optical-clearing-labeling-for-light-sheet-fluorescence-microscopy

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its ...

University of Florence

The combination of tissue clearing techniques with advanced optical microscopy facilitates the achievement of three-dimensional (3D) reconstruction of macroscopic specimens at high resolution. Whole mouse organs or even bodies have been analyzed, while the reconstruction of the human nervous system remains a challenge. Although several tissue protocols have been proposed, the high autofluorescence and variable post-mortem conditions of human specimens negatively affect the quality of the images in terms of achievable transparency and staining contrast. Moreover, homogeneous staining of high-density epitopes, such as neuronal nuclear antigen (NeuN), creates an additional challenge. Here, we evaluated different tissue transformation approaches to find the best solution to uniformly clear and label all neurons in the human cerebral cortex using anti-NeuN antibodies in combination with confocal and light-sheet fluorescence microscopy (LSFM). Finally, we performed mesoscopic high-resolution 3D reconstruction of the successfully clarified and stained samples with LSFM.

Comparison of Different Tissue Clearing Methods for Three-Dimensional Reconstruction of Human Brain Cellular Anatomy Using Advanced Imaging Techniques.

The combination of optical tissue transparency with immunofluorescence allows the molecular characterization of biological tissues in 3D. However, adult human organs are particularly challenging to become transparent because of the autofluorescence contributions of aged tissues. To meet this challenge, we optimized SHORT (SWITCH-HO-antigen Retrieval-TDE), a procedure based on standard histological treatments in combination with a refined clearing procedure to clear and label portions of the human brain. 3D histological characterization with multiple molecules is performed on cleared samples with a combination of multi-colors and multi-rounds labeling. By performing fast 3D imaging of the samples with a custom-made inverted light-sheet fluorescence microscope (LSFM), we reveal fine details of intact human brain slabs at subcellular resolution. Overall, we proposed a scalable and versatile technology that in combination with LSFM allows mapping the cellular and molecular architecture of the human brain, paving the way to reconstruct the entire organ.

3D molecular phenotyping of cleared human brain tissues with light-sheet fluorescence microscopy.

Accurate labeling of specific layers in the human cerebral cortex is crucial for advancing our understanding of neurodevelopmental and neurodegenerative disorders. Leveraging recent advancements in ultra-high resolution  MRI, we present a novel semi-supervised segmentation model capable of identifying supragranular and infragranular layers in  MRI with unprecedented precision. On a dataset consisting of 17 whole-hemisphere  scans at 120 m, we propose a multi-resolution U-Nets framework (MUS) that integrates global and local structural information, achieving reliable segmentation maps of the entire hemisphere, with Dice scores over 0.8 for supra- and infragranular layers. This enables surface modeling, atlas construction, anomaly detection in disease states, and cross-modality validation, while also paving the way for finer layer segmentation. Our approach offers a powerful tool for comprehensive neuroanatomical investigations and holds promise for advancing our mechanistic understanding of progression of neurodegenerative diseases.

Article	Year
Comparison of Different Tissue Clearing Methods for Three-Dimensional Reconstruction of Human Brain Cellular Anatomy Using Advanced Imaging Techniques. Frontiers in neuroanatomy\| PubMed ID: 34867215	2021
3D molecular phenotyping of cleared human brain tissues with light-sheet fluorescence microscopy. Communications biology\| PubMed ID: 35551498	2022
Segmentation of supragranular and infragranular layers in ultra-high resolution 7T MRI of the human cerebral cortex. bioRxiv : the preprint server for biology\| PubMed ID: 38106176	2023

Niamh Brady

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