Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation
Abstract Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is p...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-08-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-17825-5 |
| _version_ | 1811340482148564992 |
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| author | Mariano Gonzalez Pisfil Iliya Nadelson Brigitte Bergner Sonja Rottmeier Andreas W. Thomae Steffen Dietzel |
| author_facet | Mariano Gonzalez Pisfil Iliya Nadelson Brigitte Bergner Sonja Rottmeier Andreas W. Thomae Steffen Dietzel |
| author_sort | Mariano Gonzalez Pisfil |
| collection | DOAJ |
| description | Abstract Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is preferable for multi-color analyses. However, this limits the number of usable spectral channels. Using cultured cells, common staining protocols, and commercially available fluorochromes and microscopes we exploit that the number of fluorochromes in STED or confocal microscopy can be increased by phasor based fluorescence lifetime separation of two dyes with similar emission spectra but different fluorescent lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near red (exc. 594 nm, em. 600–630) and two in the far red channel (633/641–680), supplemented by a single further redshifted fluorochrome (670/701–750) were all depleted with a single laser at 775 nm thus avoiding potential alignment issues. Generally, this approach doubles the number of fully distinguishable colors in laser scanning microscopy. We provide evidence that eight color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified and we confirm that a fluorochrome may have different lifetimes depending on the molecules to which it is coupled. |
| first_indexed | 2024-04-13T18:42:36Z |
| format | Article |
| id | doaj.art-7bc4a5cbc9924ec0bf212eeed0d614ff |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-13T18:42:36Z |
| publishDate | 2022-08-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-7bc4a5cbc9924ec0bf212eeed0d614ff2022-12-22T02:34:40ZengNature PortfolioScientific Reports2045-23222022-08-0112111510.1038/s41598-022-17825-5Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separationMariano Gonzalez Pisfil0Iliya Nadelson1Brigitte Bergner2Sonja Rottmeier3Andreas W. Thomae4Steffen Dietzel5Core Facility Bioimaging and Walter-Brendel-Centre of Experimental Medicine, Biomedical Center, Ludwig-Maximilians-Universität MünchenCore Facility Bioimaging and Walter-Brendel-Centre of Experimental Medicine, Biomedical Center, Ludwig-Maximilians-Universität MünchenCore Facility Bioimaging and Walter-Brendel-Centre of Experimental Medicine, Biomedical Center, Ludwig-Maximilians-Universität MünchenCore Facility Bioimaging and Walter-Brendel-Centre of Experimental Medicine, Biomedical Center, Ludwig-Maximilians-Universität MünchenCore Facility Bioimaging and Walter-Brendel-Centre of Experimental Medicine, Biomedical Center, Ludwig-Maximilians-Universität MünchenCore Facility Bioimaging and Walter-Brendel-Centre of Experimental Medicine, Biomedical Center, Ludwig-Maximilians-Universität MünchenAbstract Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is preferable for multi-color analyses. However, this limits the number of usable spectral channels. Using cultured cells, common staining protocols, and commercially available fluorochromes and microscopes we exploit that the number of fluorochromes in STED or confocal microscopy can be increased by phasor based fluorescence lifetime separation of two dyes with similar emission spectra but different fluorescent lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near red (exc. 594 nm, em. 600–630) and two in the far red channel (633/641–680), supplemented by a single further redshifted fluorochrome (670/701–750) were all depleted with a single laser at 775 nm thus avoiding potential alignment issues. Generally, this approach doubles the number of fully distinguishable colors in laser scanning microscopy. We provide evidence that eight color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified and we confirm that a fluorochrome may have different lifetimes depending on the molecules to which it is coupled.https://doi.org/10.1038/s41598-022-17825-5 |
| spellingShingle | Mariano Gonzalez Pisfil Iliya Nadelson Brigitte Bergner Sonja Rottmeier Andreas W. Thomae Steffen Dietzel Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation Scientific Reports |
| title | Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation |
| title_full | Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation |
| title_fullStr | Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation |
| title_full_unstemmed | Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation |
| title_short | Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation |
| title_sort | stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation |
| url | https://doi.org/10.1038/s41598-022-17825-5 |
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