Thermal-plex: fluidic-free, rapid sequential multiplexed imaging of RNA and protein in brain tissues

项目摘要 脑细胞和组织的多路成像可以揭示关于丰度和空间的关键细节 分子靶标的组织。基于迭代标记和成像的序列成像方法 实用的更高的多路复用，但通常需要具有多轮慢速缓冲器的复杂的流体设置 交换。我们建议开发热络合法，它去除了复杂和缓慢的缓冲交换 步骤，并提供了一种无流体的快速序列成像方法。热敏复合体使用简单的DNA探针 被设计成当且仅当加热到指定温度(热)时才会顺序地发出荧光 频道)。频道切换速度很快(&lt；30秒)，而且是通过市面上可以买到的价格实惠的产品实现的 范围内加热装置。在我们的初步工作中，我们已经演示了15-plex RNA成像(5热×3 固定细胞中的荧光通道)在不到4分钟的时间内，无需使用缓冲液交换或流体。在这 在这项提议中，我们将进一步开发和验证用于成像脑组织中的RNA和蛋白质目标的热网络。 通过去除复杂和缓慢的流体，热复合体提高了实际顺序操作的简易性和速度 用于脑研究的多路成像。

Project Summary Multiplexed imaging of brain cells and tissues can reveal critical details about the abundance and spatial organization of molecular targets. Sequential imaging methods based on iterative labeling and imaging enables practical higher multiplexing, but generally require complex fluidic setup with multiple rounds of slow buffer exchange. We propose to develop the thermal-plex method which removes complex and slow buffer exchange steps, and provides a fluidic-free, rapid sequential imaging method. Thermal-plex uses simple DNA-probes that are engineered to sequentially fluoresce when and only when heated to designated temperatures (thermal channels). Channel switching is fast (<30 seconds), and is achieved with commercially available and affordable on-scope heating device. In our preliminary work, we have demonstrated 15-plex RNA imaging (5 thermal × 3 fluorescence channels) in fixed cells in less than 4 minutes, without using buffer-exchange or fluidics. In this proposal, we will further develop and validate thermal-plex for imaging RNA and protein targets in brain tissues. By removing complex and slow fluidics, thermal-plex increases the ease and speed of practical sequential multiplexed imaging for brain research.