Amphiphilic Rhodamine Fluorescent Probes Combined with Basal Imaging for Fine Structures of the Cell Membrane.

Confocal fluorescence imaging of fine structures of the cell membrane is important for understanding their biofunctions but is often neglected due to the lack of an effective method. Herein, we develop new amphiphilic rhodamine fluorescent probe RMGs in combination with basal imaging for this purpose. The probes show high signal-to-noise ratio and brightness and low internalization rate, making them suitable for imaging the fine substructures of the cell membrane. Using the representative probe RMG3, we not only observed the cell pseudopodia and intercellular nanotubes but also monitored the formation of migrasomes in real time. More importantly, in-depth imaging studies on more cell lines revealed for the first time that hepatocellular carcinoma cells secreted much more adherent extracellular vesicles than other cell lines, which might serve as a potential indicator of liver cells. We believe that RMGs may be useful for investigating the fine structures of the cell membrane.

Golgi-Targeted Fluorescent Probe for Imaging NO in Alzheimer's Disease.

Nitric oxide (NO) is a crucial neurotransmitter participating in many biological processes via nitrosylation reaction. NO produced in diverse subcellular regions also regulates the function of cells in different manners. A Golgi apparatus is rich in nitric oxide synthase and may serve as a potential therapeutic target for Alzheimer's disease (AD). However, due to the lack of an effective tool, it is difficult to reveal the relationship between Golgi-NO and AD. Herein, we report Golgi-NO as the first Golgi-targeted fluorescent probe for sensing and imaging NO in the Golgi apparatus. The probe is designed and synthesized by incorporating 4-sulfamoylphenylamide as a Golgi-targeted moiety to 6-carboxyrhodamine B, generating a fluorophore of Golgi-RhB with modifiable carboxyl, which is then combined with the NO recognition moiety of o-diaminobenzene. The probe shows superior analytical performance including accurate Golgi-targeted ability and high selectivity for NO. Moreover, using the probe, we disclose a significant increase of NO in Golgi apparatus in the AD model. This study provides a competent tool for studying the function and nitrosylation of NO in the Golgi apparatus in related diseases.

An Oxazine-Based Fluorogenic Probe with Changeable π-Conjugation to Eliminate False-Positive Interference of Albumin and Its Application to Sensing Aminopeptidase†N.

Many fluorophores/probes suffer from the interference of albumin in biosystems. Herein, we propose an effective strategy to overcome this interference by virtue of both an albumin-insensitive fluorophore and its changeable π-conjugation, and demonstrate the strategy by designing an oxazine-based fluorogenic probe for aminopeptidase N (APN). The modification on the N atom in the oxazine fluorophore with alanine through a cleavable linker locks the resulting probe in a non-conjugated, colorless and non-fluorescent state, so the non-specific interaction of albumin produces no spectroscopic response. APN can selectively cleave the alanine moiety, restoring the large π-conjugation and strong fluorescence. The capability of the probe to eliminate the albumin influence has been demonstrated by imaging APN in different cell lines, and by quantitatively determining APN in human serum and mouse urine. The present strategy may be useful for developing more specific fluorogenic probes for other enzymes.

Xanthene-Based NIR-II Dyes for In Vivo Dynamic Imaging of Blood Circulation.

Fluorescence bioimaging through the second near-infrared window (NIR-II, 1000-1700 nm) has attracted much attention due to its deep penetration and high contrast. However, exploring new fluorescent materials, especially small molecular fluorophores with long wavelength and high brightness, is still quite challenging. By expanding π-conjugation and enhancing the intramolecular charge transfer effect, herein we report a series of new xanthene-based NIR-II dyes, named VIXs. Among these dyes, VIX-4 exhibits the best performance with fluorescence emission at 1210 nm and high brightness and has been used for dynamically imaging the blood flow of mice at 200 fps. By virtue of high spatiotemporal resolution of the dynamic imaging, we can distinguish directly the artery and vein through the blood flow direction and measure the blood flow volume by the videos. This study provides not only an effective tool for high spatial and temporal resolution bioimaging but also a new and promising conjugated skeleton for NIR-II dyes.

Design, Synthesis, and Application of a Small Molecular NIR-II Fluorophore with Maximal Emission beyond 1200 nm.

Small molecular fluorophores in the second near-infrared window (NIR-II) have aroused much interest because of their excellent performance. Herein, a new small molecular NIR-II fluorophore, FM1210, with maximal emission beyond 1200 nm is reported. Compared to the corresponding control fluorophore CF1065, FM1210 exhibits an increase of 145 nm in the emission maximum, which is ascribed to the simultaneous introduction of both a Se atom and amino groups into the benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole) skeleton. This large increase in the maximal emission enables FM1210 to be capable of in vivo imaging with lower autofluorescence, higher signal-to-background ratio, and better resolution. Moreover, nanosized FM1210 encapsulated in liposomes possesses passive targeting ability and good water solubility, and is suitable for imaging a tumor and even its vasculature with high signal-to-background ratio.

Combination of changeable π-conjugation and hydrophilic groups for developing water-soluble small-molecule NIR-II fluorogenic probes.

Small-molecule probes emitting in the second near-infrared window (NIR-II) are attracting great attention because of their deep-tissue imaging ability. However, developing NIR-II fluorogenic (off-on) probes with good water solubility remains a great challenge due to the lack of a facile approach. Herein we first report the combination of changeable π-conjugation and hydrophilic groups as an effective strategy for developing water-soluble NIR-II fluorogenic probes. With the strategy, new water-soluble NIR-II fluorophores are prepared, among which NIR-II-F2 and NIR-II-F3 show superior stability and bright fluorescence in aqueous media, and are thus used to design two water-soluble NIR-II fluorogenic probes for leucine aminopeptidase (LAP). The excellent performance in real aqueous bio-environments is demonstrated by imaging mouse vasculatures and organs with NIR-II-F2, and LAP in drug-induced liver injury mice with one of the enzymatic probes; however, water-insoluble dyes cannot achieve such in vivo imaging under the same conditions. Our strategy may be helpful for further developing water-soluble organic NIR-II fluorogenic probes for in vivo imaging of other analytes.

Specific Spleen-Accumulated NIR-II Fluorescent Probe for Imaging-Guided Splenic Operation.

Spleen is a large immune organ in the body. Splenic operations such as splenectomy and intrasplenic injection are of paramount importance for immunological research and splenic diseases. Fluorescence imaging can vastly simplify these operations, but a specific spleen-targeting probe is still unavailable. Herein, the first specific spleen-accumulated fluorescent probe, VIX-S is reported, which fluoresces at 1064 nm and is highly stable. Systematic studies reveal the superior targeting and imaging performance of VIX-S for the spleen in both nude and haired mice. In vivo imaging indicates that the probe can image the morphology of spleen with a signal-background ratio of at least two-fold higher than that of the liver. Moreover, the application of VIX-S in imaging-guided splenic operation, including splenic injury and intrasplenic injection, is demonstrated, which may provide a practice tool for spleen research in the animal model.

A tumor-targeted near-infrared fluorescent probe for HNO and its application to the real-time monitoring of HNO release in vivo.

Nitroxyl (HNO) is a promising regulator for cancer therapy. Here, we develop a tumor-targeted near-infrared fluorescent probe for HNO and utilize it in the real-time imaging of HNO release in vivo.