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Development of fluorescent probes for bioimaging applications.

Nagano T - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2010)

Bottom Line: Fluorescent probes, which allow visualization of cations such as Ca(2+), Zn(2+) etc., small biomolecules such as nitric oxide (NO) or enzyme activities in living cells by means of fluorescence microscopy, have become indispensable tools for clarifying functions in biological systems.This review deals with the general principles for the design of bioimaging fluorescent probes by modulating the fluorescence properties of fluorophores, employing mechanisms such as acceptor-excited Photoinduced electron Transfer (a-PeT), donor-excited Photoinduced electron Transfer (d-PeT), and spirocyclization, which have been established by our group.The a-PeT and d-PeT mechanisms are widely applicable for the design of bioimaging probes based on many fluorophores and the spirocyclization process is also expected to be useful as a fluorescence off/on switching mechanism.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan. tlong@mol.f.u-tokyo.ac.jp

ABSTRACT
Fluorescent probes, which allow visualization of cations such as Ca(2+), Zn(2+) etc., small biomolecules such as nitric oxide (NO) or enzyme activities in living cells by means of fluorescence microscopy, have become indispensable tools for clarifying functions in biological systems. This review deals with the general principles for the design of bioimaging fluorescent probes by modulating the fluorescence properties of fluorophores, employing mechanisms such as acceptor-excited Photoinduced electron Transfer (a-PeT), donor-excited Photoinduced electron Transfer (d-PeT), and spirocyclization, which have been established by our group. The a-PeT and d-PeT mechanisms are widely applicable for the design of bioimaging probes based on many fluorophores and the spirocyclization process is also expected to be useful as a fluorescence off/on switching mechanism. Fluorescence modulation mechanisms are essential for the rational design of novel fluorescence probes for target molecules. Based on these mechanisms, we have developed more than fifty bioimaging probes, of which fourteen are commercially available. The review also describes some applications of the probes developed by our group to in vitro and in vivo systems.

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NO detection by DCl-DA Cal AM, DAF-2 DA and DAF-4 DA in living cells. Fluorescence and differential interference contrast (DIC) images of HeLa cells loaded with 10 µM (0.1% DMSO as a cosolvent) DCl-DA Cal-AM (upper panel), DAF-2 DA (middle panel), or DAF-4 DA (lower panel) for 30 min are shown. The change of fluorescence images after addition of NOC 7 (final 100 µM) was measured at 5 and 15 min.
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fig05: NO detection by DCl-DA Cal AM, DAF-2 DA and DAF-4 DA in living cells. Fluorescence and differential interference contrast (DIC) images of HeLa cells loaded with 10 µM (0.1% DMSO as a cosolvent) DCl-DA Cal-AM (upper panel), DAF-2 DA (middle panel), or DAF-4 DA (lower panel) for 30 min are shown. The change of fluorescence images after addition of NOC 7 (final 100 µM) was measured at 5 and 15 min.

Mentions: DAFs are excellent fluorescent probes for NO, but their sensitivity is sometimes insufficient to measure NO in living cells. Hence, we designed and synthesized dichlorodiaminocalcein as a novel fluorescent probe for NO, to confirm that improving the intracellular retention of fluorescent probes generally leads to enhancement of sensitivity.25) The fluorescence quantum yield of DCl-DA Cal is 0.013 at pH 7.4, indicating that the fluorescence is well quenched via the a-PeT mechanism. When DCl-DA Cal reacts with NO in air, the triazole compound, DCl-DA Cal T, is produced and emits strong fluorescence in the same manner as DAF-2. As a membrane-permeable fluorescent probe, we also prepared DCl-DA Cal-AM, in which the phenolic hydroxyl group and carboxyl group are protected as acetoxymethyl (AM) ester (DCl-DA Cal-AM, see Fig. 5 for the structure).


Development of fluorescent probes for bioimaging applications.

Nagano T - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2010)

NO detection by DCl-DA Cal AM, DAF-2 DA and DAF-4 DA in living cells. Fluorescence and differential interference contrast (DIC) images of HeLa cells loaded with 10 µM (0.1% DMSO as a cosolvent) DCl-DA Cal-AM (upper panel), DAF-2 DA (middle panel), or DAF-4 DA (lower panel) for 30 min are shown. The change of fluorescence images after addition of NOC 7 (final 100 µM) was measured at 5 and 15 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037519&req=5

fig05: NO detection by DCl-DA Cal AM, DAF-2 DA and DAF-4 DA in living cells. Fluorescence and differential interference contrast (DIC) images of HeLa cells loaded with 10 µM (0.1% DMSO as a cosolvent) DCl-DA Cal-AM (upper panel), DAF-2 DA (middle panel), or DAF-4 DA (lower panel) for 30 min are shown. The change of fluorescence images after addition of NOC 7 (final 100 µM) was measured at 5 and 15 min.
Mentions: DAFs are excellent fluorescent probes for NO, but their sensitivity is sometimes insufficient to measure NO in living cells. Hence, we designed and synthesized dichlorodiaminocalcein as a novel fluorescent probe for NO, to confirm that improving the intracellular retention of fluorescent probes generally leads to enhancement of sensitivity.25) The fluorescence quantum yield of DCl-DA Cal is 0.013 at pH 7.4, indicating that the fluorescence is well quenched via the a-PeT mechanism. When DCl-DA Cal reacts with NO in air, the triazole compound, DCl-DA Cal T, is produced and emits strong fluorescence in the same manner as DAF-2. As a membrane-permeable fluorescent probe, we also prepared DCl-DA Cal-AM, in which the phenolic hydroxyl group and carboxyl group are protected as acetoxymethyl (AM) ester (DCl-DA Cal-AM, see Fig. 5 for the structure).

Bottom Line: Fluorescent probes, which allow visualization of cations such as Ca(2+), Zn(2+) etc., small biomolecules such as nitric oxide (NO) or enzyme activities in living cells by means of fluorescence microscopy, have become indispensable tools for clarifying functions in biological systems.This review deals with the general principles for the design of bioimaging fluorescent probes by modulating the fluorescence properties of fluorophores, employing mechanisms such as acceptor-excited Photoinduced electron Transfer (a-PeT), donor-excited Photoinduced electron Transfer (d-PeT), and spirocyclization, which have been established by our group.The a-PeT and d-PeT mechanisms are widely applicable for the design of bioimaging probes based on many fluorophores and the spirocyclization process is also expected to be useful as a fluorescence off/on switching mechanism.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan. tlong@mol.f.u-tokyo.ac.jp

ABSTRACT
Fluorescent probes, which allow visualization of cations such as Ca(2+), Zn(2+) etc., small biomolecules such as nitric oxide (NO) or enzyme activities in living cells by means of fluorescence microscopy, have become indispensable tools for clarifying functions in biological systems. This review deals with the general principles for the design of bioimaging fluorescent probes by modulating the fluorescence properties of fluorophores, employing mechanisms such as acceptor-excited Photoinduced electron Transfer (a-PeT), donor-excited Photoinduced electron Transfer (d-PeT), and spirocyclization, which have been established by our group. The a-PeT and d-PeT mechanisms are widely applicable for the design of bioimaging probes based on many fluorophores and the spirocyclization process is also expected to be useful as a fluorescence off/on switching mechanism. Fluorescence modulation mechanisms are essential for the rational design of novel fluorescence probes for target molecules. Based on these mechanisms, we have developed more than fifty bioimaging probes, of which fourteen are commercially available. The review also describes some applications of the probes developed by our group to in vitro and in vivo systems.

Show MeSH