A structural remedy toward bright dipolar fluorophores in aqueous media.

The donor-acceptor (D-A) type dipolar fluorophores, an important class of luminescent dyes with two-photon absorption behaviour, generally emit strongly in organic solvents but poorly in aqueous media. To understand and enhance the poor emission behaviour of dipolar dyes in aqueous media, we undertake a rational approach that includes a systematic structure variation of the donor, amino substituent of acedan, an important two-photon dye. We identify several factors that influence the emission behaviour of the dipolar dyes in aqueous media through computational and photophysical studies on new acedan derivatives. As a result, we can make acedan dyes emit bright fluorescence under one- and two-photon excitation in aqueous media by suppressing the liable factors for poor emission: 1,3-allylic strain, rotational freedom, and hydrogen bonding with water. We also validate that these findings can be generally extended to other dipolar fluorophores, as demonstrated for naphthalimide, coumarin and (4-nitro-2,1,3-benzoxadiazol-7-yl)amine (NBD) dyes. The new acedan and naphthalimide dyes thus allow us to obtain much brighter two-photon fluorescent images in cells and tissues than in their conventional forms. As an application of these findings, a thiol probe is synthesized based on a new naphthalimide dye, which shows greatly enhanced fluorescence from the widely used N,N-dimethyl analogue. The results disclosed here provide essential guidelines for the development of efficient dipolar dyes and fluorescence probes for studying biological systems, particularly by two-photon microscopy.

A novel method using an acedan-based Zn(DPA) probe to monitor ATP localization in an in vivo system.

In in vitro and in vivo systems, understanding localization and the functional role of ATP is essential, but effective methods to monitor ATP in cells and tissues are limited. Although quinacrine dihydrochloride is a well-known fluorescent dye used to detect ATP, it is limited in its use because it shows non-specific nuclear staining both in vitro and in vivo. A commercial luciferin-luciferase bioluminescence assay has also been used to detect ATP, but it can not be easily used in vivo. Thus, to effectively monitor ATP in vivo, we employed a novel two-photon ATP fluorescent probe, acedan-based Zn(DPA). Using the acedan-based Zn(DPA) probe, we show that this probe produces high quality images of ATP in lung, spleen, liver and spinal cord tissues.

Studies on acedan-based mononuclear zinc complexes toward selective fluorescent probes for pyrophosphate.

We have demonstrated that mononuclear Zn(II)-dipicolylamine (DPA) complexes with an auxiliary ligand can fluorescently discriminate pyrophosphate over ATP with as high selectivity as the known fast responding dinuclear bis(ZnDPA) complexes.

Reaction-based two-photon probes for mercury ions: fluorescence imaging with dual optical windows.

For fluorescent imaging of mercury ions in living species, two-photon probes with dual optical windows are in high demand but remain unexplored. Several dithioacetals were evaluated, and a probe was found, which, upon reaction with mercury species, yielded a two-photon dye; this conversion accompanies ratiometric emission changes with a 97-nm shift, enabling fluorescent imaging of both the probe and mercury ions in cells by one- and two-photon microscopy for the first time.

A turn-on two-photon fluorescent probe for ATP and ADP.

An acedan derivative containing Zn(II)-DPA has been developed as a two-photon probe for nucleoside phosphates, which shows enhanced fluorescence toward ATP and ADP at physiological pH 7.4 among other competing anions including AMP; the probe is permeable to cell membranes and thus can be directly used for two-photon imaging of ATP and ADP in live cells.

Mononuclear Zn(II)- and Cu(II)-complexes of a hydroxynaphthalene-derived dipicolylamine: fluorescent sensing behaviours toward pyrophosphate ions.

Mononuclear Zn(II)-DPA and Cu(II)-DPA complexes crafted on 2-hydroxy-6-cyanonaphthalene fluorophore selectively recognize PPi over ATP and other anions including inorganic phosphates in aqueous medium, showing turn-on type fluorescence enhancements. Coordination of a hydroxyl group of the fluorophore, directly or in alkoxy form, to the central metal ion is crucial for the sensing processes. Both the complexes elicit a fluorescence increase in a time-dependent fashion.