In contrast to one-photon microscopy, two-photon probe-based fluorescent imaging can provide improved three-dimensional spatial localization and increased imaging depth. Consequently, it has become one of the most attractive techniques for studying biological events in living cells and tissues. However, the quantitation of these probes is primarily based on single-emission intensity change, which tends to be affected by a variety of environmental factors. Ratiometric probes, on the other hand, can eliminate these interferences by the built-in correction of the dual emission bands, resulting in a more favorable system for imaging living cells and tissues. Herein, for the first time, we adopted a through-bond energy transfer (TBET) strategy to design and synthesize a small molecular ratiometric two-photon fluorescent probe for imaging living cells and tissues in real time. Specifically, a two-photon fluorophore (D-π-A-structured naphthalene derivative) and a rhodamine B fluorophore are directly connected by electronically conjugated bond to form a TBET probe, or Np-Rh, which shows a target-modulated ratiometric two-photon fluorescence response with highly efficient energy transfer (93.7%) and two well-resolved emission peaks separated by 100 nm. This novel probe was then applied for two-photon imaging of living cells and tissues and showed high ratiometric imaging resolution and deep-tissue imaging depth of 180 μm, thus demonstrating its practical application in biological systems.

H2S is the third endogenously generated gaseous signaling compound and has also been known to involve a variety of physiological processes. To better understand its physiological and pathological functions, efficient methods for monitoring of H2S in living systems are desired. Although quite a few one photon fluorescence probes have been reported for H2S, two-photon (TP) probes are more favorable for intracellular imaging. In this work, by employing a donor-π-acceptor-structured naphthalene derivative as the two-photon fluorophore and an azide group as the recognition unit, we reported a new two-photon bioimaging probe 6-(benzo[d]thiazol-2'-yl)-2-azidonaphthalene (NHS1) for H2S with improved sensitivity. The probe shows very low background fluorescence in the absence of H2S. In the presence of H2S, however, a significant enhancement for both one photon and TP excited fluorescence were observed, resulting in a high sensitivity to H2S in aqueous solutions with a detection limit of 20 nM observed, much lower than the previously reported TP probe. The probe also exhibits a wide linear response concentration range (0-5 μM) to H2S with high selectivity. All these features are favorable for direct monitoring of H2S in complex biological samples. It was then applied for direct TP imaging of H2S in living cells with satisfactory sensitivity, demonstrating its value of practical application in biological systems.

A universal sensing platform with significantly enhanced image contrast and apparent brightness in biological imaging applications.

Diabetes is one of the metabolic diseases marked by hyperglycemia and is often accompanied by the occurrence of some complications. As a biomarker of oxidative stress, hydrogen peroxide (H 2 O 2 ) has close association with the occurrence and development of diabetes and its complications. Unfortunately, there is no fluorescent probe reported for imaging H 2 O 2 in diabetic mice. Here, a novel near-infrared (NIR) fluorescent probe named QX-B was designed and synthesized to detect H 2 O 2 . For the probe, the quinolinium-xanthene dye is used as the fluorophore and borate ester is chosen as the response group. After the addition of H 2 O 2 , a strong NIR fluorescence signal at 772 nm is observed. The probe not only shows high sensitivity with 10-fold enhancement but also displays excellent selectivity to H 2 O 2 over other possible interfering species. In the meantime, the possible response mechanism of QX-B toward H 2 O 2 was proposed and verified by the high-performance liquid chromatography (HPLC) experiment, mass spectra (MS) experiment, and density functional theory (DFT) calculation. Furthermore, based on the low cell cytotoxicity of QX-B, it has been applied in imaging exogenous and endogenous H 2 O 2 in HeLa cells, HCT116 cells, 4T1 cells, and zebrafish successfully. More importantly, inspired by the performance of NIR fluorescence, QX-B has been used in monitoring H 2 O 2 in diabetic mice for the first time. This provides very important information for the diagnosis and treatment of diabetes and its complications.

A novel coumarin-based fluorescent probe, P-CM, for quantitative detection of nitroxyl (HNO) was developed. P-CM exhibits a selective response to HNO over other biological reductants and was also applied for quantitative detection of HNO in bovine serum with satisfactory results.