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Invadopodia Structure in 3D Environment Resolved by Near-Infrared Branding Protocol Combining Correlative Confocal and FIB-SEM Microscopy

Cancer cell invasion through tissue barriers is the intrinsic feature of metastasis, the most life-threatening aspect of cancer. Detailed observation and analysis of cancer cell behaviour in a 3D environment is essential for a full understanding of the mechanisms of cancer cell invasion. The inheren...

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Autores principales: Dalecká, Markéta, Sabó, Ján, Backová, Lenka, Rösel, Daniel, Brábek, Jan, Benda, Aleš, Tolde, Ondřej
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8346040/
https://www.ncbi.nlm.nih.gov/pubmed/34360570
http://dx.doi.org/10.3390/ijms22157805
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author Dalecká, Markéta
Sabó, Ján
Backová, Lenka
Rösel, Daniel
Brábek, Jan
Benda, Aleš
Tolde, Ondřej
author_facet Dalecká, Markéta
Sabó, Ján
Backová, Lenka
Rösel, Daniel
Brábek, Jan
Benda, Aleš
Tolde, Ondřej
author_sort Dalecká, Markéta
collection PubMed
description Cancer cell invasion through tissue barriers is the intrinsic feature of metastasis, the most life-threatening aspect of cancer. Detailed observation and analysis of cancer cell behaviour in a 3D environment is essential for a full understanding of the mechanisms of cancer cell invasion. The inherent limits of optical microscopy resolution do not allow to for in-depth observation of intracellular structures, such as invadopodia of invading cancer cells. The required resolution can be achieved using electron microscopy techniques such as FIB-SEM. However, visualising cells in a 3D matrix using FIB-SEM is challenging due to difficulties with localisation of a specific cell deep within the resin block. We have developed a new protocol based on the near-infrared branding (NIRB) procedure that extends the pattern from the surface grid deep inside the resin. This 3D burned pattern allows for precise trimming followed by targeted 3D FIB-SEM. Here we present detailed 3D CLEM results combining confocal and FIB-SEM imaging of cancer cell invadopodia that extend deep into the collagen meshwork.
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spelling pubmed-83460402021-08-07 Invadopodia Structure in 3D Environment Resolved by Near-Infrared Branding Protocol Combining Correlative Confocal and FIB-SEM Microscopy Dalecká, Markéta Sabó, Ján Backová, Lenka Rösel, Daniel Brábek, Jan Benda, Aleš Tolde, Ondřej Int J Mol Sci Article Cancer cell invasion through tissue barriers is the intrinsic feature of metastasis, the most life-threatening aspect of cancer. Detailed observation and analysis of cancer cell behaviour in a 3D environment is essential for a full understanding of the mechanisms of cancer cell invasion. The inherent limits of optical microscopy resolution do not allow to for in-depth observation of intracellular structures, such as invadopodia of invading cancer cells. The required resolution can be achieved using electron microscopy techniques such as FIB-SEM. However, visualising cells in a 3D matrix using FIB-SEM is challenging due to difficulties with localisation of a specific cell deep within the resin block. We have developed a new protocol based on the near-infrared branding (NIRB) procedure that extends the pattern from the surface grid deep inside the resin. This 3D burned pattern allows for precise trimming followed by targeted 3D FIB-SEM. Here we present detailed 3D CLEM results combining confocal and FIB-SEM imaging of cancer cell invadopodia that extend deep into the collagen meshwork. MDPI 2021-07-21 /pmc/articles/PMC8346040/ /pubmed/34360570 http://dx.doi.org/10.3390/ijms22157805 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Dalecká, Markéta
Sabó, Ján
Backová, Lenka
Rösel, Daniel
Brábek, Jan
Benda, Aleš
Tolde, Ondřej
Invadopodia Structure in 3D Environment Resolved by Near-Infrared Branding Protocol Combining Correlative Confocal and FIB-SEM Microscopy
title Invadopodia Structure in 3D Environment Resolved by Near-Infrared Branding Protocol Combining Correlative Confocal and FIB-SEM Microscopy
title_full Invadopodia Structure in 3D Environment Resolved by Near-Infrared Branding Protocol Combining Correlative Confocal and FIB-SEM Microscopy
title_fullStr Invadopodia Structure in 3D Environment Resolved by Near-Infrared Branding Protocol Combining Correlative Confocal and FIB-SEM Microscopy
title_full_unstemmed Invadopodia Structure in 3D Environment Resolved by Near-Infrared Branding Protocol Combining Correlative Confocal and FIB-SEM Microscopy
title_short Invadopodia Structure in 3D Environment Resolved by Near-Infrared Branding Protocol Combining Correlative Confocal and FIB-SEM Microscopy
title_sort invadopodia structure in 3d environment resolved by near-infrared branding protocol combining correlative confocal and fib-sem microscopy
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8346040/
https://www.ncbi.nlm.nih.gov/pubmed/34360570
http://dx.doi.org/10.3390/ijms22157805
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