Three-dimensional single-cell transcriptome imaging of thick tissues

Multiplexed error-robust fluorescence in-situ hybridization (MERFISH) allows genome-scale imaging of RNAs in individual cells in intact tissues. To date, MERFISH has been applied to image thin tissue samples of ∼10-µm thickness. Here, we present a method to enable three-dimensional (3D) single-cell transcriptome imaging of thick tissue specimens by integrating MERFISH with confocal microscopy for optical sectioning and deep learning to increase imaging speed and quality. We demonstrated 3D MERFISH on mouse brain tissue sections of up to 200 µm thickness with high detection efficiency and accuracy. We anticipate that 3D thick-tissue MERFISH imaging will broaden the scope of questions that can be addressed by spatial genomics.