A structural remedy toward bright dipolar fluorophores in aqueous mediaElectronic supplementary information (ESI) available: Materials and procedures regarding the synthesis of all the dyes and the biothiol probes, one-photon and two-photon spectroscopic analysis, tissue and cell imaging experiments, and theoretical computations. See DOI: 10.1039/c5sc01076d

• Main Authors: Singha, Subhankar, Kim, Dokyoung, Roy, Basab, Sambasivan, Sunderraman, Moon, Hyunsoo, Rao, Alla Sreenivasa, Kim, Jin Yong, Joo, Taiha, Park, Jae Woo, Rhee, Young Min, Wang, Taejun, Kim, Ki Hean, Shin, Youn Ho, Jung, Junyang, Ahn, Kyo Han
• Format: Article
• Language: English
• ISSN: 2041-6520; 2041-6539
• DOI: 10.1039/c5sc01076d

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. Structural factors governing the poor emission of dipolar dyes in aqueous media are identified, leading to new acedan derivatives with brighter fluorescence and enhanced two-photon properties.