Peak emission wavelength and fluorescence lifetime are coupled in far-red, GFP-like fluorescent proteins.
The discovery and use of fluorescent proteins revolutionized cell biology by allowing the visualization of proteins in living cells. Advances in fluorescent proteins, primarily through genetic engineering, have enabled more advanced analyses, including Förster resonance energy transfer (FRET) and fl...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2018-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0208075 |
| _version_ | 1818590791834009600 |
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| author | Laura Canty Santosh Hariharan Qian Liu Steven A Haney David W Andrews |
| author_facet | Laura Canty Santosh Hariharan Qian Liu Steven A Haney David W Andrews |
| author_sort | Laura Canty |
| collection | DOAJ |
| description | The discovery and use of fluorescent proteins revolutionized cell biology by allowing the visualization of proteins in living cells. Advances in fluorescent proteins, primarily through genetic engineering, have enabled more advanced analyses, including Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) and the development of genetically encoded fluorescent biosensors. These fluorescence protein-based sensors are highly effective in cells grown in monolayer cultures. However, it is often desirable to use more complex models including tissue explants, organoids, xenografts, and whole animals. These types of samples have poor light penetration owing to high scattering and absorption of light by tissue. Far-red light with a wavelength between 650-900nm is less prone to scatter, and absorption by tissues and can thus penetrate more deeply. Unfortunately, there are few fluorescent proteins in this region of the spectrum, and they have sub-optimal fluorescent properties including low brightness and short fluorescence lifetimes. Understanding the relationships between the amino-acid sequences of far-red fluorescence proteins and their photophysical properties including peak emission wavelengths and fluorescence lifetimes would be useful in the design of new fluorescence proteins for this region of the spectrum. We used both site-directed mutagenesis and gene-shuffling between mScarlet and mCardinal fluorescence proteins to create new variants and assess their properties systematically. We discovered that for far-red, GFP-like proteins the emission maxima and fluorescence lifetime have a strong inverse correlation. |
| first_indexed | 2024-12-16T10:02:10Z |
| format | Article |
| id | doaj.art-4c805a626c314dce847f3ba9a4196a04 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-16T10:02:10Z |
| publishDate | 2018-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-4c805a626c314dce847f3ba9a4196a042022-12-21T22:35:45ZengPublic Library of Science (PLoS)PLoS ONE1932-62032018-01-011311e020807510.1371/journal.pone.0208075Peak emission wavelength and fluorescence lifetime are coupled in far-red, GFP-like fluorescent proteins.Laura CantySantosh HariharanQian LiuSteven A HaneyDavid W AndrewsThe discovery and use of fluorescent proteins revolutionized cell biology by allowing the visualization of proteins in living cells. Advances in fluorescent proteins, primarily through genetic engineering, have enabled more advanced analyses, including Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) and the development of genetically encoded fluorescent biosensors. These fluorescence protein-based sensors are highly effective in cells grown in monolayer cultures. However, it is often desirable to use more complex models including tissue explants, organoids, xenografts, and whole animals. These types of samples have poor light penetration owing to high scattering and absorption of light by tissue. Far-red light with a wavelength between 650-900nm is less prone to scatter, and absorption by tissues and can thus penetrate more deeply. Unfortunately, there are few fluorescent proteins in this region of the spectrum, and they have sub-optimal fluorescent properties including low brightness and short fluorescence lifetimes. Understanding the relationships between the amino-acid sequences of far-red fluorescence proteins and their photophysical properties including peak emission wavelengths and fluorescence lifetimes would be useful in the design of new fluorescence proteins for this region of the spectrum. We used both site-directed mutagenesis and gene-shuffling between mScarlet and mCardinal fluorescence proteins to create new variants and assess their properties systematically. We discovered that for far-red, GFP-like proteins the emission maxima and fluorescence lifetime have a strong inverse correlation.https://doi.org/10.1371/journal.pone.0208075 |
| spellingShingle | Laura Canty Santosh Hariharan Qian Liu Steven A Haney David W Andrews Peak emission wavelength and fluorescence lifetime are coupled in far-red, GFP-like fluorescent proteins. PLoS ONE |
| title | Peak emission wavelength and fluorescence lifetime are coupled in far-red, GFP-like fluorescent proteins. |
| title_full | Peak emission wavelength and fluorescence lifetime are coupled in far-red, GFP-like fluorescent proteins. |
| title_fullStr | Peak emission wavelength and fluorescence lifetime are coupled in far-red, GFP-like fluorescent proteins. |
| title_full_unstemmed | Peak emission wavelength and fluorescence lifetime are coupled in far-red, GFP-like fluorescent proteins. |
| title_short | Peak emission wavelength and fluorescence lifetime are coupled in far-red, GFP-like fluorescent proteins. |
| title_sort | peak emission wavelength and fluorescence lifetime are coupled in far red gfp like fluorescent proteins |
| url | https://doi.org/10.1371/journal.pone.0208075 |
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