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Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing Matrix

Two-photon microscopy enables monitoring cellular dynamics and communication in complex systems, within a genuine environment, such as living tissues and, even, living organisms. Particularly, its application to understand cellular interactions in the immune system has brought unique insights into p...

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Detalles Bibliográficos
Autores principales: Rakhymzhan, Asylkhan, Acs, Andreas, Hauser, Anja E., Winkler, Thomas H., Niesner, Raluca A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8199868/
https://www.ncbi.nlm.nih.gov/pubmed/34205072
http://dx.doi.org/10.3390/ijms22116046
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author Rakhymzhan, Asylkhan
Acs, Andreas
Hauser, Anja E.
Winkler, Thomas H.
Niesner, Raluca A.
author_facet Rakhymzhan, Asylkhan
Acs, Andreas
Hauser, Anja E.
Winkler, Thomas H.
Niesner, Raluca A.
author_sort Rakhymzhan, Asylkhan
collection PubMed
description Two-photon microscopy enables monitoring cellular dynamics and communication in complex systems, within a genuine environment, such as living tissues and, even, living organisms. Particularly, its application to understand cellular interactions in the immune system has brought unique insights into pathophysiologic processes in vivo. Simultaneous multiplexed imaging is required to understand the dynamic orchestration of the multiple cellular and non-cellular tissue compartments defining immune responses. Here, we present an improvement of our previously developed method, which allowed us to achieve multiplexed dynamic intravital two-photon imaging, by using a synergistic strategy. This strategy combines a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an unmixing algorithm based on the calculation of spectral similarities with previously measured fluorophore fingerprints. The improvement of the similarity spectral unmixing algorithm here described is based on dimensionality reduction of the mixing matrix. We demonstrate its superior performance in the correct pixel-based assignment of probes to tissue compartments labeled by single fluorophores with similar spectral fingerprints, as compared to the full-dimensional similarity spectral unmixing approach.
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spelling pubmed-81998682021-06-14 Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing Matrix Rakhymzhan, Asylkhan Acs, Andreas Hauser, Anja E. Winkler, Thomas H. Niesner, Raluca A. Int J Mol Sci Communication Two-photon microscopy enables monitoring cellular dynamics and communication in complex systems, within a genuine environment, such as living tissues and, even, living organisms. Particularly, its application to understand cellular interactions in the immune system has brought unique insights into pathophysiologic processes in vivo. Simultaneous multiplexed imaging is required to understand the dynamic orchestration of the multiple cellular and non-cellular tissue compartments defining immune responses. Here, we present an improvement of our previously developed method, which allowed us to achieve multiplexed dynamic intravital two-photon imaging, by using a synergistic strategy. This strategy combines a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an unmixing algorithm based on the calculation of spectral similarities with previously measured fluorophore fingerprints. The improvement of the similarity spectral unmixing algorithm here described is based on dimensionality reduction of the mixing matrix. We demonstrate its superior performance in the correct pixel-based assignment of probes to tissue compartments labeled by single fluorophores with similar spectral fingerprints, as compared to the full-dimensional similarity spectral unmixing approach. MDPI 2021-06-03 /pmc/articles/PMC8199868/ /pubmed/34205072 http://dx.doi.org/10.3390/ijms22116046 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 Communication
Rakhymzhan, Asylkhan
Acs, Andreas
Hauser, Anja E.
Winkler, Thomas H.
Niesner, Raluca A.
Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing Matrix
title Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing Matrix
title_full Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing Matrix
title_fullStr Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing Matrix
title_full_unstemmed Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing Matrix
title_short Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing Matrix
title_sort improvement of the similarity spectral unmixing approach for multiplexed two-photon imaging by linear dimension reduction of the mixing matrix
topic Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8199868/
https://www.ncbi.nlm.nih.gov/pubmed/34205072
http://dx.doi.org/10.3390/ijms22116046
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