Pan-Optical Shadow Imaging of Brain Microanatomy

Understanding the fine-scale organization of brain tissue is critical for elucidating the principles of neural circuit function and dysfunction. The SUper-resolution SHadow Imaging (SUSHI) technique was developed to provide panoramic yet high-resolution visualization of brain microarchitecture by labeling the extracellular space with membrane-impermeant fluorescent dyes, rendering surrounding cellular structures visible in a pan-optical way with negative contrast. Initially established in combination with STED microscopy, SUSHI enables unbiased nanoscale imaging of live brain tissue without relying on cell-type-specific labeling. Recent adaptations have extended the method to in vivo applications using confocal and light-sheet microscopy, broadening its accessibility and utility.This chapter presents comprehensive protocols for implementing shadow imaging in acute brain slices, organotypic slice cultures, and the intact mouse brain in vivo, including both acute and chronic preparations. We provide detailed instructions on sample preparation, dye delivery, imaging setup, and data acquisition using multiple imaging modalities (2P, STED, confocal). Additionally, we describe shadow imaging in fixed tissue using extracellular matrix labeling. Together, these protocols offer a flexible and scalable approach to resolve the structural interplay between neurons, glia, and extracellular space in diverse experimental models, including those relevant to neurological diseases.