Pan-Optical Shadow Imaging of Brain Microanatomy

Yulia Dembitskaya, Kosuke Okuda, Thibault Brugiere, Jordan Girard, Pranay Gaikwad, U. Valentin Nägerl
Methods in Molecular Biology. 2026-01-01; : 239-259
DOI: 10.1007/978-1-0716-5268-8_11

Dembitskaya Y(1)(2), Okuda K(1)(2), Brugiere T(1), Girard J(1), Gaikwad P(1)(2), Nägerl UV(3)(4).

Author information:
(1)Interdisciplinary Institute for Neuroscience, CNRS UMR 5297 and University of
Bordeaux, Bordeaux, France.
(2)Institut für Anatomie und Zellbiologie, Universitätsmedizin Göttingen,
Göttingen, Germany.
(3)Interdisciplinary Institute for Neuroscience, CNRS UMR 5297 and University of
Bordeaux, Bordeaux, France. .
(4)Institut für Anatomie und Zellbiologie, Universitätsmedizin Göttingen,
Göttingen, Germany. .

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.