Biology subjects

Laurino, A.

Publications and source records attributed to Laurino, A..

4 recordsLinked to original sources

bioRxiv2021

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

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 - H2O2 - 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.

neuroscience↗

bioRxiv2021

Exploring the human cerebral cortex using confocal microscopy

Cover-all mapping of the distribution of neurons in the human brain would have a significant impact on the deep understanding of brain function. Therefore, complete knowledge of the structural organization of different human brain regions at the cellular level would allow understanding their role in the functions of specific neural networks. Recent advances in tissue clearing techniques have allowed important advances towards this goal. These methods use specific chemicals capable of dissolving lipids, making the tissue completely transparent by homogenizing the refractive index. However, labeling and clearing human brain samples is still challenging. Here, we present an approach to perform the cellular mapping of the human cerebral cortex coupling immunostaining with SWITCH/TDE clearing and confocal microscopy. A specific evaluation of the contributions of the autofluorescence signals generated from the tissue fixation is provided as well as an assessment of lipofuscin pigments interference. Our evaluation demonstrates the possibility of obtaining an efficient clearing and labeling process of parts of adult human brain slices, making it an excellent method for morphological classification and antibody validation of neuronal and non-neuronal markers.

neuroscience↗

bioRxiv2020

Fast volumetric mapping of human brain slices

We still lack a detailed map of the anatomical disposition of neurons in the human brain. A complete map would be an important step for deeply understanding the brain function, providing anatomical information useful to decipher the neuronal pattern in healthy and diseased conditions. Here, we present several important advances towards this goal, obtained by combining a new clearing method, advanced Light Sheet Microscopy and automated machinelearning based image analysis. We perform volumetric imaging of large sequentially stained human brain slices, labelled for two different neuronal markers NeuN and GAD67, discriminating the inhibitory population and reconstructing the brain connectivity.

biophysics↗

bioRxiv2020

A combined pipeline for quantitative analysis of human brain cytoarchitecture

The 3D analysis of the human brain architecture at cellular resolution is still a big challenge. In this work, we propose a pipeline that solves the problem of performing neuronal mapping in large human brain samples at micrometer resolution. First, we introduce the SWITCH/TDE protocol: a robust methodology to clear and label human brain tissue. Then, we implement the 2.5D method based on a Convolutional Neural Network, to automatically detect and segment all neurons. Our method proved to be highly versatile and was applied successfully on specimens from different areas of the cortex originating from different subjects (young, adult and elderly, both healthy and pathological). We quantitatively evaluate the density and, more importantly, the mean volume of the thousands of neurons identified within the specimens. In conclusion, our pipeline makes it possible to study the structural organization of the brain and expands the histopathological studies to the third dimension.

neuroscience↗