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

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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.