Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

作者： Felix A. Kyere*1,2, Ian Curtin*1,2, Oleh Krupa1,2, Carolyn M. McCormick1,2, Mustafa Dere3, Sarah Khan3,7, Minjeong Kim7, Tzu-Wen Winnie Wang4,5,6, Qiuhong He4,5,6, Guorong Wu3, Yen-Yu Ian Shih4,5,6, Jason L. Stein1,2 1UNC Neuroscience Center,University of North Carolina, Chapel Hill, 2Department of Genetics,University of North Carolina, Chapel Hill, 3Department of Psychiatry,University of North Carolina, Chapel Hill, 4Center for Animal Magnetic Resonance Imaging,The University of North Carolina at Chapel Hill, 5Biomedical Research Imaging Center,The University of North Carolina at Chapel Hill, 6Department of Neurology,The University of North Carolina at Chapel Hill, 7Department of Computer Science,The University of North Carolina at Greensboro

简介：

Overview

This study outlines a protocol for imaging intact mouse brains through magnetic resonance imaging, clearing, and immunolabeling using iDISCO+ and light-sheet microscopy. The method aims to facilitate accurate cell quantification in the mouse cortex with advanced image analysis tools.

Key Study Components

Area of Science

• Neuroscience
• Imaging Techniques
• Cell Quantification

Background

• The mouse brain contains approximately 100 million cells.
• High-resolution images can reach terabyte sizes, requiring advanced analysis tools.
• The iDISCO+ protocol is crucial for clearing and immunolabeling brain samples.
• Light-sheet microscopy allows for detailed imaging of complex structures.

Purpose of Study

• To improve methods for conducting 3D imaging of mouse brains.
• To present a computational pipeline for preprocessing and quantifying cellular data.
• To demonstrate the capability of revealing distinct neuron populations in various brain regions.

Methods Used

• Light-sheet microscopy was employed for imaging using the UltraMicroscope II.
• The biological model involved intact mouse brains, specifically targeted for cell quantification.
• A computational pipeline, NuMorph, was used for preprocessing and analyzing imaging data.
• Key procedural steps included mounting samples, configuring imaging settings, and using MATLAB for data processing.

Main Results

• Nuclei were detected with high precision using a trained 3D U-Net model, allowing for extensive cellular analysis.
• Around 12 million total nuclei were quantified in the isocortex and other brain regions.
• Errors in preprocessing were highlighted as critical for ensuring accurate image alignment and cell counting.
• Successful processing led to clear visualization of specific neuron populations, aiding in understanding cortical structure.

Conclusions

• This protocol advances imaging methodologies to enable detailed studies of brain architecture.
• The integrated use of iDISCO+ and light-sheet microscopy demonstrates the importance of preprocessing for accurate analyses.
• The findings hold significant implications for further research into neuronal mechanisms and disease models.

Frequently Asked Questions