Project Summary In biology, structure determines function. To understand the function of organs (i.e., physiology), we must obtain structural information on the organs’ structure (i.e., anatomy). Mapping organs’ structures must occur at (sub)cellular spatial resolution with sufficient details on cells’ molecular states. Spatial transcriptomics technologies have made significant advances in mapping thin tissue sections. However, all spatial transcriptomics approaches are restricted to sections thinner than 150 µm, with the vast majority needing to be less than 15 µm. While 2D sections are informative, sampling 3D organs with 2D sections loses information and provides a partial view of the organs’ full structure. Here we propose to develop whole organ spatial transcriptomics. Key to our approach is our recent invention (patent pending) of dimensionality reduced Fluorescent In Situ Hybridization (dredFISH). Single-molecule FISH approaches, such as smFISH, MERFISH, seqFISH+, and many other variants, label and count individual RNAs. dredFISH is not a single molecule FISH. Instead, it is designed to directly measure an approximation of cells’ transcriptional state by measuring multiple distinct weighted sums of thousands of genes. The transcriptional signatures represented by the distinct sums of genes are generated for the reference scRNAseq and compared to the ones directly measured by dredFISH. Integrating and harmonizing the directly measured FISH signatures and the reference data allows the inference of cell types (kNN classification) and gene expression reconstruction (kNN regression). By leapfrogging the need for individual gene measurements, dredFISH achieves multiple features that make it ideal for fast light-sheet microscopy of thick samples (500 µm). Specifically, i) dredFISH is performed using low magnification objectives (10x, 16x) instead of high magnification objectives (60x,100x) used for single-molecule FISH. ii) dredFISH utilizes the tissue clearing and hydrogel embedding technique used by CLARITY, making it compatible with existing light-sheet imaging protocols. iii) dredFISH signal comes from the weighted sums of fluorescence from tens of thousands of RNA molecules per cell, each stained by tens of probes, making it much brighter and easier to measure than all other FISH approaches. Our preliminary results demonstrate that dredFISH works well in thin 2D sections validating our approach. The goal of this proposal is to extend dredFISH from 2D to 3D to allow whole organ spatial transcriptomics. This goal will be accomplished by parallel development steps divided into two aims. Aim #1 will continue optimizing and validating dredFISH in 2D by changing a few aspects of the method that will ease the transition to 3D. Aim #2 will update the staining and imaging steps in the dredFISH protocol to make them compatible with thick samples. Aim #3 will demonstrate the power of dredFISH whole organ spatial transcriptomics by creating the entire mouse br...