Large-scale tissue clearing (PACT): Technical evaluation and new perspectives in immunofluorescence, histology and ultrastructure

Abstract

Novel techniques, like CLARITY and PACT, render large tissue specimens transparent and thereby suitable for microscopic analysis. We used these techniques to evaluate their potential in the intestine as an exemplary organ with a complex tissue composition. Immunohistochemistry, light sheet-, and confocal scanning-microscopy enabled us to follow complex three-dimensional structures, like nerve fibers, vessels, and epithelial barriers throughout the entire organ. Moreover, in a systematic electron microscopic study, we analyzed the morphology and preservation of tissue on ultrastructural level during the clearing process. We also connect tissue clearing with classical histology and demonstrate that cleared tissues can be stained with Hematoxylin-Eosin and Heidenhain's Azan stain, suggesting potential use in histopathology. These experiments showed that a neutral pH during the clearing process results in much better preservation of tissue ultrastructure and standard stainability. Volume changes of specimens were monitored and quantified during the course of the protocol. Additionally, we employed the technique to visualize the enteric nervous system and the epithelial barrier in post mortem human gut preparations. Our data show the high potential of tissue clearing throughout different tissue types supporting its usefulness in research and diagnosis, and contribute to the technical discussion of ultrastructural tissue-retention.In order to evaluate the three-dimensional organization of complex organs and tissues, biologists are largely restricted to the use of serial sections and post-hoc reconstruction of the cytoarchitecture. This methodology is highly time consuming, requires a lot of computational effort, and is prone to sectioning artifacts. Recently developed optical sectioning methods like confocal optics, two-photon and light sheet microscopy can overcome some of these obstacles but are limited by the diffraction properties of tissue components. Therefore, clearing and microscopical evaluation of intact tissues is a very compelling and challenging approach. In the late 19th/ early 20th century, the German anatomist Werner Spalteholz presented "Aufhellungspräparate", organs and tissues that were translucent by a combination of H 2 O 2 dependent bleaching and refractive index matching 1 . However, due to the rather limited microscopical technology at that time, these pioneering steps did not leave a deep impact on the scientific landscape. Fuelled by the development of optical sectioning methods, a number of novel techniques evolved especially in the last five years as recently reviewed by Richardson and Lichtman 2 . These new tissue clearing approaches make use of refractive index matching 3,4 , hyperhydratation 5 , lipid solving 6-8 , and hydrogel-embedding 9-12 , thereby yielding better tissue conservation and allowing for the detection of intrinsic fluorescent protein and/or immunohistochemical staining.New tissue clearing techniques were largely established and have already been frequently used for the c...

Comparative analysis reveals Ce3D as optimal clearing method for in toto imaging of the mouse intestine

“…The more recently developed protocols (SeeDB2, ScaleS, Ce3D, and UbasM) have been reported to show superior outcomes in terms of clearing capability, GFP retention, and morphological preservation . CLARITY and derived protocols compose a third category of clearing approaches, which involves the embedding of intact tissue into a hydrogel . Although these protocols aim to further improve tissue transparency with minimal protein loss, they are more time‐demanding or require additional infrastructure …”

“…As far as the GI tract is concerned, a systematic benchmark of different clearing methods is lacking. Clearing of gut tissue has been performed with variable results and for specific aims using different approaches or modifications of existing protocols including FocusClear, BABB‐based methods, urea‐based ScaleA2, and SeeDB, the PACT variant of CLARITY and RIMS . Therefore, the aim of this study was to evaluate and directly compare the performance of the most effective, state‐of‐the‐art clearing protocols) on intestinal tissue.…”

“…11,14,23,28 CLARITY and derived protocols compose a third category of clearing approaches, which involves the embedding of intact tissue into a hydrogel. [30][31][32][33] Although these protocols aim to further improve tissue transparency with minimal protein loss, they are more time-demanding or require additional infrastructure. 7,29 As far as the GI tract is concerned, a systematic benchmark of different clearing methods is lacking.…”

Advances and perspectives in tissue clearing using CLARITY

“…Cyanine-3) akin to regular ICH (unpublished observations, Jensen and Berg). CLARITY can also be combined with classical histology such as Hematoxylin-Eosin and Heidenhain's Azan stain, suggesting potential use in histopathology (Neckel et al, 2016) or combined with colorimetric (nonfluorescent) methods such as horseradish peroxidase conversion of diaminobenzidine to a coloured insoluble product .…”

“…autism and drug-resistant epilepsy due to hemimegaloencephaly (Costantini et al, 2015) have been visualised in 3D by CLARITY on formalin-fixed tissues. One frozen brain sample (PFA-fixed and cryoprotected in sucrose) has also been cleared and stained using CLARITY (Phillips et al, 2016), and the human enteric nervous system as well (Neckel et al, 2016). The speed of tissue clearing differs between CNS regions depending on the degree of myelination and the duration of formalin fixation.…”