A Highly Multiplexed, Multiomic 3D Mouse Brain Map Using MALDI-IHC

使用 MALDI-IHC 绘制高度多重、多组学 3D 小鼠脑图

• 批准号: 10705203
• 负责人: Mark Lim
• 金额: $ 91.04万
• 依托单位: AMBERGEN, INC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10705203
• 关键词: 3-Dimensional; Acceleration; Agar; Antibodies; BRAIN initiative; Biological Markers; Brain; Brain Mapping; Brain imaging; Collaborations; Computer software; Data Analyses; Data Set; Development; Disease; Fluorescence; Fourier transform ion cyclotron resonance; Freezing; Goals; Hospitals; Hour; Human; Image; Image Analysis; Imaging Device; Imaging technology; Immunohistochemistry; Individual; Label; Lectin; Length; Link; Maps; Marketing; Metals; Methods; Modality; Molecular; Mus; Neurodegenerative Disorders; Neurosciences; Pattern; Phase; Play; Polysaccharides; Procedures; Production; Proteins; Proteomics; Resolution; Scanning; Slice; Spatial Distribution; Specimen; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stains; Techniques; Technology; Tissue imaging; Tissues; United States National Institutes of Health; Validation; Visualization; Woman; brain dysfunction; brain tissue; commercialization; denoising; fluorescence imaging; glycosylation; instrumentation; mass spectrometric imaging; medical schools; metabolomics; meter; multimodality; multiple omics; neuronal circuitry; novel; preservation; protein expression; reconstruction; small molecule; tool

Summary/Abstract A central goal of the NIH Brain Initiative is to develop new imaging tools sufficiently powerful to spatially map at high resolution the neuronal circuitry and underlying molecular composition of the brain. While cutting edge imaging tools and related labeling techniques have been developed, it is still a major challenge to map the spatial distribution at different length scales of the thousands of biomolecules, including expressed proteins, which play key roles in brain function. The goal of this Phase II project is to evaluate the ability of a new tissue imaging technology developed by AmberGen, termed MALDI-IHC, to rapidly create a highly multiplexed, multiomic and multimodal 3D molecular map of the mouse brain. The development of MALDI-IHC for whole brain imaging will provide neuroscientists with an important new tool for exploring the underlying molecular basis of brain function and neurodegenerative disorders. MALDI-IHC is based on the use of novel photocleavable mass-tags (PC-MTs) developed by AmberGen which when linked to antibody or lectin probes enable targeted biomolecules to be identified in the mass spectrometric image. This approach significantly exceeds the multiplex capability of fluorescence immunohistochemistry (IHC) and previous cleavable mass-tag based methods which are generally limited to 5 biomarkers or require extensive cycling procedures. It also exceeds the capability of metal-tagged antibody techniques such as IMC and MIBI which can probe small mm2 regions at subcellular resolution but are limited to approximately 40 antibody probes and require several days to scan a whole tissue section. In contrast, MALDI-IHC can image an entire mouse brain FFPE section for over 100 targeted proteins at 40 µm resolution in less than one hour. The ability of MALDI-IHC to perform label-free, untargeted small molecule mass spectrometric imaging (MSI), fluorescence imaging using unique dual-labeled fluorescent-PC-MT probes and high-plex imaging of intact expressed proteins including glycosylation patterns on the same tissue section greatly extends the power of this approach. During Phase I, we demonstrated the feasibility of this combined approach on mouse brain FF and FFPE tissue specimens. During Phase II, we will develop methods using MALDI-IHC to reconstruct whole mouse brain protein expression maps at 40 µm voxel resolution. FFPE sagittal and coronal mouse brain tissue slices from mouse brain will be probed by MALDI-IHC using a panel of 50 NeuroMab PC-MT antibodies and 25 PC-MT lectins. Validation of individual PC-MT probes will be performed by comparing MALDI-IHC and fluorescence IHC images. A 3D tri-modal map of the mouse brain merging both metabolites and expressed proteins will also be reconstructed based on a demonstrated workflow that involves MSI of unlabeled small molecules from successive FF specimens, IHC staining with a 75-plex panel of PC-MT probes including some dual-labeled PC-MT antibodies, and fluorescence imaging followed by MSI of the PC-MTs. Reconstruction of 3D maps, visualization and image analysis will be performed using Bruker SCiLS™ software. Commercialization of MALDI-IHC technology will be accelerated by a close collaboration with Bruker Daltonics, the market leader of MALDI-MSI instrumentation.

摘要/摘要 美国国立卫生研究院大脑计划的一个中心目标是开发新的成像工具，其功能足够强大，可以在空间上绘制地图 高分辨率的神经元回路和大脑的底层分子组成。在切削刃的同时 成像工具和相关标记技术已经开发出来，但绘制空间图仍然是一个重大挑战 数千种生物分子（包括表达的蛋白质）在不同长度尺度上的分布，这些生物分子发挥着 在大脑功能中发挥关键作用。该二期项目的目标是评估新组织成像的能力 AmberGen 开发的技术称为 MALDI-IHC，可快速创建高度多重、多组学和 小鼠大脑的多模态 3D 分子图。全脑成像 MALDI-IHC 的发展将 为神经科学家提供了探索大脑功能的分子基础的重要新工具 和神经退行性疾病。 MALDI-IHC 基于新型光裂解质量标签 (PC-MT) 的使用 由 AmberGen 开发，当与抗体或凝集素探针连接时，能够将目标生物分子 在质谱图像中识别。这种方法大大超过了多路复用能力 荧光免疫组织化学 (IHC) 和以前基于可切割质量标签的方法通常是 限于 5 个生物标志物或需要大量的循环程序。它也超出了金属标记的能力 抗体技术，如 IMC 和 MIBI，可以以亚细胞分辨率探测小 mm2 区域，但 仅限于大约 40 个抗体探针，并且需要几天时间才能扫描整个组织切片。相比之下， MALDI-IHC 可以对整个小鼠大脑 FFPE 切片进行 40 µm 分辨率的 FFPE 切片成像 不到一小时。 MALDI-IHC 能够进行无标记、非靶向小分子质谱分析 成像 (MSI)、使用独特的双标记荧光 PC-MT 探针的荧光成像和高复数成像 完整表达的蛋白质（包括同一组织切片上的糖基化模式）大大扩展了能力 这种方法。在第一阶段，我们证明了这种组合方法在小鼠大脑 FF 上的可行性 和 FFPE 组织标本。在第二阶段，我们将开发使用 MALDI-IHC 重建整体的方法 小鼠脑蛋白表达图谱分辨率为 40 µm。 FFPE 矢状和冠状小鼠脑组织切片 将使用一组 50 个 NeuroMab PC-MT 抗体和 25 个 PC-MT 通过 MALDI-IHC 探测小鼠大脑中的 凝集素。将通过比较 MALDI-IHC 和荧光 IHC 来验证各个 PC-MT 探针 图像。合并代谢物和表达蛋白质的小鼠大脑 3D 三模态图也将被 基于经过验证的工作流程重建，该工作流程涉及来自连续序列的未标记小分子的 MSI FF 标本，使用 75 重 PC-MT 探针（包括一些双标记 PC-MT 抗体）进行 IHC 染色， 和荧光成像，然后是 PC-MT 的 MSI。 3D 地图、可视化和图像重建 将使用 Bruker SCiLS™ 软件进行分析。 MALDI-IHC 技术的商业化将 与 MALDI-MSI 仪器市场领导者 Bruker Daltonics 的密切合作加速了这一进程。