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有关脑细胞的丰度和空间分布的关键细节。 分子目标的组织。基于迭代标记和成像的顺序成像方法使得能够 实际上更高的多路复用，但通常需要具有多轮缓慢缓冲液的复杂流体设置 交易所我们建议开发热复合方法，该方法消除了复杂和缓慢的缓冲液交换 步骤，并提供了一种无流体的快速顺序成像方法。Thermal-plex使用简单的DNA探针， 被设计成当且仅当被加热到指定温度（热）时顺序地发出荧光 渠道）。通道切换速度快（<30秒），并且可以通过市售和经济实惠的 内窥镜加热装置。在我们的初步工作中，我们已经证明了15重RNA成像（5热× 3 荧光通道）在固定的细胞中在少于4分钟内，而不使用缓冲液交换或射流。在这 根据该提案，我们将进一步开发和验证用于成像脑组织中RNA和蛋白质靶点的热复合物。 通过去除复杂和缓慢的流体，热复合物增加了实际顺序的容易性和速度。 用于大脑研究的多重成像。

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.