Simultaneous quantitative live cell imaging of multiple FRET-based biosensors.
We have developed a novel method for multi-color spectral FRET analysis which is used to study a system of three independent FRET-based molecular sensors composed of the combinations of only three fluorescent proteins. This method is made possible by a novel routine for computing the 3-D excitation/...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3628763?pdf=render |
| _version_ | 1811205219774627840 |
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| author | Andrew Woehler |
| author_facet | Andrew Woehler |
| author_sort | Andrew Woehler |
| collection | DOAJ |
| description | We have developed a novel method for multi-color spectral FRET analysis which is used to study a system of three independent FRET-based molecular sensors composed of the combinations of only three fluorescent proteins. This method is made possible by a novel routine for computing the 3-D excitation/emission spectral fingerprint of FRET from reference measurements of the donor and acceptor alone. By unmixing the 3D spectrum of the FRET sample, the total relative concentrations of the fluorophores and their scaled FRET efficiencies are directly measured, from which apparent FRET efficiencies can be computed. If the FRET sample is composed of intramolecular FRET sensors it is possible to determine the total relative concentration of the sensors and then estimate absolute FRET efficiency of each sensor. Using multiple tandem constructs with fixed FRET efficiency as well as FRET-based calcium sensors with novel fluorescent protein combinations we demonstrate that the computed FRET efficiencies are accurate and changes in these quantities occur without crosstalk. We provide an example of this method's potential by demonstrating simultaneous imaging of spatially colocalized changes in [Ca(2+)], [cAMP], and PKA activity. |
| first_indexed | 2024-04-12T03:27:11Z |
| format | Article |
| id | doaj.art-5708b1b672644d898cacd8ce2f7929c0 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-12T03:27:11Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-5708b1b672644d898cacd8ce2f7929c02022-12-22T03:49:38ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0184e6109610.1371/journal.pone.0061096Simultaneous quantitative live cell imaging of multiple FRET-based biosensors.Andrew WoehlerWe have developed a novel method for multi-color spectral FRET analysis which is used to study a system of three independent FRET-based molecular sensors composed of the combinations of only three fluorescent proteins. This method is made possible by a novel routine for computing the 3-D excitation/emission spectral fingerprint of FRET from reference measurements of the donor and acceptor alone. By unmixing the 3D spectrum of the FRET sample, the total relative concentrations of the fluorophores and their scaled FRET efficiencies are directly measured, from which apparent FRET efficiencies can be computed. If the FRET sample is composed of intramolecular FRET sensors it is possible to determine the total relative concentration of the sensors and then estimate absolute FRET efficiency of each sensor. Using multiple tandem constructs with fixed FRET efficiency as well as FRET-based calcium sensors with novel fluorescent protein combinations we demonstrate that the computed FRET efficiencies are accurate and changes in these quantities occur without crosstalk. We provide an example of this method's potential by demonstrating simultaneous imaging of spatially colocalized changes in [Ca(2+)], [cAMP], and PKA activity.http://europepmc.org/articles/PMC3628763?pdf=render |
| spellingShingle | Andrew Woehler Simultaneous quantitative live cell imaging of multiple FRET-based biosensors. PLoS ONE |
| title | Simultaneous quantitative live cell imaging of multiple FRET-based biosensors. |
| title_full | Simultaneous quantitative live cell imaging of multiple FRET-based biosensors. |
| title_fullStr | Simultaneous quantitative live cell imaging of multiple FRET-based biosensors. |
| title_full_unstemmed | Simultaneous quantitative live cell imaging of multiple FRET-based biosensors. |
| title_short | Simultaneous quantitative live cell imaging of multiple FRET-based biosensors. |
| title_sort | simultaneous quantitative live cell imaging of multiple fret based biosensors |
| url | http://europepmc.org/articles/PMC3628763?pdf=render |
| work_keys_str_mv | AT andrewwoehler simultaneousquantitativelivecellimagingofmultiplefretbasedbiosensors |