A Scanning Electron Microscopy-Compatible Optical Imaging Method for Mesoscopic All-Cell Brain Mapping

Summary

We introduced a novel imaging technique called Optical Multilayer Interference Tomography (OMLIT), which enables unbiased imaging of all cells in brain specimens at the mesoscale and can be seamlessly integrated into the imaging workflow of tape-based serial scanning electron microscopy on the same sample.

Abstract

Understanding the structural and functional relationships within complex neural networks in the brain requires the construction of brain atlases that possess both a broad field of view and subcellular resolution. However, current optical and electron microscopy imaging methods each have limitations, making it difficult to image all cells within a single specimen. This protocol introduces an imaging technique called Optical Multilayer Interference Tomography (OMLIT), which enables indiscriminate optical imaging of all cells in brain specimens made following electron microscopy sample preparation, thereby reconstructing a complete brain atlas of all neural cells. In addition, OMLIT imaging can be seamlessly integrated with the imaging workflow of automated tape-collecting ultramicrotomy scanning electron microscopy (ATUM-SEM). This allows researchers to obtain mesoscale structural information of cells prior to electron microscopy imaging, facilitating the precise selection of regions of interest and significantly reducing the area and data volume required for high-resolution electron microscopy (EM) imaging. We validated the accuracy and compatibility of this method in actual samples from the adult mouse cerebral cortex, demonstrating its broad application prospects in multi-scale brain atlas construction.

Introduction

Comprehensive mapping of neural circuits at cellular and subcellular resolution is essential for revealing the structure and function of the brain. Traditional optical imaging methods, such as two-photon microscopy¹, fluorescence micro-optical sectioning tomography (fMOST)^2,3,4, and the VISoR system⁵, have enabled mesoscale neuronal imaging and in vivo functional imaging. However, due to their reliance on sparse labeling, they fail to capture the complete cellular population. On the other hand, label-free optical imagin....

Protocol

All animal procedures were performed in accordance with the institutional guidelines of the University of Science and Technology of China and relevant national regulations. The sample preparation for OMLIT imaging follows the same protocol as that used in conventional EM, and the specific procedure we employed has been described elsewhere²¹.In brief, after anesthesia, mice were transcardially perfused sequentially with sodium cacodylate buffer, artificial cerebrospinal fluid (ACSF), and finally a fixative containing glutaraldehyde and paraformaldehyde. The brain was carefully removed, and around 1 × 1 × 1 mm³ blocks of brain tissue were section....

Results

The overall workflow of the protocol (Figure 1) begins with the preparation of different collection tapes. Figure 1 shows the three types of tapes mentioned in the protocol (from left to right: Kapton, D-50, and CNT-coated PET), which exhibit distinct optical properties when placed on the same background substrate. After sample preparation and block trimming, a few sections should be first collected for electron microscopy imaging to ensure the preparation meets.......

Discussion

Here, we developed an optical imaging approach, termed OMLIT, that enables mesoscopic imaging and is compatible with tape-based serial scanning electron microscopy workflows. By using the OMLIT method, optical microscopy can be employed to capture mesoscopic structural features from brain samples, including blood vessels, cell bodies, nuclei, major dendritic branches, and some large myelinated axons. In addition, OMLIT can be seamlessly integrated into the serial SEM pipeline, providing structural information prior to el.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adhesive tape	3M	B5005094008	Double-sided adhesive
Atomic Force Microscope	Bruker	Dimension Icon
Automatic ultrathin section collecting system	Lehua	AutoCUTS II
Conductive adhesive tape	Ted Pella	FP16084-8
Dektak Stylus Profilers	Bruker	DektakXT
Diamond knife for sectioning	Diatome	DUJ3530	Diatome Jumbo knife
Diamond knife for trimming	Diatome	DTB90	Glass knife
Electron microscope	Zeiss	MultiSEM505	Alternative: GeminiSEM 300, Zeiss
FIJI (v1.54p, 64-bit)	Open source	https://fiji.sc
Glass trimming knife	Selfmade
Lead Citrate	Leica	T534/2
Light microscope	Olympus	VS200	Alternative: Axio Imager. A2 Vario, Zeiss
Light microscope	Zeiss	Axio Imager.A2 Vario
Plasma cleaner	Yidon Technologies	Hydro-S4	Alternatives: Ted Pella Pelco or other benchtop plasma cleaner
Polymer tape	Melton	Kapton	The website of the company is no longer accessible. We recommend that researchers try locally available KAPTON tapes.
Polymer tape	Teijin	PET	https://www.teijin.com/
Polymer tape	Teijin	D-50	https://www.teijin.com/
Silicon wafer	Saichi	912303	Wafer is polished on one side.
Sputter Coater	Leica	ACE600	Alternative:Double-head Sputter, Yujie
Ultramicrotome	Leica	UC7	Alternative: RMC PT-PC
Uranylacetate	EMS	22400
VAST (v1.5.0, 64-bit)	Howard Hughes Medical Institute	https://software.dvid.io/vast/	VAST A1:D32Lite is a free tool for manual annotation and segmentation of large 3D microscopy datasets.
ZEN software (v3.2, 64-bit)	Zeiss	https://www.zeiss.com/microscopy/zh/products/software/zeiss-zen.html