Tissue clearing may alter emission and absorption properties of common fluorophores
Abstract In recent years, 3D cell culture has been gaining a more widespread following across many fields of biology. Tissue clearing enables optical analysis of intact 3D samples and investigation of molecular and structural mechanisms by homogenizing the refractive indices of tissues to make them...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-04-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-09303-9 |
| _version_ | 1828733814732488704 |
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| author | Farsam Eliat Rebecca Sohn Henrik Renner Theresa Kagermeier Stefan Volkery Heike Brinkmann Nils Kirschnick Friedemann Kiefer Martha Grabos Katharina Becker Ivan Bedzhov Hans R. Schöler Jan M. Bruder |
| author_facet | Farsam Eliat Rebecca Sohn Henrik Renner Theresa Kagermeier Stefan Volkery Heike Brinkmann Nils Kirschnick Friedemann Kiefer Martha Grabos Katharina Becker Ivan Bedzhov Hans R. Schöler Jan M. Bruder |
| author_sort | Farsam Eliat |
| collection | DOAJ |
| description | Abstract In recent years, 3D cell culture has been gaining a more widespread following across many fields of biology. Tissue clearing enables optical analysis of intact 3D samples and investigation of molecular and structural mechanisms by homogenizing the refractive indices of tissues to make them nearly transparent. Here, we describe and quantify that common clearing solutions including benzyl alcohol/benzyl benzoate (BABB), PEG-associated solvent system (PEGASOS), immunolabeling-enabled imaging of solvent-cleared organs (iDISCO), clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC), and ScaleS4 alter the emission spectra of Alexa Fluor fluorophores and fluorescent dyes. Clearing modifies not only the emitted light intensity but also alters the absorption and emission peaks, at times to several tens of nanometers. The resulting shifts depend on the interplay of solvent, fluorophore, and the presence of cells. For biological applications, this increases the risk for unexpected channel crosstalk, as filter sets are usually not optimized for altered fluorophore emission spectra in clearing solutions. This becomes especially problematic in high throughput/high content campaigns, which often rely on multiband excitation to increase acquisition speed. Consequently, researchers relying on clearing in quantitative multiband excitation experiments should crosscheck their fluorescent signal after clearing in order to inform the proper selection of filter sets and fluorophores for analysis. |
| first_indexed | 2024-04-12T22:37:20Z |
| format | Article |
| id | doaj.art-73f74211564f4e0996b0fbd8dae25874 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-12T22:37:20Z |
| publishDate | 2022-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-73f74211564f4e0996b0fbd8dae258742022-12-22T03:13:50ZengNature PortfolioScientific Reports2045-23222022-04-0112111410.1038/s41598-022-09303-9Tissue clearing may alter emission and absorption properties of common fluorophoresFarsam Eliat0Rebecca Sohn1Henrik Renner2Theresa Kagermeier3Stefan Volkery4Heike Brinkmann5Nils Kirschnick6Friedemann Kiefer7Martha Grabos8Katharina Becker9Ivan Bedzhov10Hans R. Schöler11Jan M. Bruder12Max Planck Institute for Molecular BiomedicineMax Planck Institute for Molecular BiomedicineMax Planck Institute for Molecular BiomedicineMax Planck Institute for Molecular BiomedicineMax Planck Institute for Molecular BiomedicineEmbryonic Self-Organization research group, Max Planck Institute for Molecular BiomedicineMax Planck Institute for Molecular BiomedicineMax Planck Institute for Molecular BiomedicineMax Planck Institute for Molecular BiomedicineMax Planck Institute for Molecular BiomedicineEmbryonic Self-Organization research group, Max Planck Institute for Molecular BiomedicineMax Planck Institute for Molecular BiomedicineMax Planck Institute for Molecular BiomedicineAbstract In recent years, 3D cell culture has been gaining a more widespread following across many fields of biology. Tissue clearing enables optical analysis of intact 3D samples and investigation of molecular and structural mechanisms by homogenizing the refractive indices of tissues to make them nearly transparent. Here, we describe and quantify that common clearing solutions including benzyl alcohol/benzyl benzoate (BABB), PEG-associated solvent system (PEGASOS), immunolabeling-enabled imaging of solvent-cleared organs (iDISCO), clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC), and ScaleS4 alter the emission spectra of Alexa Fluor fluorophores and fluorescent dyes. Clearing modifies not only the emitted light intensity but also alters the absorption and emission peaks, at times to several tens of nanometers. The resulting shifts depend on the interplay of solvent, fluorophore, and the presence of cells. For biological applications, this increases the risk for unexpected channel crosstalk, as filter sets are usually not optimized for altered fluorophore emission spectra in clearing solutions. This becomes especially problematic in high throughput/high content campaigns, which often rely on multiband excitation to increase acquisition speed. Consequently, researchers relying on clearing in quantitative multiband excitation experiments should crosscheck their fluorescent signal after clearing in order to inform the proper selection of filter sets and fluorophores for analysis.https://doi.org/10.1038/s41598-022-09303-9 |
| spellingShingle | Farsam Eliat Rebecca Sohn Henrik Renner Theresa Kagermeier Stefan Volkery Heike Brinkmann Nils Kirschnick Friedemann Kiefer Martha Grabos Katharina Becker Ivan Bedzhov Hans R. Schöler Jan M. Bruder Tissue clearing may alter emission and absorption properties of common fluorophores Scientific Reports |
| title | Tissue clearing may alter emission and absorption properties of common fluorophores |
| title_full | Tissue clearing may alter emission and absorption properties of common fluorophores |
| title_fullStr | Tissue clearing may alter emission and absorption properties of common fluorophores |
| title_full_unstemmed | Tissue clearing may alter emission and absorption properties of common fluorophores |
| title_short | Tissue clearing may alter emission and absorption properties of common fluorophores |
| title_sort | tissue clearing may alter emission and absorption properties of common fluorophores |
| url | https://doi.org/10.1038/s41598-022-09303-9 |
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