Project Summary The quest to understand the brain’s complex structure has become more challenging as the high degree of molecular heterogeneity among brain cells has become evident in recent years. Mapping the brain in detail will require incorporating large amounts of molecular information into high-resolution imaging. Current imaging methods are limited by the number of distinguishable detection channels, so greater degrees of multiplexing entail repeated cycles of stripping and reapplying probes. These methods degrade tissue integrity and impair sensitivity, and do not address the other major challenge of multiplexing- incompatibility between protein and RNA labeling methods and the need to compromise both for simultaneous detection. We propose a novel imaging approach, Serial-section parallel immuno/ Fluorescence In Situ Hybridization (SpiFISH), whose core strategy is to physically subdivide specimens into sections two orders of magnitude smaller than a neuronal cell body. Each section is treated as a separate sample for labeling and imaging, so hundreds of discrete labeling experiments can be performed in parallel on a given neuron. The method is based on ultrathin sectioning, but unlike existing ultrathin sectioning methods such as electron microscopy (EM) and array tomography, SpiFISH does not use EM embedding resins. Without resin interfering, sensitive immunolabeling and RNA detection are possible. Each section is labeled and imaged separately, so that any given cell can be labeled with many different antibodies and RNA probes under conditions optimized for each. Sections are shelf-stable, so large datasets can be built up across time and even across laboratories. The method allows multiplexing of techniques as well as labels, so the same sample can be used with multiple imaging and staining platforms. The goal of this project is to develop robust, reproducible protocols and workflows from sample preparation through data analysis across scales. This will include small samples through whole rodent brains and streamlined methods for fully manual through fully automated data collection and analysis.

Project Narrative Creating a map of the immensely complicated structure of the brain is a major goal in neuroscience, but a key challenge is that brain cells have their own individual combinations of molecules that must be mapped as well. Current imaging methods can only detect two or three molecules at a time. This project will develop a novel method for incorporating large numbers of molecules in high-resolution microscope images of brain tissue, allowing for unprecedented richness in the next generation of brain maps.