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Multiphoton Photochemistry of Red Fluorescent Proteins in Solution and Live Cells
[Image: see text] Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function. Ultimately, the goal of the bioimaging community is to use these probes deep in tissues and even in entire organisms, and this will require...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical
Society
2014
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4126731/ https://www.ncbi.nlm.nih.gov/pubmed/25004113 http://dx.doi.org/10.1021/jp502477c |
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author | Drobizhev, Mikhail Stoltzfus, Caleb Topol, Igor Collins, Jack Wicks, Geoffrey Mikhaylov, Alexander Barnett, Lauren Hughes, Thomas E. Rebane, Aleksander |
author_facet | Drobizhev, Mikhail Stoltzfus, Caleb Topol, Igor Collins, Jack Wicks, Geoffrey Mikhaylov, Alexander Barnett, Lauren Hughes, Thomas E. Rebane, Aleksander |
author_sort | Drobizhev, Mikhail |
collection | PubMed |
description | [Image: see text] Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function. Ultimately, the goal of the bioimaging community is to use these probes deep in tissues and even in entire organisms, and this will require two-photon laser scanning microscopy (TPLSM), with its greater tissue penetration, lower autofluorescence background, and minimum photodamage in the out-of-focus volume. However, the extremely high instantaneous light intensities of femtosecond pulses in the focal volume dramatically increase the probability of further stepwise resonant photon absorption, leading to highly excited, ionizable and reactive states, often resulting in fast bleaching of fluorescent proteins in TPLSM. Here, we show that the femtosecond multiphoton excitation of red FPs (DsRed2 and mFruits), both in solution and live cells, results in a chain of consecutive, partially reversible reactions, with individual rates driven by a high-order (3–5 photon) absorption. The first step of this process corresponds to a three- (DsRed2) or four-photon (mFruits) induced fast isomerization of the chromophore, yielding intermediate fluorescent forms, which then subsequently transform into nonfluorescent products. Our experimental data and model calculations are consistent with a mechanism in which ultrafast electron transfer from the chromophore to a neighboring positively charged amino acid residue triggers the first step of multiphoton chromophore transformations in DsRed2 and mFruits, consisting of decarboxylation of a nearby deprotonated glutamic acid residue. |
format | Online Article Text |
id | pubmed-4126731 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | American Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-41267312015-07-08 Multiphoton Photochemistry of Red Fluorescent Proteins in Solution and Live Cells Drobizhev, Mikhail Stoltzfus, Caleb Topol, Igor Collins, Jack Wicks, Geoffrey Mikhaylov, Alexander Barnett, Lauren Hughes, Thomas E. Rebane, Aleksander J Phys Chem B [Image: see text] Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function. Ultimately, the goal of the bioimaging community is to use these probes deep in tissues and even in entire organisms, and this will require two-photon laser scanning microscopy (TPLSM), with its greater tissue penetration, lower autofluorescence background, and minimum photodamage in the out-of-focus volume. However, the extremely high instantaneous light intensities of femtosecond pulses in the focal volume dramatically increase the probability of further stepwise resonant photon absorption, leading to highly excited, ionizable and reactive states, often resulting in fast bleaching of fluorescent proteins in TPLSM. Here, we show that the femtosecond multiphoton excitation of red FPs (DsRed2 and mFruits), both in solution and live cells, results in a chain of consecutive, partially reversible reactions, with individual rates driven by a high-order (3–5 photon) absorption. The first step of this process corresponds to a three- (DsRed2) or four-photon (mFruits) induced fast isomerization of the chromophore, yielding intermediate fluorescent forms, which then subsequently transform into nonfluorescent products. Our experimental data and model calculations are consistent with a mechanism in which ultrafast electron transfer from the chromophore to a neighboring positively charged amino acid residue triggers the first step of multiphoton chromophore transformations in DsRed2 and mFruits, consisting of decarboxylation of a nearby deprotonated glutamic acid residue. American Chemical Society 2014-07-08 2014-08-07 /pmc/articles/PMC4126731/ /pubmed/25004113 http://dx.doi.org/10.1021/jp502477c Text en Copyright © 2014 American Chemical Society Terms of Use (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) |
spellingShingle | Drobizhev, Mikhail Stoltzfus, Caleb Topol, Igor Collins, Jack Wicks, Geoffrey Mikhaylov, Alexander Barnett, Lauren Hughes, Thomas E. Rebane, Aleksander Multiphoton Photochemistry of Red Fluorescent Proteins in Solution and Live Cells |
title | Multiphoton Photochemistry
of Red Fluorescent Proteins
in Solution and Live Cells |
title_full | Multiphoton Photochemistry
of Red Fluorescent Proteins
in Solution and Live Cells |
title_fullStr | Multiphoton Photochemistry
of Red Fluorescent Proteins
in Solution and Live Cells |
title_full_unstemmed | Multiphoton Photochemistry
of Red Fluorescent Proteins
in Solution and Live Cells |
title_short | Multiphoton Photochemistry
of Red Fluorescent Proteins
in Solution and Live Cells |
title_sort | multiphoton photochemistry
of red fluorescent proteins
in solution and live cells |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4126731/ https://www.ncbi.nlm.nih.gov/pubmed/25004113 http://dx.doi.org/10.1021/jp502477c |
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