Clearing-induced tisssue shrinkage: A novel observation of a thickness size effect

Vulders, Roland C. M.; Hoogenhuizen, R. C. van; Giessen, E. van der; Zaag, P. J. van der

doi:10.1371/journal.pone.0261417

Cited by 5 publications

(5 citation statements)

References 25 publications

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“…In many clearing protocols, such as iDISCO+, the solvents result in the expansion or shrinkage of the tissue samples ( 1 ). While some studies report shrinkage in all dimensions hereby maintaining similar spatial distribution, others suggest different amounts of shrinkage for different tissues, which could warp the original tissue and organ structure ( 19 , 25 , 26 ). On the other hand, tissue shrinkage can also benefit imaging depth as it renders larger samples smaller.…”

Section: Discussionmentioning

confidence: 94%

Tissue clearing and immunostaining to visualize the spatial organization of vasculature and tumor cells in mouse liver

et al. 2023

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The liver has a complex and hierarchical segmental organization of arteries, portal veins, hepatic veins and lymphatic vessels. In-depth imaging of liver vasculature and malignancies could improve knowledge on tumor micro-environment, local tumor growth, invasion, as well as metastasis. Non-invasive imaging techniques such as computed tomography (CT), magnetic resonance imaging (MRI) and positron-emission transmission (PET) are routine for clinical imaging, but show inadequate resolution at cellular and subcellular level. In recent years, tissue clearing – a technique rendering tissues optically transparent allowing enhanced microscopy imaging – has made great advances. While mainly used in the neurobiology field, recently more studies have used clearing techniques for imaging other organ systems as well as tumor tissues. In this study, our aim was to develop a reproducible tissue clearing and immunostaining model for visualizing intrahepatic blood microvasculature and tumor cells in murine colorectal liver metastases. CLARITY and 3DISCO/iDISCO+ are two established clearing methods that have been shown to be compatible with immunolabelling, most often in neurobiology research. In this study, CLARITY unfortunately resulted in damaged tissue integrity of the murine liver lobes and no specific immunostaining. Using the 3DISCO/iDISCO+ method, liver samples were successfully rendered optically transparent. After which, successful immunostaining of the intrahepatic microvasculature using panendothelial cell antigen MECA-32 and colorectal cancer cells using epithelial cell adhesion molecule (EpCAM) was established. This approach for tumor micro-environment tissue clearing would be especially valuable for allowing visualization of spatial heterogeneity and complex interactions of tumor cells and their environment in future studies.

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Section: Discussionmentioning

confidence: 94%

Tissue clearing and immunostaining to visualize the spatial organization of vasculature and tumor cells in mouse liver

et al. 2023

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“…In the present cases, although minor morphological changes due to shrinkage were confirmed, we observed no significant effects in terms of histopathological evaluation and diagnosis. This may have been because our sample sizes were relatively large, as Vulders et al have recently suggested that large samples tend to shrink less after solvent‐based tissue clearing [24]. Regarding immunohistochemical staining, although we did not perform such examinations on cleared tissues, it has been shown that ethyl cinnamate‐based TOC is compatible with immunohistochemical labeling [15].…”

Section: Discussionmentioning

confidence: 99%

Usefulness of a tissue optical clearing technique for forensic autopsy

Takahashi

Hayakawa

Sano

et al. 2022

Journal of Forensic Sciences

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Forensic pathologists are required to investigate lethal trauma or disease at autopsy. In addition to massive contusions of various organs, a number of small features with potentially fatal implications also need to be sought. Since such lesions may need microscopic examinations for detailed evaluation, it is important to select suitable anatomic locations for tissue sampling. For practical screening of small lesions, we have developed a tissue optical clearing (TOC) technique for forensic autopsy. The technique involves clearing with a non‐toxic organic solvent, ethyl cinnamate, which renders excised organs transparent, while hemorrhages or blood‐containing vessels remain opaque. Using this technique, tiny hemorrhages in the spinal cord were able to be identified by gross examination, allowing proper selection of locations for tissue sampling. Subsequent histopathological evaluation was successfully performed with no apparent artifacts related with the TOC procedure. In addition, a combination of TOC and targeted CT angiography allowed feasible examination of the arterial occlusive lesion in the superior mesenteric artery, and when combined with micro‐CT scanning it was useful for evaluating the lumen of the coronary artery with stent implantation. The results obtained so far indicated that TOC could complement routine forensic autopsy procedures when detailed evaluation of small lesions is required.

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“…Large microglial image datasets can also be obtained via light-sheet microscopy used in conjunction with methods to clear brains containing fluorescently labeled microglia. This approach ensures intact microglial architecture and enables global analysis of microglial morphotypes without sectioning artifacts; however, with the caveat that the shrinkage which occurs during sample preparation will alter microglial profiles ( Bekkouche et al, 2020 ; Vulders et al, 2021 ). Furthermore, light-sheet technology has modest spatial resolution which does pose an issue for researchers wanting to capture finer microglial processes in their morphometric analysis ( Watkins and St. Croix, 2018 ; Chen et al, 2022 ).…”

Section: Tools For Imaging Stained/labeled Microglia In Situmentioning

confidence: 99%

Microglial morphometric analysis: so many options, so little consistency

Reddaway,

Richardson,

Bevan

et al. 2023

Front. Neuroinform.

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Quantification of microglial activation through morphometric analysis has long been a staple of the neuroimmunologist’s toolkit. Microglial morphological phenomics can be conducted through either manual classification or constructing a digital skeleton and extracting morphometric data from it. Multiple open-access and paid software packages are available to generate these skeletons via semi-automated and/or fully automated methods with varying degrees of accuracy. Despite advancements in methods to generate morphometrics (quantitative measures of cellular morphology), there has been limited development of tools to analyze the datasets they generate, in particular those containing parameters from tens of thousands of cells analyzed by fully automated pipelines. In this review, we compare and critique the approaches using cluster analysis and machine learning driven predictive algorithms that have been developed to tackle these large datasets, and propose improvements for these methods. In particular, we highlight the need for a commitment to open science from groups developing these classifiers. Furthermore, we call attention to a need for communication between those with a strong software engineering/computer science background and neuroimmunologists to produce effective analytical tools with simplified operability if we are to see their wide-spread adoption by the glia biology community.

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Clearing-induced tisssue shrinkage: A novel observation of a thickness size effect

Cited by 5 publications

References 25 publications

Tissue clearing and immunostaining to visualize the spatial organization of vasculature and tumor cells in mouse liver

Tissue clearing and immunostaining to visualize the spatial organization of vasculature and tumor cells in mouse liver

Usefulness of a tissue optical clearing technique for forensic autopsy

Microglial morphometric analysis: so many options, so little consistency

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