序，以便人们可以很容易地找到目标细胞。最后一种是将 FM 图像与 EM 图像对齐的方法。在这里，我们详细介绍了 FM 和 EM 图像之间的配准。在该协议中，我们在 VGLUT2 神经元中表达 mScarlet，并证明 mScarlet 可以使用 Epon 嵌入后 CLEM 靶向二级溶酶体。我们提供了 Epon 嵌入后 CLEM 的分步细节，而不会影响荧光和超微结构。
Protocol
Representative Results
Discussion
这里介绍的方案是一种通用成像方法，它可以结合光学显微镜 （LM） 中靶蛋白的定位信息和电子显微镜 （EM） 中靶蛋白周围的背景6。由于目前荧光蛋白的局限性，广泛使用的方法是预嵌入相关光和电子显微镜 （CLEM），这意味着 LM 成像是在 EM 样品制备之前完成的。几乎所有现有的荧光蛋白都可以在预包埋CLEM中进行检查。然而，由于不可避免的失真和收缩，最终图像的精确对?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
该项目得到了国家自然科学基金（32201235傅志飞）、福建省自然科学基金（2022J01287致傅志飞）、福建医科大学先进人才研究基金（XRCZX2021013傅志飞）、福建省金融专项科学基金（22SCZZX002致傅志飞）的支持。 国家卫健委非人灵长类动物生育力调控技术评价重点实验室、福建省妇幼保健院成立（2022-NHP-04致傅志飞）。我们感谢福建医科大学公共技术服务中心的Linying 周、Minxia Wu、习 Lin和Yan 胡在EM样品制备和EM成像方面的支持。
Materials
| 0.2 M Phosphate Buffer (PB) | NaH2PO4 · 2H2O+Na2HPO4 · 12H2O | ||
| 0.2 M Tris-Cl (pH 8.5) | Shanghai yuanye Bio-Technology | R26284 | |
| 25% Glutaraldehyde (GA) | Alfa Aesar | A17876 | Hazardous chemical |
| Abbelight 3D | Nanolnsights | ||
| Acetone | SCR | 10000418 | |
| Ammonium hydroxide | J&K Scientific | 335213 | |
| BioPhotometer D30 | eppendorf | ||
| Cleaning buffer of cover glasses | 50 mL Ammonium hydroxide, 50 mL Hydrogen peroxide, 250 mL H2O | ||
| Coverglass | Warner | 64-0715 | |
| DABCO | Sigma | 290734 | Hazardous chemical |
| DDSA | SPI company | GS02827 | Hazardous chemical |
| Desktop centrifuge | WIGGENS | MINICEN 10E | |
| Diamond knife | DiATOME | MX6353 | |
| DMP-30 | SPI company | GS02823 | Hazardous chemical |
| DNA transfection reagent | Thermo Fisher | 2696953 | Lipofectamine 3000 Transfection Kit |
| Epon 812 | SPI company | GS02659 | Hazardous chemical |
| Ethanol | SCR | 10009218 | |
| Fiji image J | National Institutes of Health | ||
| Fixative solution | 4% PFA+0.25% GA+0.02 M PB | ||
| Formvar | Sigma | 9823 | |
| Glycerol | SCR | 10010618 | |
| Gold nanoparticles | Corpuscular | 790120-010 | |
| Gradient resin | Acetone to resin 3:1, 1:1, 1:3 | ||
| Hydrofluoric acid | SCR | 10011118 | |
| Hydrogen peroxide | SCR | 10011218 | |
| ICY (https://icy.bioimageanalysis.org/about/) | Easy CLEMv0 Plugin | ||
| Imaging chamber | Thermo Fisher | A7816 | |
| Large gelatin capsules | Electron Microscopy Sciences | 70117 | |
| Mounting buffer | Mowiol 4-88, Glycerol, 0.2 M Tris-Cl (pH 8.5), DABCO | ||
| Mowiol 4-88 | Sigma | 9002-89-5 | |
| Na2HPO4 ž12H2O | SCR | 10020318 | |
| NaH2PO4 ž2H2O | SCR | 20040718 | |
| NMA | SPI company | GS02828 | Hazardous chemical |
| Oligonucleotide primers | Takara Biomedical Technology (Beijing) | Three oligonucleotides primers were used to detect Vglut2-ires-Cre and wild-type simultaneously. The primers 5,-ATCGACCGGTAATGCAGGCAA-3, and 5,-CGGTACCACCAAATCTTACGG-3, aimed to detect Vglut2-ires-Cre. The primers 5,-CGGTACCACCAAATCTTACGG-3, and 5,-CATGGTCTGTTTTGAATTCAG-3, aimed to detect wild-type. | |
| Oscillating microtome | Leica | VT1000S | |
| Osmium tetroxide | SCR | L01210302 | Hazardous chemical |
| OsO4 solution | 1% Osmium tetroxide+1.5% K4Fe (CN)6·3H2O | ||
| Parafilm | Amcor | PM-996 | |
| Paraformaldehyde (PFA) | SCR | 80096618 | Hazardous chemical |
| Perfusion buffer | 4% PFA+0.1 M PB | ||
| Pioloform | Sigma | 63148-65-2 | Hazardous chemical |
| Poly-L-lysine | Sigma | 25986-63-0 | |
| Potassium ferrocyanide (K4Fe (CN)6·3H2O) | SCR | 10016818 | |
| Scalpel blades | Merck | S2771 | |
| Scalpel handles | Merck | S2896-1EA | |
| Stereomicroscope | OLYMPUS | MVX10 | |
| Transgenic mice | The Jackson Laboratory | Vglut2-ires-Cre mice (strain: 129S6/SvEvTac) were housed in standard conditions (25 °C, a 12 h light/dark cycle, with water and food given ad libitum. Male and Female mice were used at 2–3 months old, weight range 20-30 g. | |
| Transmission electron microscope (TEM) | FEI | TECNAL G2 | |
| UA solution (2% UA) | Aqueous solution | ||
| Ultramicrotome | Leica | LEICA EM UC6 | |
| Uranyl acetate (UA) | TED PELLA | 19481 | Hazardous chemical |
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