A quantitative metric for the comparative evaluation of optical clearing protocols for 3D multicellular spheroids
3D multicellular spheroids quickly emerged as in vitro models because they represent the in vivo tumor environment better than standard 2D cell cultures. However, with current microscopy technologies, it is difficult to visualize individual cells in the deeper layers of 3D samples mainly because of...
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doaj-4594fabdb03e41c1b1081bda60ba2fac2021-02-25T04:17:41ZengElsevierComputational and Structural Biotechnology Journal2001-03702021-01-011912331243A quantitative metric for the comparative evaluation of optical clearing protocols for 3D multicellular spheroidsAkos Diosdi0Dominik Hirling1Maria Kovacs2Timea Toth3Maria Harmati4Krisztian Koos5Krisztina Buzas6Filippo Piccinini7Peter Horvath8Synthetic and Systems Biology Unit, Biological Research Centre (BRC), H-6726 Szeged, Hungary; Doctoral School of Biology, University of Szeged, H-6726 Szeged, HungarySynthetic and Systems Biology Unit, Biological Research Centre (BRC), H-6726 Szeged, Hungary; Doctoral School of Computer Science, University of Szeged, H-6701 Szeged, HungarySynthetic and Systems Biology Unit, Biological Research Centre (BRC), H-6726 Szeged, HungarySynthetic and Systems Biology Unit, Biological Research Centre (BRC), H-6726 Szeged, Hungary; Doctoral School of Biology, University of Szeged, H-6726 Szeged, HungarySynthetic and Systems Biology Unit, Biological Research Centre (BRC), H-6726 Szeged, HungarySynthetic and Systems Biology Unit, Biological Research Centre (BRC), H-6726 Szeged, HungarySynthetic and Systems Biology Unit, Biological Research Centre (BRC), H-6726 Szeged, Hungary; Department of Immunology, Faculty of Medicine, Faculty of Science and Informatics, University of Szeged, H-6720 Szeged, HungaryIRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, Via Piero Maroncelli 40, I-47014 Meldola, FC, ItalySynthetic and Systems Biology Unit, Biological Research Centre (BRC), H-6726 Szeged, Hungary; Institute for Molecular Medicine Finland, University of Helsinki, FI-00014 Helsinki, Finland; Single-Cell Technologies Ltd., H-6726 Szeged, Hungary; Corresponding author at: Synthetic and Systems Biology Unit, Biological Research Centre (BRC), H-6726 Szeged, Hungary.3D multicellular spheroids quickly emerged as in vitro models because they represent the in vivo tumor environment better than standard 2D cell cultures. However, with current microscopy technologies, it is difficult to visualize individual cells in the deeper layers of 3D samples mainly because of limited light penetration and scattering. To overcome this problem several optical clearing methods have been proposed but defining the most appropriate clearing approach is an open issue due to the lack of a gold standard metric. Here, we propose a guideline for 3D light microscopy imaging to achieve single-cell resolution. The guideline includes a validation experiment focusing on five optical clearing protocols. We review and compare seven quality metrics which quantitatively characterize the imaging quality of spheroids. As a test environment, we have created and shared a large 3D dataset including approximately hundred fluorescently stained and optically cleared spheroids. Based on the results we introduce the use of a novel quality metric as a promising method to serve as a gold standard, applicable to compare optical clearing protocols, and decide on the most suitable one for a particular experiment.http://www.sciencedirect.com/science/article/pii/S2001037021000441Optical tissue clearingSpheroidLight-sheet microscopyFocus metrics |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Akos Diosdi Dominik Hirling Maria Kovacs Timea Toth Maria Harmati Krisztian Koos Krisztina Buzas Filippo Piccinini Peter Horvath |
spellingShingle |
Akos Diosdi Dominik Hirling Maria Kovacs Timea Toth Maria Harmati Krisztian Koos Krisztina Buzas Filippo Piccinini Peter Horvath A quantitative metric for the comparative evaluation of optical clearing protocols for 3D multicellular spheroids Computational and Structural Biotechnology Journal Optical tissue clearing Spheroid Light-sheet microscopy Focus metrics |
author_facet |
Akos Diosdi Dominik Hirling Maria Kovacs Timea Toth Maria Harmati Krisztian Koos Krisztina Buzas Filippo Piccinini Peter Horvath |
author_sort |
Akos Diosdi |
title |
A quantitative metric for the comparative evaluation of optical clearing protocols for 3D multicellular spheroids |
title_short |
A quantitative metric for the comparative evaluation of optical clearing protocols for 3D multicellular spheroids |
title_full |
A quantitative metric for the comparative evaluation of optical clearing protocols for 3D multicellular spheroids |
title_fullStr |
A quantitative metric for the comparative evaluation of optical clearing protocols for 3D multicellular spheroids |
title_full_unstemmed |
A quantitative metric for the comparative evaluation of optical clearing protocols for 3D multicellular spheroids |
title_sort |
quantitative metric for the comparative evaluation of optical clearing protocols for 3d multicellular spheroids |
publisher |
Elsevier |
series |
Computational and Structural Biotechnology Journal |
issn |
2001-0370 |
publishDate |
2021-01-01 |
description |
3D multicellular spheroids quickly emerged as in vitro models because they represent the in vivo tumor environment better than standard 2D cell cultures. However, with current microscopy technologies, it is difficult to visualize individual cells in the deeper layers of 3D samples mainly because of limited light penetration and scattering. To overcome this problem several optical clearing methods have been proposed but defining the most appropriate clearing approach is an open issue due to the lack of a gold standard metric. Here, we propose a guideline for 3D light microscopy imaging to achieve single-cell resolution. The guideline includes a validation experiment focusing on five optical clearing protocols. We review and compare seven quality metrics which quantitatively characterize the imaging quality of spheroids. As a test environment, we have created and shared a large 3D dataset including approximately hundred fluorescently stained and optically cleared spheroids. Based on the results we introduce the use of a novel quality metric as a promising method to serve as a gold standard, applicable to compare optical clearing protocols, and decide on the most suitable one for a particular experiment. |
topic |
Optical tissue clearing Spheroid Light-sheet microscopy Focus metrics |
url |
http://www.sciencedirect.com/science/article/pii/S2001037021000441 |
work_keys_str_mv |
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