sample mounting for light-sheet fluorescence microscopy and stripping for multi-round labeling of the same specimens

High-Resolution 3D Reconstruction of Human Brain with SHORT and LSFM

Analyzing the 3D molecular organization and cytoarchitecture of large volumes of the human brain requires optical transparency of specimens, achieved through protocols with extensive processing time. Optical clearing techniques were developed to minimize heterogeneity in refractive index (RI) within the tissues, thereby reducing light scattering and increasing the light penetration depth for high-resolution imaging1,2,3,4,5. Current advances in clearing and deep tissue-labeling methods allow volumetric imaging of intact rodent organs and embryos by exploiting cutting-edge microscopy techniques6,7,8,9,10,11,12.
However, volumetric 3D reconstruction of large areas of the postmortem human brain still represents a challenging task compared with model organisms. The complex biological composition and the variable postmortem fixation and storage conditions can compromise the tissue clearing efficiency, the antibody penetration depth, and the epitope recognition13,14,15,16,17,18,19. Moreover, mechanical tissue sectioning and subsequent clearing and labeling of each slice is still required to achieve an efficient clearing and uniform labeling of large human brain areas, resulting in long processing times and the need for sophisticated custom equipment,&#160;compared with model organisms15,20,21,22.
The SWITCH - H2O2 - antigen Retrieval -TDE (SHORT) tissue transformation technique has been developed specifically to analyze large volumes of the human brain18,23. This method employs the tissue structural preservation of the SWITCH protocol11 and high concentrations of peroxide hydrogen to decrease tissue autofluorescence, in combination with epitope restoration. SHORT allows uniform staining of human brain slices with markers for different neuronal subtypes, glial cells, vasculature, and myelinated fibers18,24. Its results are compatible with the analysis of both low- and high-density proteins. The resulting high transparency levels and uniform labeling enable volumetric reconstruction of thick slices with fluorescence microscopy, in particular, for fast acquisition light-sheet apparatus can be used18,24,25,26,27.
In this work, we describe how the SHORT tissue transformation technique can be used for simultaneous clearing and multi-round labeling of tens of formalin-fixed human brain sections. Four different fluorescent markers can be used together, leading to the identification of different cellular sub-populations. After clearing, high-resolution volumetric imaging can be performed with fluorescence microscopy. Here, we used a custom-made inverted LSFM18,24,25,26,27, which enables fast optical sectioning of the sample and rapid acquisition of multiple channels in parallel. With this routinely high-throughput protocol, it is possible to obtain a comprehensive cellular and structural characterization with a sub-cellular resolution of large areas of the human brain as already demonstrated in the mapping of an entire Broca&#39;s area23.

Author Spotlight: Advancing 3D Cytoarchitecture Analysis - Rapid Volumetric Reconstruction of the Human Brain 

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

The human brain is a complex organ spanning different skills. To understand its functionality, it's essential to construct a detailed cell sensors across the whole brain. Our routinely high-throughput protocol permits the analysis of 3D cytoarchitecture of volumetric areas of the human brain with micrometer resolution, enabling its structural characterization.Volumetric reconstruction of large areas of the human brain presents several experimental challenges linked to the massive dimensions of brain samples, the complex biological composition, the variable postmortem fixation and storage conditions, and the autofluorescent signals from lipofuscin-type pigments. All these can compromise the optical clearing efficiency and demonstrating quality. Compared to other clearing techniques, the short tissue transformation method combined with light sheet microscopy imaging can be used for rapid and simultaneous processing of multiple human brain sections.These result in 3D brain reconstruction with a subcellular resolution.

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Sample Mounting for Light-Sheet Fluorescence Microscopy and Stripping for Multi-Round Labeling of the Same Specimens  

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Neuroscience

99 次观看.  University of Florence. Sample Mounting for Light-Sheet Fluorescence Microscopy and Stripping for Multi-Round Labeling of the Same Specimens

视频: Sample Mounting for Light-Sheet Fluorescence Microscopy and Stripping for Multi-Round Labeling of the Same Specimens  

optical clearing and labeling for light-sheet fluorescence microscopy in large-scale human brain imaging

The present protocol provides a step-by-step procedure for rapid and simultaneous optical clearing, muti-round labeling, and 3D volumetric reconstruction of tens of postmortem human brain sections by combining the (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT tissue transformation technique with light-sheet fluorescence microscopy imaging in a routinely high-throughput...

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light sheet microscopy sample preparation: mounting live zebrafish embryos for long-term imaging

This article describes sample preparation for light-sheet fluorescence microscopy. It also demonstrates a method to mount live zebrafish embryos for long term imaging, which allows recording of long time-lapse movies of live embryos in near-physiological...

Light Sheet Microscopy Sample Preparation: Mounting Live Zebrafish Embryos for Long-Term Imaging

multilayer mounting for long-term light sheet microscopy of zebrafish

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration...

light sheet microscopy imaging and mounting strategies for early zebrafish embryos

Author Spotlight: Strategies for Mounting Zebrafish Embryos for High-Resolution Multiview Light-Sheet Microscopy &#8212; Techniques for Imaging and Image Reconstruction

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet...

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

light-sheet fluorescence microscopy for the study of the murine heart

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine...

imaging the aging cochlea with light-sheet fluorescence microscopy

Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy

A light-sheet microscope was developed to image and digitize whole...

preparation of rodent heart sample for light-sheet microscopy

Preparation of Rodent Heart Sample for Light-Sheet Microscopy

using light sheet fluorescence microscopy to image zebrafish eye development

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview...

light-sheet imaging to reveal cardiac structure in rodent hearts

Author Spotlight: Customized Light-Sheet Imaging for Investigating Myocardial Structures in Rodent Hearts

The protocol utilizes advanced light-sheet microscopy along with adapted tissue clearing methods to investigate intricate cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and...

Light-Sheet Microscopy and Tissue Clearing for Comprehensive Cardiac Imaging

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light sheet fluorescence microscopy of plant roots growing on the surface of a gel

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized...

light sheet-based fluorescence microscopy of living or fixed and stained tribolium castaneum embryos

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

simultaneous live imaging of multiple insect embryos in sample chamber-based light sheet fluorescence microscopes

Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

Light sheet-based fluorescence microscopy is the most valuable tool in developmental biology. A major issue in comparative studies is ambient variance. Our protocol describes an experimental framework for simultaneous live imaging of multiple specimens and, therefore, addresses this issue...

zebrafish larva preparation, mounting, and light-sheet imaging for cardiac development studies

Zebrafish Larva Preparation, Mounting, and Light-Sheet Imaging for Cardiac Development Studies

three-dimensional imaging of the vertebral lymphatic vasculature and drainage using idisco+ and light sheet fluorescence microscopy

Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy

A protocol is presented combining tissue clearing with light sheet fluorescence microscopy (LSFM) to obtain three-dimensional and cellular resolution images of the lymphatic vessels and lymph nodes (LNs) collecting the cerebrospinal fluid (CSF) and spinal epidural...

Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy

light-sheet microscopy for three-dimensional visualization of human immune cells

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D...

Refined Multiview Light Sheet Microscopy Protocol for Zebrafish Embryos

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confocal and light sheet fluorescence microscopy (lsfm) imaging of cleared mouse kidneys

Confocal and Light Sheet Fluorescence Microscopy (LSFM) Imaging of Cleared Mouse Kidneys  

design and building of a customizable, single-objective, light-sheet fluorescence microscope for the visualization of cytoskeleton networks

Author Spotlight: Advancing Knowledge in Far-From-Equilibrium Materials Through Light-Sheet Microscopy

This protocol describes in detail how to build a single-objective, light-sheet fluorescence microscope and its usage for visualizing cytoskeleton networks.

Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with light-sheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi-labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high-throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.

The present protocol provides a step-by-step procedure for rapid and simultaneous optical clearing, muti-round labeling, and 3D volumetric reconstruction of tens of postmortem human brain sections by combining the (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT tissue transformation technique with light-sheet fluorescence microscopy imaging in a routinely high-throughput protocol.