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Two-photon antenna-core oxygen probe with enhanced performance.

Roussakis E, Spencer JA, Lin CP, Vinogradov SA - Anal. Chem. (2014)

Bottom Line: Recent development of two-photon phosphorescence lifetime microscopy (2PLM) of oxygen enabled first noninvasive high-resolution measurements of tissue oxygenation in vivo in 3D, providing valuable physiological information.The key improvements include significant increase in the phosphorescence quantum yield, higher efficiency of the antenna-core energy transfer, minimized quenching of the phosphorescence by electron transfer and increased signal dynamic range.Performance of PtTCHP-C307 was demonstrated in vivo in pO2 measurements through the intact mouse skull into the bone marrow, where all blood cells are made from hematopoietic stem cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.

ABSTRACT
Recent development of two-photon phosphorescence lifetime microscopy (2PLM) of oxygen enabled first noninvasive high-resolution measurements of tissue oxygenation in vivo in 3D, providing valuable physiological information. The so far developed two-photon-enhanced phosphorescent probes comprise antenna-core constructs, in which two-photon absorbing chromophores (antenna) capture and channel excitation energy to a phosphorescent core (metalloporphyrin) via intramolecular excitation energy transfer (EET). These probes allowed demonstration of the methods' potential; however, they suffer from a number of limitations, such as partial loss of emissivity to competing triplet state deactivation pathways (e.g., electron transfer) and suboptimal sensitivity to oxygen, thereby limiting spatial and temporal resolution of the method. Here we present a new probe, PtTCHP-C307, designed to overcome these limitations. The key improvements include significant increase in the phosphorescence quantum yield, higher efficiency of the antenna-core energy transfer, minimized quenching of the phosphorescence by electron transfer and increased signal dynamic range. For the same excitation flux, the new probe is able to produce up to 6-fold higher signal output than previously reported molecules. Performance of PtTCHP-C307 was demonstrated in vivo in pO2 measurements through the intact mouse skull into the bone marrow, where all blood cells are made from hematopoietic stem cells.

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(a) State energydiagram of the processes occurring in antenna-coretwo-photon-enhanced phosphorescent probes. EET, excitation energytransfer; ic, internal conversion; isc, intersystem crossing; ET,electron transfer; CR, charge recombination. See text for definitionof all involved states. (b) Chromophores used in construction of probePtTCHP-C307: coumarin-307 (C307) and PtTCHP (2, 3).
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fig1: (a) State energydiagram of the processes occurring in antenna-coretwo-photon-enhanced phosphorescent probes. EET, excitation energytransfer; ic, internal conversion; isc, intersystem crossing; ET,electron transfer; CR, charge recombination. See text for definitionof all involved states. (b) Chromophores used in construction of probePtTCHP-C307: coumarin-307 (C307) and PtTCHP (2, 3).

Mentions: The statediagram underlying the principles of 2P-enhanced antenna-corephosphorescent probes is shown in Figure 1a.Two-photon excitation first populates a 2P-accessible state of theantenna (AS2P), which may or may not be thesame as its first excited singlet state (AS1). State AS2P, if different from AS1, rapidly internally converts to AS1. The antenna chromophore(s) is positioned sufficiently close tothe phosphorescent core, so that the excitation energy transfer (EET),presumably via the Förster dipole–dipole mechanism,efficiently populates the excited singlet state of the core (CS1). The following intersystem crossing withinthe core (CS1→CT1) produces the triplet state (CT1), which eitheremits phosphorescence or undergoes quenching by molecular oxygen.For imaging applications, it is essential that minimal losses areencountered in the energy cascade leading to the final emissive state(CT1). It is also important that the quantumyield of phosphorescence from the CT1 stateis not diminished due to competing deactivation processes [e.g., tripletelectron transfer (ET) and subsequent charge recombination (CR),38,46 shown in Figure 1a with dashed lines].


Two-photon antenna-core oxygen probe with enhanced performance.

Roussakis E, Spencer JA, Lin CP, Vinogradov SA - Anal. Chem. (2014)

(a) State energydiagram of the processes occurring in antenna-coretwo-photon-enhanced phosphorescent probes. EET, excitation energytransfer; ic, internal conversion; isc, intersystem crossing; ET,electron transfer; CR, charge recombination. See text for definitionof all involved states. (b) Chromophores used in construction of probePtTCHP-C307: coumarin-307 (C307) and PtTCHP (2, 3).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4066907&req=5

fig1: (a) State energydiagram of the processes occurring in antenna-coretwo-photon-enhanced phosphorescent probes. EET, excitation energytransfer; ic, internal conversion; isc, intersystem crossing; ET,electron transfer; CR, charge recombination. See text for definitionof all involved states. (b) Chromophores used in construction of probePtTCHP-C307: coumarin-307 (C307) and PtTCHP (2, 3).
Mentions: The statediagram underlying the principles of 2P-enhanced antenna-corephosphorescent probes is shown in Figure 1a.Two-photon excitation first populates a 2P-accessible state of theantenna (AS2P), which may or may not be thesame as its first excited singlet state (AS1). State AS2P, if different from AS1, rapidly internally converts to AS1. The antenna chromophore(s) is positioned sufficiently close tothe phosphorescent core, so that the excitation energy transfer (EET),presumably via the Förster dipole–dipole mechanism,efficiently populates the excited singlet state of the core (CS1). The following intersystem crossing withinthe core (CS1→CT1) produces the triplet state (CT1), which eitheremits phosphorescence or undergoes quenching by molecular oxygen.For imaging applications, it is essential that minimal losses areencountered in the energy cascade leading to the final emissive state(CT1). It is also important that the quantumyield of phosphorescence from the CT1 stateis not diminished due to competing deactivation processes [e.g., tripletelectron transfer (ET) and subsequent charge recombination (CR),38,46 shown in Figure 1a with dashed lines].

Bottom Line: Recent development of two-photon phosphorescence lifetime microscopy (2PLM) of oxygen enabled first noninvasive high-resolution measurements of tissue oxygenation in vivo in 3D, providing valuable physiological information.The key improvements include significant increase in the phosphorescence quantum yield, higher efficiency of the antenna-core energy transfer, minimized quenching of the phosphorescence by electron transfer and increased signal dynamic range.Performance of PtTCHP-C307 was demonstrated in vivo in pO2 measurements through the intact mouse skull into the bone marrow, where all blood cells are made from hematopoietic stem cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.

ABSTRACT
Recent development of two-photon phosphorescence lifetime microscopy (2PLM) of oxygen enabled first noninvasive high-resolution measurements of tissue oxygenation in vivo in 3D, providing valuable physiological information. The so far developed two-photon-enhanced phosphorescent probes comprise antenna-core constructs, in which two-photon absorbing chromophores (antenna) capture and channel excitation energy to a phosphorescent core (metalloporphyrin) via intramolecular excitation energy transfer (EET). These probes allowed demonstration of the methods' potential; however, they suffer from a number of limitations, such as partial loss of emissivity to competing triplet state deactivation pathways (e.g., electron transfer) and suboptimal sensitivity to oxygen, thereby limiting spatial and temporal resolution of the method. Here we present a new probe, PtTCHP-C307, designed to overcome these limitations. The key improvements include significant increase in the phosphorescence quantum yield, higher efficiency of the antenna-core energy transfer, minimized quenching of the phosphorescence by electron transfer and increased signal dynamic range. For the same excitation flux, the new probe is able to produce up to 6-fold higher signal output than previously reported molecules. Performance of PtTCHP-C307 was demonstrated in vivo in pO2 measurements through the intact mouse skull into the bone marrow, where all blood cells are made from hematopoietic stem cells.

Show MeSH