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 thick-tissue three-dimensional (3D) MERFISH imaging method, which uses confocal microscopy for optical sectioning, deep learning for increasing imaging speed and quality, as well as sample preparation and imaging protocol optimized for thick samples. 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.

Chronic back pain (CBP) is a global health concern with significant societal and economic burden. While various predictors of back pain chronicity have been proposed, including demographic and psychosocial factors, neuroimaging studies have pointed to brain characteristics as predictors of CBP. However, large-scale, multisite validation of these predictors is currently lacking. In two independent longitudinal studies, we examined white matter diffusion imaging data and pain characteristics in patients with subacute back pain (SBP) over 6- and 12-month periods. Diffusion data from individuals with CBP and healthy controls (HC) were analyzed for comparison. Whole-brain tract-based spatial statistics analyses revealed that a cluster in the right superior longitudinal fasciculus (SLF) tract had larger fractional anisotropy (FA) values in patients who recovered (SBPr) compared to those with persistent pain (SBPp), and predicted changes in pain severity. The SLF FA values accurately classified patients at baseline and follow-up in a third publicly available dataset (Area under the Receiver Operating Curve ~0.70). Notably, patients who recovered had FA values larger than those of HC suggesting a potential role of SLF integrity in resilience to CBP. Structural connectivity-based models also classified SBPp and SBPr patients from the three data sets (validation accuracy 67%). Our results validate the right SLF as a robust predictor of CBP development, with potential for clinical translation. Cognitive and behavioral processes dependent on the right SLF, such as proprioception and visuospatial attention, should be analyzed in subacute stages as they could prove important for back pain chronicity.