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Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy
Three-photon excitation has recently been demonstrated as an effective method to perform intravital microscopy in deep, previously inaccessible regions of the mouse brain. The applicability of 3-photon excitation for deep imaging of other, more heterogeneous tissue types has been much less explored....
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8849342/ https://www.ncbi.nlm.nih.gov/pubmed/35166669 http://dx.doi.org/10.7554/eLife.63776 |
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author | Bakker, Gert-Jan Weischer, Sarah Ferrer Ortas, Júlia Heidelin, Judith Andresen, Volker Beutler, Marcus Beaurepaire, Emmanuel Friedl, Peter |
author_facet | Bakker, Gert-Jan Weischer, Sarah Ferrer Ortas, Júlia Heidelin, Judith Andresen, Volker Beutler, Marcus Beaurepaire, Emmanuel Friedl, Peter |
author_sort | Bakker, Gert-Jan |
collection | PubMed |
description | Three-photon excitation has recently been demonstrated as an effective method to perform intravital microscopy in deep, previously inaccessible regions of the mouse brain. The applicability of 3-photon excitation for deep imaging of other, more heterogeneous tissue types has been much less explored. In this work, we analyze the benefit of high-pulse-energy 1 MHz pulse-repetition-rate infrared excitation near 1300 and 1700 nm for in-depth imaging of tumorous and bone tissue. We show that this excitation regime provides a more than 2-fold increased imaging depth in tumor and bone tissue compared to the illumination conditions commonly used in 2-photon excitation, due to improved excitation confinement and reduced scattering. We also show that simultaneous 3- and 4-photon processes can be effectively induced with a single laser line, enabling the combined detection of blue to far-red fluorescence together with second and third harmonic generation without chromatic aberration, at excitation intensities compatible with live tissue imaging. Finally, we analyze photoperturbation thresholds in this excitation regime and derive setpoints for safe cell imaging. Together, these results indicate that infrared high-pulse-energy low-repetition-rate excitation opens novel perspectives for intravital deep-tissue microscopy of multiple parameters in strongly scattering tissues and organs. |
format | Online Article Text |
id | pubmed-8849342 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-88493422022-02-17 Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy Bakker, Gert-Jan Weischer, Sarah Ferrer Ortas, Júlia Heidelin, Judith Andresen, Volker Beutler, Marcus Beaurepaire, Emmanuel Friedl, Peter eLife Cancer Biology Three-photon excitation has recently been demonstrated as an effective method to perform intravital microscopy in deep, previously inaccessible regions of the mouse brain. The applicability of 3-photon excitation for deep imaging of other, more heterogeneous tissue types has been much less explored. In this work, we analyze the benefit of high-pulse-energy 1 MHz pulse-repetition-rate infrared excitation near 1300 and 1700 nm for in-depth imaging of tumorous and bone tissue. We show that this excitation regime provides a more than 2-fold increased imaging depth in tumor and bone tissue compared to the illumination conditions commonly used in 2-photon excitation, due to improved excitation confinement and reduced scattering. We also show that simultaneous 3- and 4-photon processes can be effectively induced with a single laser line, enabling the combined detection of blue to far-red fluorescence together with second and third harmonic generation without chromatic aberration, at excitation intensities compatible with live tissue imaging. Finally, we analyze photoperturbation thresholds in this excitation regime and derive setpoints for safe cell imaging. Together, these results indicate that infrared high-pulse-energy low-repetition-rate excitation opens novel perspectives for intravital deep-tissue microscopy of multiple parameters in strongly scattering tissues and organs. eLife Sciences Publications, Ltd 2022-02-15 /pmc/articles/PMC8849342/ /pubmed/35166669 http://dx.doi.org/10.7554/eLife.63776 Text en © 2022, Bakker et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited. |
spellingShingle | Cancer Biology Bakker, Gert-Jan Weischer, Sarah Ferrer Ortas, Júlia Heidelin, Judith Andresen, Volker Beutler, Marcus Beaurepaire, Emmanuel Friedl, Peter Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy |
title | Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy |
title_full | Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy |
title_fullStr | Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy |
title_full_unstemmed | Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy |
title_short | Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy |
title_sort | intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy |
topic | Cancer Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8849342/ https://www.ncbi.nlm.nih.gov/pubmed/35166669 http://dx.doi.org/10.7554/eLife.63776 |
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