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

SHORT ve LSFM ile İnsan Beyninin Yüksek Çözünürlüklü 3D Rekonstrüksiyonu

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.

Yazar Spotlight: 3D Sitomimari Analizini Geliştirme - İnsan Beyninin Hızlı Hacimsel Rekonstrüksiyonu 

Büyük Ölçekli İnsan Beyni Görüntülemede Işık Tabakası Floresan Mikroskobu için Optik Temizleme ve Etiketleme

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.

Watch this Scientific Journal Video about Sample Mounting for Light-Sheet Fluorescence Microscopy and Stripping for Multi-Round Labeling of the Same Specimens at JoVE.com

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

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Research

JoVE Journal

Neuroscience

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