Choosing the Probe for Single-Molecule Fluorescence Microscopy
Probe choice in single-molecule microscopy requires deeper evaluations than those adopted for less sensitive fluorescence microscopy studies. Indeed, fluorophore characteristics can alter or hide subtle phenomena observable at the single-molecule level, wasting the potential of the sophisticated ins...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-11-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/23/14949 |
| _version_ | 1797463059745210368 |
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| author | Chiara Schirripa Spagnolo Stefano Luin |
| author_facet | Chiara Schirripa Spagnolo Stefano Luin |
| author_sort | Chiara Schirripa Spagnolo |
| collection | DOAJ |
| description | Probe choice in single-molecule microscopy requires deeper evaluations than those adopted for less sensitive fluorescence microscopy studies. Indeed, fluorophore characteristics can alter or hide subtle phenomena observable at the single-molecule level, wasting the potential of the sophisticated instrumentation and algorithms developed for advanced single-molecule applications. There are different reasons for this, linked, e.g., to fluorophore aspecific interactions, brightness, photostability, blinking, and emission and excitation spectra. In particular, these spectra and the excitation source are interdependent, and the latter affects the autofluorescence of sample substrate, medium, and/or biological specimen. Here, we review these and other critical points for fluorophore selection in single-molecule microscopy. We also describe the possible kinds of fluorophores and the microscopy techniques based on single-molecule fluorescence. We explain the importance and impact of the various issues in fluorophore choice, and discuss how this can become more effective and decisive for increasingly demanding experiments in single- and multiple-color applications. |
| first_indexed | 2024-03-09T17:45:20Z |
| format | Article |
| id | doaj.art-3b71424fecb04ac8af47423743c0b4a5 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T17:45:20Z |
| publishDate | 2022-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-3b71424fecb04ac8af47423743c0b4a52023-11-24T11:10:38ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-11-0123231494910.3390/ijms232314949Choosing the Probe for Single-Molecule Fluorescence MicroscopyChiara Schirripa Spagnolo0Stefano Luin1National Enterprise for Nanoscience and Nanotechnology (NEST) Laboratory, Scuola Normale Superiore, Piazza San Silvestro 12, I-56127 Pisa, ItalyNational Enterprise for Nanoscience and Nanotechnology (NEST) Laboratory, Scuola Normale Superiore, Piazza San Silvestro 12, I-56127 Pisa, ItalyProbe choice in single-molecule microscopy requires deeper evaluations than those adopted for less sensitive fluorescence microscopy studies. Indeed, fluorophore characteristics can alter or hide subtle phenomena observable at the single-molecule level, wasting the potential of the sophisticated instrumentation and algorithms developed for advanced single-molecule applications. There are different reasons for this, linked, e.g., to fluorophore aspecific interactions, brightness, photostability, blinking, and emission and excitation spectra. In particular, these spectra and the excitation source are interdependent, and the latter affects the autofluorescence of sample substrate, medium, and/or biological specimen. Here, we review these and other critical points for fluorophore selection in single-molecule microscopy. We also describe the possible kinds of fluorophores and the microscopy techniques based on single-molecule fluorescence. We explain the importance and impact of the various issues in fluorophore choice, and discuss how this can become more effective and decisive for increasingly demanding experiments in single- and multiple-color applications.https://www.mdpi.com/1422-0067/23/23/14949fluorophoresfluorescent probefluorescent labelingsingle-molecule imagingfluorescent microscopy |
| spellingShingle | Chiara Schirripa Spagnolo Stefano Luin Choosing the Probe for Single-Molecule Fluorescence Microscopy International Journal of Molecular Sciences fluorophores fluorescent probe fluorescent labeling single-molecule imaging fluorescent microscopy |
| title | Choosing the Probe for Single-Molecule Fluorescence Microscopy |
| title_full | Choosing the Probe for Single-Molecule Fluorescence Microscopy |
| title_fullStr | Choosing the Probe for Single-Molecule Fluorescence Microscopy |
| title_full_unstemmed | Choosing the Probe for Single-Molecule Fluorescence Microscopy |
| title_short | Choosing the Probe for Single-Molecule Fluorescence Microscopy |
| title_sort | choosing the probe for single molecule fluorescence microscopy |
| topic | fluorophores fluorescent probe fluorescent labeling single-molecule imaging fluorescent microscopy |
| url | https://www.mdpi.com/1422-0067/23/23/14949 |
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