Imaging the transcriptome in situ with high accuracy has been a major challenge in single-cell biology, which is particularly hindered by the limits of optical resolution and the density of transcripts in single cells . Here we demonstrate an evolution of sequential fluorescence in situ hybridization (seqFISH+). We show that seqFISH+ can image mRNAs for 10,000 genes in single cells-with high accuracy and sub-diffraction-limit resolution-in the cortex, subventricular zone and olfactory bulb of mouse brain, using a standard confocal microscope. The transcriptome-level profiling of seqFISH+ allows unbiased identification of cell classes and their spatial organization in tissues. In addition, seqFISH+ reveals subcellular mRNA localization patterns in cells and ligand-receptor pairs across neighbouring cells. This technology demonstrates the ability to generate spatial cell atlases and to perform discovery-driven studies of biological processes in situ.