Influence of ClearT and ClearT2 Agitation Conditions in the Fluorescence Imaging of 3D Spheroids
Autor(a) principal: | |
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Data de Publicação: | 2020 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10316/104807 https://doi.org/10.3390/ijms22010266 |
Resumo: | 3D tumor spheroids have arisen in the last years as potent tools for the in vitro screening of novel anticancer therapeutics. Nevertheless, to increase the reproducibility and predictability of the data originated from the spheroids it is still necessary to develop or optimize the techniques used for spheroids' physical and biomolecular characterization. Fluorescence microscopy, such as confocal laser scanning microscopy (CLSM), is a tool commonly used by researchers to characterize spheroids structure and the antitumoral effect of novel therapeutics. However, its application in spheroids' analysis is hindered by the limited light penetration in thick samples. For this purpose, optical clearing solutions have been explored to increase the spheroids' transparency by reducing the light scattering. In this study, the influence of agitation conditions (i.e., static, horizontal agitation, and rotatory agitation) on the ClearT and ClearT2 methods' clearing efficacy and tumor spheroids' imaging by CLSM was characterized. The obtained results demonstrate that the ClearT method results in the improved imaging of the spheroids interior, whereas the ClearT2 resulted in an increased propidium iodide mean fluorescence intensity as well as a higher signal depth in the Z-axis. Additionally, for both methods, the best clearing results were obtained for the spheroids treated under the rotatory agitation. In general, this work provides new insights on the ClearT and ClearT2 clearing methodologies and their utilization for improving the reproducibility of the data obtained through the CLSM, such as the analysis of the cell death in response to therapeutics administration. |
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Influence of ClearT and ClearT2 Agitation Conditions in the Fluorescence Imaging of 3D SpheroidsClearTClearT2confocal microcopypropidium iodidetumor spheroidsCell Line, TumorHumansMicroscopy, ConfocalSpheroids, CellularMicroscopy, FluorescenceOptical Imaging3D tumor spheroids have arisen in the last years as potent tools for the in vitro screening of novel anticancer therapeutics. Nevertheless, to increase the reproducibility and predictability of the data originated from the spheroids it is still necessary to develop or optimize the techniques used for spheroids' physical and biomolecular characterization. Fluorescence microscopy, such as confocal laser scanning microscopy (CLSM), is a tool commonly used by researchers to characterize spheroids structure and the antitumoral effect of novel therapeutics. However, its application in spheroids' analysis is hindered by the limited light penetration in thick samples. For this purpose, optical clearing solutions have been explored to increase the spheroids' transparency by reducing the light scattering. In this study, the influence of agitation conditions (i.e., static, horizontal agitation, and rotatory agitation) on the ClearT and ClearT2 methods' clearing efficacy and tumor spheroids' imaging by CLSM was characterized. The obtained results demonstrate that the ClearT method results in the improved imaging of the spheroids interior, whereas the ClearT2 resulted in an increased propidium iodide mean fluorescence intensity as well as a higher signal depth in the Z-axis. Additionally, for both methods, the best clearing results were obtained for the spheroids treated under the rotatory agitation. In general, this work provides new insights on the ClearT and ClearT2 clearing methodologies and their utilization for improving the reproducibility of the data obtained through the CLSM, such as the analysis of the cell death in response to therapeutics administration.MDPI AG2020-12-29info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://hdl.handle.net/10316/104807http://hdl.handle.net/10316/104807https://doi.org/10.3390/ijms22010266eng1422-0067Silva, Daniel N.Costa, Elisabete C.Rodrigues, Carolina F.de Melo-Diogo, DuarteCorreia, Ilídio J.Moreira, André F.info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-01-25T21:59:11Zoai:estudogeral.uc.pt:10316/104807Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T21:21:27.235780Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Influence of ClearT and ClearT2 Agitation Conditions in the Fluorescence Imaging of 3D Spheroids |
title |
Influence of ClearT and ClearT2 Agitation Conditions in the Fluorescence Imaging of 3D Spheroids |
spellingShingle |
Influence of ClearT and ClearT2 Agitation Conditions in the Fluorescence Imaging of 3D Spheroids Silva, Daniel N. ClearT ClearT2 confocal microcopy propidium iodide tumor spheroids Cell Line, Tumor Humans Microscopy, Confocal Spheroids, Cellular Microscopy, Fluorescence Optical Imaging |
title_short |
Influence of ClearT and ClearT2 Agitation Conditions in the Fluorescence Imaging of 3D Spheroids |
title_full |
Influence of ClearT and ClearT2 Agitation Conditions in the Fluorescence Imaging of 3D Spheroids |
title_fullStr |
Influence of ClearT and ClearT2 Agitation Conditions in the Fluorescence Imaging of 3D Spheroids |
title_full_unstemmed |
Influence of ClearT and ClearT2 Agitation Conditions in the Fluorescence Imaging of 3D Spheroids |
title_sort |
Influence of ClearT and ClearT2 Agitation Conditions in the Fluorescence Imaging of 3D Spheroids |
author |
Silva, Daniel N. |
author_facet |
Silva, Daniel N. Costa, Elisabete C. Rodrigues, Carolina F. de Melo-Diogo, Duarte Correia, Ilídio J. Moreira, André F. |
author_role |
author |
author2 |
Costa, Elisabete C. Rodrigues, Carolina F. de Melo-Diogo, Duarte Correia, Ilídio J. Moreira, André F. |
author2_role |
author author author author author |
dc.contributor.author.fl_str_mv |
Silva, Daniel N. Costa, Elisabete C. Rodrigues, Carolina F. de Melo-Diogo, Duarte Correia, Ilídio J. Moreira, André F. |
dc.subject.por.fl_str_mv |
ClearT ClearT2 confocal microcopy propidium iodide tumor spheroids Cell Line, Tumor Humans Microscopy, Confocal Spheroids, Cellular Microscopy, Fluorescence Optical Imaging |
topic |
ClearT ClearT2 confocal microcopy propidium iodide tumor spheroids Cell Line, Tumor Humans Microscopy, Confocal Spheroids, Cellular Microscopy, Fluorescence Optical Imaging |
description |
3D tumor spheroids have arisen in the last years as potent tools for the in vitro screening of novel anticancer therapeutics. Nevertheless, to increase the reproducibility and predictability of the data originated from the spheroids it is still necessary to develop or optimize the techniques used for spheroids' physical and biomolecular characterization. Fluorescence microscopy, such as confocal laser scanning microscopy (CLSM), is a tool commonly used by researchers to characterize spheroids structure and the antitumoral effect of novel therapeutics. However, its application in spheroids' analysis is hindered by the limited light penetration in thick samples. For this purpose, optical clearing solutions have been explored to increase the spheroids' transparency by reducing the light scattering. In this study, the influence of agitation conditions (i.e., static, horizontal agitation, and rotatory agitation) on the ClearT and ClearT2 methods' clearing efficacy and tumor spheroids' imaging by CLSM was characterized. The obtained results demonstrate that the ClearT method results in the improved imaging of the spheroids interior, whereas the ClearT2 resulted in an increased propidium iodide mean fluorescence intensity as well as a higher signal depth in the Z-axis. Additionally, for both methods, the best clearing results were obtained for the spheroids treated under the rotatory agitation. In general, this work provides new insights on the ClearT and ClearT2 clearing methodologies and their utilization for improving the reproducibility of the data obtained through the CLSM, such as the analysis of the cell death in response to therapeutics administration. |
publishDate |
2020 |
dc.date.none.fl_str_mv |
2020-12-29 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10316/104807 http://hdl.handle.net/10316/104807 https://doi.org/10.3390/ijms22010266 |
url |
http://hdl.handle.net/10316/104807 https://doi.org/10.3390/ijms22010266 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
1422-0067 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.publisher.none.fl_str_mv |
MDPI AG |
publisher.none.fl_str_mv |
MDPI AG |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
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1799134105125781504 |