使用拉曼染料进行高度多重组织成像
Summary
彩虹状拉曼染料的电子预共振受激拉曼散射（epr-SRS）成像是高度多重表位蛋白质成像的新平台。在这里，我们提供了一个实用指南，包括抗体制备，组织样品染色，SRS显微镜组装和epr-SRS组织成像。
Abstract
可视化组织中广泛的特定生物标志物在探索复杂生物系统的复杂组织方面起着至关重要的作用。因此，高度多路复用的成像技术越来越受到赞赏。在这里，我们描述了一个新兴的平台，通过彩虹状拉曼染料的电子预共振激发拉曼散射（epr-SRS）成像，对特定蛋白质进行高度多重振动成像，其灵敏度与标准免疫荧光相当。该方法规避了传统免疫荧光中光谱可分辨通道的极限，并提供了一种一次性光学方法，以亚细胞分辨率询问组织中的多个标记物。它通常与标准组织制剂相容，包括多聚甲醛固定组织，冷冻组织和福尔马林固定石蜡包埋（FFPE）人体组织。我们设想该平台将提供生物标本蛋白质相互作用的更全面图片，特别是对于厚的完整组织。该协议提供了从抗体制备到组织样品染色，再到SRS显微镜组装，再到epr-SRS组织成像的工作流程。
Introduction
复杂的组织系统由不同的细胞亚群组成，其空间位置和相互作用网络与其功能和功能障碍深深交织在一起1，2。为了揭示组织结构并质疑其复杂性，了解蛋白质在单细胞分辨率下的空间位置至关重要。因此，高度多重的蛋白质成像技术越来越受到赞赏，并可能成为研究组织生物学的基石3，4，5。目前常用的多重蛋白质成像方法可分为两大类。一种是依靠多轮组织染色和成像的连续免疫荧光成像，另一种是成像大量细胞术与重金属标记抗体6，7，8，9，10，11，12。
在这里，介绍了基于多重抗体的蛋白质成像的替代策略。与流行的荧光成像模式不同，由于激发和发射光谱宽（半最大值全宽（FWHM）~500 cm-1），拉曼显微镜只能同时显示4-5个通道，拉曼显微镜表现出更窄的光谱线宽（FWHM~10 cm-1），因此提供可扩展的多重性。最近，通过利用窄光谱，开发了一种名为电子预共振激发拉曼散射（epr-SRS）显微镜的拉曼显微镜新方案，为多路复用成像提供了强大的策略13。通过探测拉曼染料的电子耦合振动模式，epr-SRS在拉曼横截面上实现了10倍的大幅增强效果，并克服了传统拉曼显微镜的灵敏度瓶颈（图1A）13，14，15。因此，epr-SRS的检测限已被推至亚μM，这使得拉曼能够检测出有趣的分子标记物，例如细胞内的特定蛋白质和细胞器13，16。特别是，利用拉曼染料偶联抗体，细胞和组织中特定蛋白质的epr-SRS成像（称为免疫-eprSRS）被证明具有与标准免疫荧光相当的灵敏度（图1B）13，17。通过仅将泵浦波长调谐2 nm，epr-SRS信号将完全关闭（图1B），这显示出高振动对比度。
在探针侧，已经开发了一组称为曼哈顿拉曼散射（MARS）染料的彩虹状拉曼探针，用于抗体偶联13，18，19，20。这种独特的拉曼调色板由具有π共轭三键（补充材料）的新型染料组成，每种染料在生物正交拉曼光谱范围内显示单个窄的epr-SRS峰（图1C）。通过修改核心发色团的结构并同位素编辑三键的两个原子（补充材料），已经开发了光谱分离的拉曼探针。利用可扩展的多重性，epr-SRS显微镜与MARS染料调色板相结合，为细胞和组织中的一次性多重蛋白质成像提供了一种光学策略。
Immuno-eprSRS为当前具有独特优势的多重蛋白质成像方法提供了一种替代策略。与具有循环染色、成像和信号去除功能的荧光方法相比，这种基于拉曼的平台可确保单轮染色和成像。因此，它规避了循环程序的实际复杂性，并在很大程度上简化了方案，从而开辟了多重蛋白质成像的新领域。例如，利用拉曼染料定制的组织清除方案，immuno-eprSRS已扩展到三维，用于在厚完整组织中进行高度多重的蛋白质图谱17。沿着毫米厚的小鼠脑组织观察超过10个蛋白质靶标17。最近，将免疫-eprSRS与优化的生物分子保留膨胀显微镜（ExM）方案21偶联，还证明了多个靶标的一次性纳米级成像22。与成像质谱4，9相比，epr-SRS是无损的，并且具有固有的光学切片能力。此外，epr-SRS在组织扫描上更省时。通常，对于单个epr-SRS通道，像素尺寸为0.5μm的0.25 mm 2 的组织区域只需几分钟即可成像。例如， 图4 中四个SRS通道加四个荧光通道的总成像时间约为10分钟。
Protocol
该协议是根据哥伦比亚大学机构动物护理和使用委员会批准的动物实验方案（AC-AABD1552）进行的。 1. 拉曼染料偶联抗体的制备 将偶联缓冲液制备为PBS缓冲液中〜0.1M NaHCO3 ，pH = 8.3，储存在4°C。 在无水DMSO中制备N-羟基琥珀酰亚胺（NHS）酯功能MARS探针（补充材料）溶液作为3mM。MARS探测器的合成可参考先前的报告13</su…
Representative Results
图3显示了不同样品中epr-SRS的示例图像，包括固定细胞（图3A），多聚甲醛（PFA）固定小鼠组织（图3B）和福尔马林固定石蜡包埋（FFPE）人类标本（图3C）。SRS显微镜的空间分辨率受衍射限制，典型的横向分辨率约为300 nm，使用近红外光进行激发的轴向分辨率为1-2 μm。结果，通过α微管蛋白的免疫eprSRS成像，?…
Discussion
在这里，我们提出了免疫-eprSRS方案，该方案广泛适用于常见的组织类型，包括新鲜保存的小鼠组织，FFPE人体组织和冷冻小鼠组织。免疫-eprSRS已经验证了细胞和组织中的一组表位，如 表1所示。这种一次性平台特别适用于循环策略不能很好地运行的应用程序。例如，循环荧光对厚组织的要求很高，因为多轮3D免疫标记是不切实际的冗长17。由于非线性3D组织学变化<sup c…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Ruth A. Singer和Richard K.P. Benninger提供小鼠胰腺组织。W.M.感谢NIH R01（GM128214），R01（GM132860），R01（EB029523）和美国陆军（W911NF-19-1-0214）的支持。
Materials
| 16% Paraformaldehyde, EM Grade | Electron Microscopy Sciences | 15710 | |
| α-tubulin | Abcam | ab18251 | Primary antibodies |
| α-tubulin | BioLegend | 625902 | Primary antibodies |
| β-III-tubulin | BioLegend | 657402 | Primary antibodies |
| β-III-tubulin | Abcam | ab41489 | Primary antibodies |
| β-tubulin | Abcam | ab131205 | Primary antibodies |
| Agarose, low gellling temperature | Sigma Aldrich | A9414 | For brain embedding |
| Anti-a-tubulin antibody produced in rabbit (α-tubulin) | Abcam | ab52866 | Primary antibodies |
| Anti-Calbindin antibody produced in mouse (Calbindin) | Abcam | ab82812 | Primary antibodies |
| Anti-GABA B receptor R2 antibody produced in guinea pig (GABA B receptor R2) | Millipore Sigma | AB2255 | Primary antibodies |
| Anti-GFAP antibody produced in goat (GFAP) | Thermo Scientific | PA5-18598 | Primary antibodies |
| Anti-Glucagon antibody produced in mouse (Glucagon) | Santa Cruz Biotechnology | sc-514592 | Primary antibodies |
| Anti-insulin antibody produced in guinea pig (insulin) | DAKO | IR00261-2 | Primary antibodies |
| Anti-MBP antibody produced in rat (MBP) | Abcam | ab7349 | Primary antibodies |
| Anti-NeuN antibody produced in rabbit (NeuN) | Thermo Scientific | PA5-78639 | Primary antibodies |
| Anti-Pancreatic polypeptide (PP) antibody produced in goat- Pancreatic polypeptide (PP) | Sigma Aldrich | SAB2500747 | Primary antibodies |
| Anti-Pdx1 antibody produced in rabbit (Pdx1) | Milipore | 06-1379 | Primary antibodies |
| Anti-Somatostatin antibody produced in rat (Somatostatin) | Abcam | ab30788 | Primary antibodies |
| Anti-Vimentin antibody produced in chicken (Vimentin) | Abcam | ab24525 | Primary antibodies |
| Band-pass filter | KR Electronics | KR2724 | 8 MHz |
| BNC 50 Ohm Terminator | Mini Circuits | STRM-50 | |
| BNC cable | Thorlabs | 2249-C | Coaxial Cable, BNC Male / Male |
| Broadband dielectric mirror | Thorlabs | BB1-E03 | 750 – 1100 nm |
| C57BL/6J mice | Jackson Laboratory | 000664 | |
| Centrifuge | |||
| Condenser | Olympus | oil immersion, 1.4 N.A. | |
| Cytokeratin 18 | Abcam | ab7797 | Primary antibodies |
| Cytokeratin 18 | Abcam | ab24561 | Primary antibodies |
| DC power supply | TopWard | 6302D | Bias voltage is 64 V |
| Dichroic mount | Thorlabs | KM100CL | Kinematic Mount for up to 1.3" (33 mm) Tall Rectangular Optics, Left Handed |
| Donkey anti-Chicken IgY (H+L) | Jackson ImmunoResearch | 703-005-155 | Secondary antibodies for MARS conjugation |
| Donkey anti-Goat IgG (H+L) | Jackson ImmunoResearch | 705-005-147 | Secondary antibodies for MARS conjugation |
| Donkey anti-Guinea Pig IgG (H+L) | Jackson ImmunoResearch | 706-005-148 | Secondary antibodies for MARS conjugation |
| Donkey anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 715-005-151 | Secondary antibodies for MARS conjugation |
| Donkey anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | 711-005-152 | Secondary antibodies for MARS conjugation |
| Donkey anti-Rat IgG (H+L) | Jackson ImmunoResearch | 712-005-153 | Secondary antibodies for MARS conjugation |
| Donkey anti-Sheep IgG (H+L) | Jackson ImmunoResearch | 713-005-147 | Secondary antibodies for MARS conjugation |
| DPBS | Fisher Scientific | 14-190-250 | |
| EpCAM | Abcam | ab71916 | Primary antibodies |
| Ethanol | Sigma Aldrich | 443611 | |
| Fast-speed look-in amplifier | Zurich Instruments | HF2LI | DC – 50 MHz |
| FFPE Kidney Sample | USBiomax | HuFPT072 | |
| Fibrillarin | Abcam | ab5821 | Primary antibodies |
| Giantin | Abcam | ab24586 | Primary antibodies |
| Glucagon | Santa Cruz Biotechnology | sc-514592 | Primary antibodies |
| H2B | Abcam | ab1790 | Primary antibodies |
| HeLa | ATCC | ATCC CCL-2 | |
| High O.D. bandpass filter | Chroma Technology | ET890/220m | Filter the Stokes beam and transmit the pump beam |
| Hydrophobic pen | Fisher Scientific | NC1384846 | |
| Insulin | ThermoFisher | 701265 | Primary antibodies |
| Integrated SRS laser system | Applied Physics & Electronics, Inc. | picoEMERALD | picoEMERALD provides an output pulse train at 1,064 nm with 6-ps pulse width and 80-MHz repetition rate, which serves as the Stokes beam. The frequency doubled beam at 532 nm is used to synchronously seed a picosecond optical parametric oscillator (OPO) to produce a mode-locked pulse train with five~6 ps pulse width (the idler beam of the OPO is blocked with an interferometric filter). The output wavelength of the OPO is tunable from 720–950 nm, which serves as the pump beam. The intensity of the 1,064-nm Stokes beam is modulated sinusoidally by a built-in EOM at 8 MHz with a modulation depth of more than 90%. The pump beam is spatially overlapped with the Stokes beam by using a dichroic mirror inside picoEMERALD. The temporal overlap between pump and Stokes pulse trains is achieved with a built-in delay stage and optimized by the SRS signal of pure D2O at the microscope. |
| Inverted laser-scanning microscope | Olympus | FV1200MPE | |
| Kinematic mirror mount | Thorlabs | POLARIS-K1-2AH | 2 Low-Profile Hex Adjusters |
| Lectin from Triticum vulgaris (wheat) | Sigma Aldrich | L0636-5 mg | |
| Long-pass dichroic beam splitter | Semrock | Di02-R980-25×36 | 980 nm laser BrightLine single-edge laser-flat dichroic beamsplitter |
| MAP2 | BioLegend | 801810 | Primary antibodies |
| Microscopy imaging software | Olympus | FluoView | |
| NanoQuant Plate | Tecan | For absorbance-based, small volume analyses in a plate reader. | |
| Normal donkey serum | Jackson ImmunoResearch | 017-000-121 | |
| NucBlue Fixed Cell ReadyProbes Reagent (DAPI) | Thermo Scientific | R37606 | |
| Nunc 4-Well Dishes | Fisher Scientific | 12-566-300 | |
| Objective lens | Olympus | XLPlan N | x25, 1.05-NA, MP, working distance = 2 mm |
| Paint brush | |||
| Periscope assembly | Thorlabs | RS99 | includes the top and bottom units, Ø1" post, and clamping fork. |
| pH meter | |||
| Plate reader | Tecan | Infinite 200 PRO | An easy-to-use multimode plate reader. Absorbance measurement capabilities over a spectral range of 230–1000 nm. |
| ProLong Gold antifade reagent | Thermo Scientific | P36930 | |
| PSD95 | Invitrogen | 51-6900 | Primary antibodies |
| Sephadex G-25 Medium | GE Life Sciences | 17-0033-01 | gel filtration resin for desalting and buffer exchange |
| Shielded box with BNC connectors | Pomona Electronics | 2902 | Aluminum Box With Cover, BNC Female/Female |
| Si photodiode | Thorlabs | FDS1010 | 350–1100 nm, 10 mm x 10 mm Active Area |
| Synapsin 2 | ThermoFisher | OSS00073G | Primary antibodies |
| Tissue Path Superfrost Plus Gold Slides | Fisher Scientific | 22-035813 | Adhesive slide to attract and chemically bond fresh or formalin-fixed tissue sections firmly to the slide surface (tiisue bindling glass slides) |
| Triton X-100 | Fisher Scientific | BP151-500 | |
| Vibratome | Leica | VT1000 | |
| Vimentin | Abcam | ab8069 | Primary antibodies |
| Xylenes | Sigma Aldrich | 214736 |
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Cite This Article
Shi, L., Wei, M., Min, W. Highly-Multiplexed Tissue Imaging with Raman Dyes. J. Vis. Exp. (182), e63547, doi:10.3791/63547 (2022).