Organic fluorophores have been widely used for biological imaging in the visible and the first near-infrared windows. However, their application in the second near-infrared window (NIR-II, 1000-1700 nm) is still limited mainly due to low fluorescence quantum yields (QYs). Here, we explore molecular engineering on the donor unit to develop high performance NIR-II fluorophores. The fluorophores are constructed by a shielding unit-donor(s)-acceptor-donor(s)-shielding unit structure. Thiophene is introduced as the second donor connected to the shielding unit, which can increase the conjugation length and red-shift the fluorescence emission. Alkyl thiophene is employed as the first donor connected to the acceptor unit. The bulky and hydrophobic alkyl thiophene donor affords larger distortion of the conjugated backbone and fewer interactions with water molecules compared to other donor units studied before. The molecular fluorophore IR-FTAP with octyl thiophene as the first donor and thiophene as the second donor exhibits fluorescence emission peaked at 1048 nm with a QY of 5.3% in aqueous solutions, one of the highest for molecular NIR-II fluorophore reported so far. Superior temporal and spatial resolutions have been demonstrated with IR-FTAP fluorophore for NIR-II imaging of the blood vessels of a mouse hindlimb.

Tumor‐lymph node (LN) metastasis is the dominant prognostic factor for tumor staging and therapeutic decision‐making. However, concurrently visualizing metastasis and performing imaging‐guided lymph node surgery is challenging. Here, a multiplexed‐near‐infrared‐II (NIR‐II) in vivo imaging system using nonoverlapping NIR‐II probes with markedly suppressed photon scattering and zero‐autofluorescence is reported, which enables visualization of the metastatic tumor and the tumor metastatic proximal LNs resection. A bright and tumor‐seeking donor–acceptor–donor (D‐A‐D) dye, IR‐FD, is screened for primary/metastatic tumor imaging in the NIR‐IIa (1100–1300 nm) window. This optimized D‐A‐D dye exhibits greatly improved quantum yield of organic D‐A‐D fluorophores in aqueous solutions (≈6.0%) and good in vivo performance. Ultrabright PbS/CdS core/shell quantum dots (QDs) with dense polymer coating are used to visualize cancer‐invaded sentinel LNs in the NIR‐IIb (>1500 nm) window. Compared to clinically used indocyanine green, the QDs show superior brightness and photostability (no obvious bleaching even after continuous laser irradiation for 5 h); thus, only a picomolar dose is required for sentinel LNs detection. This combination of dual‐NIR‐II image‐guided surgery can be performed under bright light, adding to its convenience and appeal in clinical use.

Deep-tissue three-dimensional (3D) optical imaging of live mammals with high spatiotemporal resolution in non-invasive manners has been challenging due to light scattering. Here, we developed near-infrared II (NIR-II, 1000–1700 nm) light sheet microscopy (LSM) with excitation and emission up to ~ 1320 nm and ~ 1700 nm respectively for optical sectioning through live tissues at ~ 750-μm penetration depth without any invasive surgery. Suppressed light scattering allowed imaging at ~ 2 mm depth in glycerol-cleared brain tissues. NIR-II LSM in normal and oblique configurations enabled in vivo imaging of live mice through intact tissue, revealing abnormal blood flow and T cell motion in tumor microcirculation and mapping out programmed-death ligand 1 (PD-L1) and programmed cell death protein 1 (PD-1) in tumors with cellular resolution. 3D imaging through intact mouse head resolved vascular channels between skull and brain cortex, and monitored recruitment of macrophages/microglia to traumatic brain injury site post injury.

The selenoprotein thioredoxin reductase (TrxR) plays a pivotal role in regulating cellular redox homeostasis and has attracted increasing attention as a promising anticancer drug target. We report here that 2-(4-aminophenyl)-1,3,2-dithiarsinane (PAO-PDT, 4), a potent and highly selective small molecule inhibitor of TrxR, stoichiometrically binds to the C-terminal selenocysteine/cysteine pair in the enzyme in vitro and induces oxidative stress-mediated apoptosis in HL-60 cells. The molecular action of 4 in cells involves inhibition of TrxR, elevation of reactive oxygen species, depletion of cellular thiols, and activation of caspase-3. Knockdown of TrxR sensitizes the cells to 4 treatment, whereas overexpression of the functional enzyme alleviates the cytotoxicity, providing physiological relevance for targeting TrxR by 4 in cells. The simplicity of the structure and the presence of an easily manipulated amine group will facilitate the further development of 4 as a potential cancer chemotherapeutic agent.

A super-contrast NIR-II fluorophore IR-BEMC6P with enhanced quantum yield is developed and the excretion mechanism is identified.

Metrics & MoreArticle Recommendations CONSPECTUS: Fluorescence bioimaging in the second nearinfrared window (NIR-II, 1000−1700 nm) is a superior visualization tool with deeper penetration and higher spatial resolution than the traditional vis/NIR window (400−1000 nm). Developing desirable NIR-II agents with high brightness (high quantum yield (QY) and absorption coefficient), longer emission wavelengths, light stability, and good biocompatibility is a bottleneck that must be crossed in the process of clinical transformation. NIR-II organic agents attract more attention because of their good biocompatibility. Researchers are committed to develop small organic molecules with an adjustable narrow band gap to emit longer NIR-II wavelengths. However, the reduced band gap of molecular fluorophores generally triggered interactions between the conjugated skeleton and other molecules, leading to a decreased fluorescence QY, especially quenching in aqueous systems.Aiming to enhance NIR-II fluorophores' brightness in aqueous systems, the molecular engineering of a shielding and donor unit is introduced in NIR-II fluorophores, and these molecules are composed of a shielding unit, donor(s), acceptor, donor(s), shielding unit (S-D-A-D-S) structure. We found that the donor's steric hindrance brings about a molecular twist that can be tuned with electrostatic potential surfaces and the donor's hydrophobic effects, which can reduce water molecules approaching the excitation center. In addition to the protective effect of the electron shielding units, these changes can be weakened by the interactions between water and fluorophore molecules, which is beneficial to the stability of excited states for fluorophore brightness maintained in watersoluble environments. However, the molecular torsion will reduce intramolecular charge transfer (ICT), the weakened transition from the ground state to the excited state (S 0 −S 1 ), leading to an undesired decrease in the absorption coefficient. Thus, it is necessary to develop molecular engineering to improve emission QY without sacrificing the absorption coefficient. Therefore, bright NIR-II fluorophores need to have a rational balanced donor unit, which can have decreased backbone distortion to strengthen ICT and better protect the conjugated backbone to reduce the water molecule effect, resulting in improvements to the QY and absorption coefficient simultaneously.In this Account, we will present a concise summary and analysis of the rational molecular design of S-D-A-D-S structural NIR-II fluorophores for tunable enhanced QY and absorption coefficients based on the shielding and donor engineering strategy. We expect that this Account will trigger more research interest to explore the inspiring performance of S-D-A-D-S structural NIR-II fluorophores and employ them in bioimaging to promote the process of clinical transformation.

Developing molecular fluorophores with high brightness is of considerable importance to achieve superior biological imaging quality in the second near-infrared (NIR-II) window. It has been demonstrated that the improved fluorescence quantum yield (QY) can be obtained for NIR-II molecular fluorophores with S−D−A−D−S (S, shielding unit; D, donor; A, acceptor)structures. However, their absorption coefficient is relatively low, limiting their brightness for imaging. Here, 3,4-propylenedioxy thiophene (PDOT) is introduced as a donor unit to construct NIR-II fluorophores with better protection of the conjugated backbone and decreased backbone distortion, eventually leading to simultaneously improved QY and absorption coefficient. Thus, the new fluorophore IR-FP8P shows fluorescence emission with a peak of 1040 nm, a QY of 0.6% (with reference to IR-26 with a QY of 0.05% in dichloroethane), and a peak absorption coefficient of 1.3 × 10 4 M −1 •cm −1 in aqueous solutions. The higher brightness at 808 nm excitation endows IR-FP8P with superior imaging quality in the NIR-II window. When conjugated with a specific hormone, the targeting probe FSH@FP8 enables fast and unambiguous ovary imaging in mice, revealing the potential of this bright fluorophore for visualizing complicated biological systems in the NIR-II window.

In vivo molecular imaging of tumors targeting a specific cancer cell marker is a promising strategy for cancer diagnosis and imaging guided surgery and therapy. While targeted imaging often relies on antibody-modified probes, peptides can afford targeting probes with small sizes, high penetrating ability, and rapid excretion. Recently, in vivo fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) shows promise in reaching sub-centimeter depth with microscale resolution. Here, a novel peptide (named CP) conjugated NIR-II fluorescent probe is reported for molecular tumor imaging targeting a tumor stem cell biomarker CD133. The click chemistry derived peptide-dye (CP-IRT dye) probe afforded efficient in vivo tumor targeting in mice with a high tumor-to-normal tissue signal ratio (T/NT > 8). Importantly, the CP-IRT probes are rapidly renal excreted (≈87% excretion within 6 h), in stark contrast to accumulation in the liver for typical antibody-dye probes. Further, with NIR-II emitting CP-IRT probes, urethra of mice can be imaged fluorescently for the first time noninvasively through intact tissue. The NIR-II fluorescent, CD133 targeting imaging probes are potentially useful for human use in the clinic for cancer diagnosis and therapy.