Abstract Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed E xpansion- As sisted Iterative F luorescence In S itu H ybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 µm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine novel spatially and molecularly defined subregions. EASI-FISH also facilitates iterative re-analysis of scRNA-Seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization. Highlights - EASI-FISH enables robust gene expression profiling in thick brain slices - A turnkey analysis pipeline for facile analysis of large EASI-FISH image datasets - EASI-FISH reveals novel subregions of the lateral hypothalamus - Identification of rare cell types based on morphological and spatial heterogeneity

Expansion-Assisted Iterative-FISH defines lateral hypothalamus spatio-molecular organization