GPU-accelerated, self-optimizing processing for 3D multiplexed iterative RNA-FISH experiments

Imaging-based spatial transcriptomic approaches rely on iterative labeling and imaging of carefully prepared samples, followed by solving a computational inverse problem to determine the location and identity of the targeted RNA. Because these approaches require high-resolution optics, the Nyquist-Shannon determined voxel size is small relative to typical tissue sample footprints. A common solution to speed up both experiments and computation is to increase the distance in between focal planes, trading off local information content to sample a larger imaging area in a reasonable time. In this work we introduce a GPU-accelerated computational framework, merfish3d-analysis , designed to speed up the computational processing of barcoded, in situ imaging-based spatial transcriptomics. Using this framework, we quantify the information lost due to axial sampling changes in simulated imaging-based spatial transcriptomic experiments, robustly reprocess publicly available multiplexed error-robust fluorescence in situ hybridization (MERFISH) datasets, and analyze new MERFISH experiments performed on a post-mortem human olfactory bulb sample. To improve the quality of experimental data in the post-mortem human sample, we designed a multi-step autofluorescence quenching protocol specific for in situ imaging-based spatial transcriptomic strategies. Taken together, we hope that the sample preparation protocols and single workstation, GPUaccelerated processing will further democratize imaging-based spatial transcriptomic experiments.