A Protocol for Activated Bioorthogonal Fluorescence Labeling and Imaging of 4-Hydroxyphenylpyruvate Dioxygenase in Plants.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) plays a crucial role in the synthesis of nutrients needed to maintain optimal plant growth. Its level is closely linked to the extent of abiotic stress experienced by plants. Moreover, it is also the target of commercial herbicides. Therefore, labeling of HPPD in plants not only enables visualization of its tissue distribution and cellular uptake, it also facilitates assessment of abiotic stress of plants and provides information needed for the development of effective environmentally friendly herbicides. In this study, we created a method for fluorescence labeling of HPPD that avoids interference with the normal growth of plants. In this strategy, a perylene-linked dibenzyl-cyclooctyne undergoes strain-promoted azide-alkyne cycloaddition with an azide-containing HPPD ligand. The activation-based labeling process results in a significant emission enhancement caused by the change in the fluorescent forms from an excimer to a monomer. Notably, this activated bioorthogonal strategy is applicable to visualizing HPPD in Arabidopsis thaliana, and assessing its response to multiple abiotic stresses. Also, it can be employed to monitor in vivo levels and locations of HPPD in crops. Consequently, the labeling strategy will be a significant tool in investigations of HPPD-related abiotic stress mechanisms, discovering novel herbicides, and uncovering unknown biological functions.

The Aggregates of Near-Infrared Cyanine Dyes in Phototherapy.

Cyanines in the near-infrared region are a typical example of a classic fluorescent dye that has garnered significant attention and widespread use in the life sciences and biotechnology. Their character to form assemblies or aggregates has inspired the development of various functional cyanine dye aggregates in phototherapy. This article provides a brief summary of the strategies used to prepare these cyanine dye aggregates. The reports in this concept suggest that the self-assembly of cyanine dyes can enhance their photostability, opening up new possibilities for their application in phototherapy. This concept may encourage researchers to explore the development of functional fluorescent dye aggregates further.

In vivo deep-brain 2-photon fluorescent microscopy labeled with near-infrared dyes excited at the 1700 nm window.

2-Photon fluorescence microscopy (2PFM) is an indispensable imaging technology for neuroscience. However, the imaging depth is usually limited to the cortical layer in mouse brain in vivo. Here, we demonstrate deep brain 2PFM in vivo excited at the 1700 nm window, using IR780 and aza-IR780 as fluorescent labels. Our detailed characterization of the multiphoton excitation and emission properties of IR780 and aza-IR780 show that: (1) IR780 or aza-IR780 generate 2-photon fluorescence excited at the 1700 nm window and are promising for 2PFM; (2) aza-IR780 exhibits a larger ησ2 with better anti-photobleaching property compared to IR780; The 2-photon action cross-sections of IR780 and aza-IR780 in plasma are an order-of-magnitude larger than those in PBS; (3) In vivo 2-photon emission spectra for both dyes show a notable red shift compared to those in vitro. Based on these characterization results, we demonstrate deep brain 2PFM labeled by them. A maximum imaging depth of 1585 µm (labeled by IR780) and 1800 µm (labeled by aza-IR780) into the mouse brain in vivo readily penetrates the subcortical region of hippocampus. Besides, a maximum of 1528 µm hemodynamic imaging depth is realized via 2PFM with aza-IR780 labeling, enabling us to measure blood flow speed in the hippocampus.

Bright Asymmetric Shielding Strategy-Based NIR-II Probes for Angiography and Localized Photothermal Therapy.

Fluorescent probes with fluorescence emission in the NIR-II window have been widely studied due to increased imaging depth. However, the currently reported NIR-II fluorescent probes present some disadvantages, such as complicated synthesis routes and low fluorescence quantum yields (QYs). The shielding strategy has been used in the development of NIR-II probes to improve their QYs. So far, this strategy has only been used for the symmetric NIR-II probes, especially those based on the benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole) (BBTD) skeleton. This work reports the synthesis of a series of asymmetric NIR-II probes based on shielding strategies accompanied by simple synthetic routes, high synthetic yields (above 90%), high QYs, and large Stoke shifts. Furthermore, the use of d-α-tocopheryl polyethylene glycol succinate (TPGS) as a surfactant for an NIR-II fluorescence probe (NT-4) improved its water solubility. In vivo studies showed that TPGS-NT-4 NPs with a high QY (3.46%) achieve high-resolution angiography and efficient local photothermal therapy, while displaying good biocompatibility. Hence, we combined angiography and local photothermal therapy to improve the tumor uptake of nanophotothermal agents while reducing their damage to normal tissues.

J-Aggregates Formed by NaCl Treatment of Aza-Coating Heptamethine Cyanines and Their Application to Monitoring Salt Stress of Plants and Promoting Photothermal Therapy of Tumors.

The cationic nature of heptamethine cyanines gives them the capacity to form aggregates with salts by electrostatic interactions. In this work, NaCl promoted J-aggregate formation of aza-coating heptamethine cyanines is explored. NaCl can induce the N-benzyloxycarbonyl Cy-CO2 Bz to assemble into a J-aggregate having an absorption at 890 nm. Its excellent fluorescence response to NaCl implies that it has great potential for use as a probe for tracing salt stress in plants. Moreover, NaCl also promotes formation of J-aggregates from the N-ethyloxycarbonyl Cy-CO2 Et. The aggregate shows an intense absorption at 910 nm compared to the monomer which absorbs at 766 nm. Its J-aggregated form can serve as a photothermal agent. And the photothermal conversion efficiency is increased from 29.37 % to 57.59 %. This effort leads to the development of two applications of new cyanine J-aggregates including one for tracing salt stress of plants and the other for promoting photothermal therapy of tumors.

Near-infrared heptamethine cyanines (Cy7): from structure, property to application.

Heptamethine cyanine dyes (Cy7) have attracted much attention in the field of biological application due to their unique structure and attractive near infrared (NIR) photophysical properties. In this review, the influences of different modification sites on the absorption characteristics, photostability, Stokes shift, fluorescence characteristics, water solubility, and singlet oxygen generation efficiency of this class of dyes are summarized, and the application development of the corresponding dyes in the field of biological application is introduced, which will provide a reference for the optimization and improvement of heptamethine cyanine dyes in the future.

Construction and bioimaging application of novel indole heptamethine cyanines containing functionalized tetrahydropyridine rings.

IR780 as a commercially available dye with near-infrared emission has been extensively applied in fluorescent probes and bioimaging. In this work, to further intensify the optical behavior, a tetrahydropyridine ring was used to replace the cyclohexene ring at the center of IR780, forming a cyanine dye Cy-NH with near-infrared emission. Photophysical properties demonstrated that Cy-NH exhibits good optical performance. In particular, Cy-NH contains two functional reaction sites (e.g. Cl and NH sites on the tetrahydropyridine ring) and can be used to construct functional cyanine dyes. Investigation on imaging showed that these cyanines can be used as near-infrared fluorescent imaging agents in living cells and in vivo.