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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| Format: | Article |
| Language: | English |
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MDPI AG
2021-06-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/11/6046 |
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| author | Asylkhan Rakhymzhan Andreas Acs Anja E. Hauser Thomas H. Winkler Raluca A. Niesner |
| author_facet | Asylkhan Rakhymzhan Andreas Acs Anja E. Hauser Thomas H. Winkler Raluca A. Niesner |
| author_sort | Asylkhan Rakhymzhan |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-10T10:44:49Z |
| format | Article |
| id | doaj.art-5d049c66536c4c73be654627e08dbb1e |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T10:44:49Z |
| publishDate | 2021-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-5d049c66536c4c73be654627e08dbb1e2023-11-21T22:41:09ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-06-012211604610.3390/ijms22116046Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing MatrixAsylkhan Rakhymzhan0Andreas Acs1Anja E. Hauser2Thomas H. Winkler3Raluca A. Niesner4Biophysical Analytics, Deutsches Rheuma-Forschungszentrum, Berlin, a Leibniz Institute, 10117 Berlin, GermanyNicolaus-Fiebiger-Zentrum, Department of Biology, Division of Genetics, Friedrich-Alexander-University of Erlangen-Nürnberg, 91054 Erlangen, GermanyCharité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Rheumatology and Clinical Immunology, Charitéplatz 1, 10117 Berlin, GermanyNicolaus-Fiebiger-Zentrum, Department of Biology, Division of Genetics, Friedrich-Alexander-University of Erlangen-Nürnberg, 91054 Erlangen, GermanyBiophysical Analytics, Deutsches Rheuma-Forschungszentrum, Berlin, a Leibniz Institute, 10117 Berlin, GermanyTwo-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.https://www.mdpi.com/1422-0067/22/11/6046two-photon laser scanning microscopymultiplexed imagingfluorescent proteinssimilarity spectral unmixing approacheslinear matrix dimension reduction |
| spellingShingle | Asylkhan Rakhymzhan Andreas Acs Anja E. Hauser Thomas H. Winkler Raluca A. Niesner Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing Matrix International Journal of Molecular Sciences two-photon laser scanning microscopy multiplexed imaging fluorescent proteins similarity spectral unmixing approaches linear matrix dimension reduction |
| 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 | two-photon laser scanning microscopy multiplexed imaging fluorescent proteins similarity spectral unmixing approaches linear matrix dimension reduction |
| url | https://www.mdpi.com/1422-0067/22/11/6046 |
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