Molecule-specific techniques such as MALDI and desorption electrospray ionization mass spectrometry imaging enable direct and simultaneous mapping of biomolecules in tissue sections in a single experiment. However, neurotransmitter imaging in the complex environment of biological samples remains challenging. Our covalent charge-tagging approach using on-tissue chemical derivatization of primary and secondary amines and phenolic hydroxyls enables comprehensive mapping of neurotransmitter networks. Here, we present robust and easy-to-use chemical derivatization protocols that facilitate quantitative and simultaneous molecular imaging of complete neurotransmitter systems and drugs in diverse biological tissue sections with high lateral resolution. This is currently not possible with any other imaging technique. The protocol, using fluoromethylpyridinium and pyrylium reagents, describes all steps from tissue preparation (~1 h), chemical derivatization (1–2 h), data collection (timing depends on the number of samples and lateral resolution) and data analysis and interpretation. The specificity of the chemical reaction can also help users identify unknown chemical identities. Our protocol can reveal the cellular locations in which signaling molecules act and thus shed light on the complex responses that occur after the administration of drugs or during the course of a disease.

分子特异性技术，如 MALDI 和解吸电喷雾电离质谱成像，可以在单个实验中直接和同步地绘制组织切片中的生物分子。然而，生物样本复杂环境中的神经递质成像仍然具有挑战性。我们的共价电荷标记方法使用伯胺和仲胺以及酚羟基的组织化学衍生化，可以全面绘制神经递质网络。在这里，我们提出了稳健且易于使用的化学衍生化协议，这些协议有助于以高横向分辨率对不同生物组织切片中的完整神经递质系统和药物进行定量和同步分子成像。这是目前任何其他成像技术都无法做到的。该协议，使用氟甲基吡啶和吡啶试剂，描述了从组织制备（~1 小时）、化学衍生化（1-2 小时）、数据收集（时间取决于样本数量和横向分辨率）以及数据分析和解释的所有步骤。化学反应的特异性也可以帮助用户识别未知的化学成分。我们的协议可以揭示信号分子作用的细胞位置，从而阐明药物给药后或疾病过程中发生的复杂反应。化学反应的特异性也可以帮助用户识别未知的化学成分。我们的协议可以揭示信号分子作用的细胞位置，从而阐明药物给药后或疾病过程中发生的复杂反应。化学反应的特异性也可以帮助用户识别未知的化学成分。我们的协议可以揭示信号分子作用的细胞位置，从而阐明药物给药后或疾病过程中发生的复杂反应。