Near-infrared Fluorophore Research Articles

A NIR-Fluorochrome for Live Cell Dual Emission and Lifetime Tracking from the First Plasma Membrane Interaction to Subcellular and Extracellular Locales.

Molecular probes with the ability to differentiate between subcellular variations in acidity levels remain important for the investigation of dynamic cellular processes and functions. In this context, a series of cyclic peptide and PEG bio-conjugated dual near-infrared emissive BF2-azadipyrromethene fluorophores with maxima emissions at 720 nm (at pH > 6) and 790 nm (at pH < 5) have been developed and their aqueous solution photophysical properties determined. Their inter-converting emissions and fluorescence lifetime characteristics were exploited to track their spatial and temporal progression from first contact with the plasma membrane to subcellular locales to their release within extracellular vesicles. A pH-dependent reversible phenolate/phenol interconversion on the fluorophore controlled the dynamic changes in dual emission responses and corresponding lifetime changes. Live-cell confocal microscopy experiments in the metastatic breast cancer cell line MDA-MB-231 confirmed the usability of the dual emissive properties for imaging over prolonged periods. All three derivatives performed as probes capable of real-time continuous imaging of fundamental cellular processes such as plasma membrane interaction, tracking endocytosis, lysosomal/large acidic vesicle accumulation, and efflux within extracellular vesicles without perturbing cellular function. Furthermore, fluorescence lifetime imaging microscopy provided valuable insights regarding fluorophore progression through intracellular microenvironments over time. Overall, the unique photophysical properties of these fluorophores show excellent potential for their use as information-rich probes.

Tetraanionic Oligo-Dioxaborines: Strongly Absorbing Near-Infrared Dyes.

Polymethine dyes of tetraanionic nature comprising 1,3,2-dioxaborine rings in the polymethine chain and end-groups of different electron-accepting abilities have been synthesized. They can be considered as oligomeric polymethines, where a linear conjugated π-system passes through three 1,3,2-dioxaborine units and a number of tri- and dimethine π-bridges between two end-groups. The obtained dyes exhibit near-infrared absorption and fluorescence, with molar absorption coefficients reaching as high as 564000 M-1 cm-1 in DMF, rendering them among the strongest absorbers known. The novel compounds are bright NIR fluorophores, with fluorescence quantum yields up to 0.13 in DMF. A comparative analysis of the electronic structure of the obtained dyes with respective dianionic and trianionic oligomers was conducted through quantum chemical calculations.

A Förster Resonance Energy Transfer (FRET)-Based Immune Assay for the Detection of Microcystin-LR in Drinking Water.

Cyanobacteria bloom is the term used to describe an abnormal and rapid growth of cyanobacteria in aquatic ecosystems such as lakes, rivers, and oceans as a consequence of anthropic factors, ecosystem degradation, or climate change. Cyanobacteria belonging to the genera Microcystis, Anabaena, Planktothrix, and Nostoc produce and release toxins called microcystins (MCs) into the water. MCs can have severe effects on human and animal health following their ingestion and inhalation. The MC structure is composed of a constant region (composed of five amino acid residues) and a variable region (composed of two amino acid residues). When the MC variable region is composed of arginine and leucine, it is named MC-LR. The most-common methods used to detect the presence of MC-LR in water are chromatographic-based methods (HPLC, LC/MS, GC/MS) and immunological-based methods (ELISA). In this work, we developed a new competitive Förster resonance energy transfer (FRET) assay to detect the presence of traces of MC-LR in water. Monoclonal antibody anti-MC-LR and MC-LR conjugated with bovine serum albumin (BSA) were labeled with the near-infrared fluorophores CF568 and CF647, respectively. Steady-state fluorescence measurements were performed to investigate the energy transfer process between anti-MC-LR 568 and MC-LR BSA 647 upon their interaction. Since the presence of unlabeled MC-LR competes with the labeled one, a lower efficiency of FRET process can be observed in the presence of an increasing amount of unlabeled MC-LR. The limit of detection (LoD) of the FRET assay is found to be 0.245 nM (0.245 µg/L). This value is lower than the provisional limit established by the World Health Organization (WHO) for quantifying the presence of MC-LR in drinking water.

A superior near infrared fluorescent probe for revealing the relationship between peroxynitrite and Alzheimer's disease

The typical neurodegenerative Alzheimer's disease (AD) poses an increasing threat to human health as the population ages. Though increasing attention has been drawn for the etiology of AD over decades, regretfully, the exact pathogenesis of AD is not fully elucidated. The elevated levels of ROS are among the major pathological alterations in AD. Among them, peroxynitrite (ONOO-) exhibits a greater impact compared to other reactive oxygen species and plays an important role in AD, but the role ONOO- plays in AD progression remains unclear. In this work, the near-infrared (NIR) fluorophore (benzopyran derivative) was optimized by molecular skeleton modification, and a fluorophore DCM-Cl-OH with better spectral properties was obtained. On this basis, a NIR fluorescence probe DCM-Cl-P with excellent detection performance was obtained. Based on the high selectivity and fast response of the probe to ONOO-, DCM-Cl-P was used to visual detection of ONOO- in living cells and zebrafish, and further demonstrated the excessive accumulation of ONOO- in AD model mice. More importantly, through analysis of the correlation between ONOO- and amyloid-β protein (Aβ), ONOO- and the aggregation of Aβ were closely related and mutually reinforcing was demonstrated and ONOO- underwent significant changes in the early stage of the change of Aβ aggregation, indicating that ONOO- may serve as an early marker of AD. These findings have greatly promoted the research on the intrinsic relationship between ONOO- and AD, and provides a powerful means for pathological research of AD.

Near-Infrared Emission Beyond 900 nm from Stable Radicals in Nonconjugated Poly(diphenylmethane).

Organic radicals with narrow energy gaps are highly sought-after for the production of near-infrared (NIR) fluorophores. However, the current repertoire of developed organic radicals is notably limited, facing challenges related to stability and low fluorescence efficiency. This study addresses these limitations by achieving stable radicals in nonconjugated poly(diphenylmethane) (PDPM). Notably, PDPM exhibits a well-balanced structural flexibility and rigidity, resulting in a robust intra-/inter-chain through-space conjugation (TSC). The stable radicals within PDPM, coupled with strong TSC, yield a remarkable full-spectrum emission spanning from blue to NIR beyond 900 nm. This extensive tunability is achieved through careful adjustments of concentration and excitation wavelength. The findings highlight the efficacy of polymerization in stabilizing radicals and introduce a novel approach for developing nonconjugated NIR emitters based on triphenylmethane subunits.

Fluorescence Imaging Using Deep-Red Indocyanine Blue, a Complementary Partner for Near-Infrared Indocyanine Green.

Indocyanine Blue (ICB) is the deep-red pentamethine analogue of the widely used clinical near-infrared heptamethine cyanine dye Indocyanine Green (ICG). The two fluorophores have the same number of functional groups and molecular charge and vary only by a single vinylene unit in the polymethine chain, which produces a predictable difference in spectral and physicochemical properties. We find that the two dyes can be employed as a complementary pair in diverse types of fundamental and applied fluorescence imaging experiments. A fundamental fluorescence spectroscopy study used ICB and ICG to test a recently proposed Förster Resonance Energy Transfer (FRET) mechanism for enhanced fluorescence brightness in heavy water (D2O). The results support two important corollaries of the proposal: (a) the strategy of using heavy water to increase the brightness of fluorescent dyes for microscopy or imaging is most effective when the dye emission band is above 650 nm, and (b) the magnitude of the heavy water florescence enhancement effect for near-infrared ICG is substantially diminished when the ICG surface is dehydrated due to binding by albumin protein. Two applied fluorescence imaging studies demonstrated how deep-red ICB can be combined with a near-infrared fluorophore for paired agent imaging in the same living subject. One study used dual-channel mouse imaging to visualize increased blood flow in a model of inflamed tissue, and a second mouse tumor imaging study simultaneously visualized the vasculature and cancerous tissue in separate fluorescence channels. The results suggest that ICB and ICG can be incorporated within multicolor fluorescence imaging methods for perfusion imaging and hemodynamic characterization of a wide range of diseases.

One-pot accessing of meso-Aryl heptamethine indocyanine NIR fluorophores and potential application in developing dye-antibody conjugate for imaging tumor

One-pot accessing of meso-Aryl heptamethine indocyanine NIR fluorophores and potential application in developing dye-antibody conjugate for imaging tumor

Construction of a double-increasing emission fluorescent probe and its application in food detection of benzoyl peroxide and biosystem imaging

In this work, based on the Förster resonance energy transfer (FRET) mechanism strategy, a new dual-increasing emission proportional near-infrared (NIR) fluorescent probe Lay-1 was designed for fast benzoyl peroxide (BPO) detection in real food samples and biosystems. Specifically, it employed a naphthylimide derivative and a NIR fluorophore dicyanoisophorone derivative as the energy transfer donor and acceptor, respectively, and a phenylboronic acid (Ph-B(OH)2) as the responding group of BPO. In addition, the results exhibited that the fluorescence color of Lay-1 was changed from red to orange in the absence and the presence of BPO with a fast response time (∼120 s), high sensitivity, and an excellent limit of detection as low as 60.8 nM. Impressively, Lay-1 has been successfully used for BPO detection in real food samples and biosystems with satisfactory results. Therefore, Lay-1 can be a robust molecular tool to further investigate the physiological and pathological function of BPO.

Butyrylcholinesterase-Activated Near-Infrared Fluorogenic Probe for In Vivo Theranostics of Alzheimer's Disease.

Butyrylcholinesterase (BChE) is a promising biomarker and effective therapeutic target for Alzheimer's disease (AD). Herein, we designed a BChE-activated near-infrared (NIR) probe, DTNP, which could be activated by BChE and inhibit its enzymatic activity. DTNP is composed of a cyclopropane moiety as the recognition unit, a NIR fluorophore hemicyanine as the NIR reporter, and a BChE inhibitor as the therapeutic unit. DTNP specifically binds BChE with high sensitivity and exhibits strong "turn-on" NIR fluorescence as well as nerve cell protection. In vivo imaging shows DTNP has favorable blood-brain barrier permeability and long-term tracking ability with preliminary competence in AD diagnosis. DTNP can significantly inhibit BChE activity, promote the release of ACh, and rescue learning deficits and cognitive impairment. Therefore, DTNP, the first reported and partially validated theranostic probe for the detection of BChE in AD, may provide a foundation and inspiration for imaging and therapy in AD.

Synthesis, characterization and revealing the sono-photochemical potential of new NIR BODIPY derivative

Although the idea of combining the light of photodynamic therapy (PDT) and the sound of sonodynamic therapy (SDT) and converting into a much more effective therapeutic treatment method, sonophotodynamic therapy (SPDT), is very wise and sensitizing agents such as phthalocyanines produce higher amounts of singlet oxygen (1O2) with this method, the usage of SPDT for boron-dipyrromethene (BODIPY)-based theranostics has put forward by our group for the first time and no studies have been published by other groups in the literature so far. Therefore, as our continuing motivation in this direction, in the present study, a novel near-infrared (NIR) BODIPY fluorophore bearing thienyl-pyridine substituent at meso-position, iodines as heavy atoms at 2-,6- positions and distyryl segments at 3-,5- positions which enlarge π-conjugation of the molecule has been synthesized successfully. The structural characterizations of the target NIR BODIPY2 has been accomplished by the conventional spectroscopic methods (1H NMR, 13C NMR, FT-IR (ATR), UV–Vis and HRMS/MS) and elemental analysis. In order to evaluate the singlet oxygen generation capacity of NIR BODIPY2, photochemical and sonophotochemical methods have been employed comparatively. The singlet oxygen quantum yield (ΦΔ) of NIR BODIPY2 was calculated 0.37 using by photochemical method, this value increased to 0.66 by sonophotochemical study. It can be said that these results will open new horizons for researchers in terms of understanding sonophotochemical measurements of BODIPY derivatives for future in vitro or in vivo studies.

Near-Infrared Perylenecarboximide Fluorophores for Live-Cell Super-Resolution Imaging.

Organic near-infrared (NIR) photoblinking fluorophores are highly desirable for live-cell super-resolution imaging based on single-molecule localization microscopy (SMLM). Herein we introduce a novel small chromophore, PMIP, through the fusion of perylenecarboximide with 2,2-dimetheylpyrimidine. PMIP exhibits an emission maximum at 732 nm with a high fluorescence quantum yield of 60% in the wavelength range of 700-1000 nm and excellent photoblinking without any additives. With resorcinol-functionalized PMIP (PMIP-OH), NIR SMLM imaging of lysosomes is demonstrated for the first time in living mammalian cells under physiological conditions. Moreover, metabolically labeled nascent DNA is site-specifically detected using azido-functionalized PMIP (PMIP-N3) via click chemistry, thereby enabling the super-resolution imaging of nascent DNA in phosphate-buffered saline with a 9-fold improvement in spatial resolution. These results indicate the potential of PMIP-based NIR blinking fluorophores for biological applications of SMLM.

Molecularly targeted protease-activated probes for visualization of glioblastoma: a comparison with 5-ALA.

The highly infiltrative growth of glioblastoma (GBM) makes distinction between the tumor and normal brain tissue challenging. Therefore, fluorescence-guided surgery is often used to improve visual identification of radiological tumor margins. The aim of this study was to evaluate the ability of recently developed molecularly targeted near-infrared (NIR) protease-activated probes to visualize GBM tissue and to compare the most promising candidate with the gold standard, 5-aminolevulinic acid (5-ALA). Single-substrate probes 6QC-ICG and 6QC-Cy5 (cysteine cathepsin cleavable), double-substrate probes AG2-FNIR and AG2-Cy5 (cysteine cathepsin and caspase 3 cleavable), and 5-ALA were administered intravenously to mice with orthotopic tumors. Activation of the probes was also evaluated in cell cultures in vitro and in biopsy material from patients with GBM ex vivo. The tumor to normal brain tissue fluorescence ratio (TNR) was quantified in brain sections using preclinical and clinical visualization platforms, and in tissue homogenates and cell suspensions using spectrofluorimetry. Subcellular localization of the fluorophores was visualized by confocal microscopy. In vitro, the single-substrate probe 6QC-ICG was cleaved in glioma cells and macrophages, and the resulting fluorophore accumulated intracellularly. In experimental GBMs, both single- and double-substrate probes visualized tumor tissue, while in healthy brain tissue the signal was minimal. TNR was highest for 6QC-ICG and AG2-FNIR, but the signal intensity was higher for 6QC-ICG. Using xenograft and syngeneic mouse models, as well as human GBM biopsy material ex vivo, the authors confirmed the ability of 6QC-ICG to specifically visualize the glioma tissue using preclinical and clinical visualization platforms. Finally, a comparison with 5-ALA in animals coadministered with both compounds revealed a higher TNR for 6QC-ICG in experimental GBMs. The cysteine cathepsin-cleavable probe 6QC-ICG is activated by glioma cells and tumor-associated macrophages, leading to a high contrast between tumor and nontumorous brain tissue that is superior to that of the current standard, 5-ALA. In addition to a well-defined mechanism of action, protease-activated probes that use NIR fluorophores (e.g., indocyanine green) have the advantage of low absorption and scattering of the NIR light and lower tissue autofluorescence. These results suggest that 6QC-ICG has the potential to become the targeted agent in intraoperative detection of GBM tissue using fluorescence imaging.

Stereochemistry-Dependent Labeling of Organelles with a Near-Infrared-Emissive Phosphorus-Bridged Rhodamine Dye in Live-Cell Imaging.

The development of near-infrared (NIR) fluorophores that have both excellent chemical stability and photostability, as well as efficient cell permeability, is highly demanded. In this study, we present phospha-rhodamine (POR) dyes which display significantly improved performance for protein labeling. This is achieved by incorporating a 2-carboxy-3-benzothiophenyl group at the 9-position of the xanthene scaffold. The resulting cis and trans isomers were successfully isolated and structurally characterized using X-ray diffraction. The HaloTag ligand conjugates of the two isomers exhibited different staining abilities in live cells. While the cis isomer showed non-specific accumulation on the organelle membranes, the trans isomer selectively labeled the HaloTag-fused proteins, enabling the long-term imaging of cell division and the 5-color imaging of cell organelles. Molecular dynamics simulations of the HaloTag ligand conjugates within the lipid membrane suggested that the cis isomer is more prone to forming oligomers in the membrane. In contrast, the oligomerization of the trans isomer is effectively suppressed by its interaction with the lipid molecules. By taking advantage of the superior labeling performance of the trans isomer and its NIR-emissive properties, multi-color time-lapse super-resolution 3D imaging, namely super-resolution 5D-imaging, of the interconnected network between the endoplasmic reticulum and microtubules was achieved in living cells.

Tuning optical properties of aromatic near-infrared fluorescent switch through engineering functional heterocycle on donor moiety

Near-infrared (NIR) emissive fluorophores with donor−acceptor−donor (D−A−D) structures are attractive for deep tissue in vivo bioimaging and sensing, owing to their low background autofluorescence and deep penetration depth. In this study, we constructed a series of nitric oxide (NO)-responsive fluorescent switches with D−A−D structure, in which functional heterocycles act as donor moiety, resulting in the modification of fluorescence wavelengths from NIR-I (750–950 nm) to NIR-II (1000–1700 nm) region. Specifically, the NIR-I fluorescence can be achieved by using furan, thiophene and selenophene as the donor moiety. By using pyrrole as the donor moiety, NIR-II fluorescence can be achieved due to the strong N–H⋯N hydrogen bond interaction and tunable intramolecular charge transfer (ICT) process. Based on our findings, tailoring electronegativity of donor moiety could controllably tune the fluorescence wavelength from NIR-I to NIR-II region, proposing a practical design strategy for developing NIR-II fluorescent switches.

New Near-Infrared Fluorescence Imaging Platform with Large Stokes Shift for Carboxylesterase 2 Detection in Thyroid Cancer and Inflammatory Diseases Diagnosis.

Development of new near-infrared fluorophores is one of the eternal themes in the field of biosensing and biological imaging. In this work, we constructed a novel fluorophore platform MOR by replacing methylindole of hemicyanine fluorophore (CyR) with benzoxazole to acquire better fluorescence characteristics. Based on the platform, a near infrared (NIR) fluorescent probe MOR-CES2 was synthesized for the specific "off-on" response to carboxylesterase 2 (CES2). The probe exhibited excellent properties including near-infrared emission (735 nm), large Stokes shift (105 nm), high sensitivity (LOD, 0.3 ng/mL), and rapid response (15 min). The successful application of MOR-CES2 in biological imaging of CES2 in mice with thyroid cancer and inflammatory bowel disease demonstrated that the probe could identify cancer cells and tissues and sensitively respond to inflammation. The results proved the potency of MOR-CES2 as an efficient imaging tool to assist in the surgical resection of CES2-related tumors.

IR780-based diffuse fluorescence tomography for cancer detection.

IR780 iodide is a commercially available targeted near-infrared contrast agent for in vivo imaging and cancer photodynamic or photothermal therapy, whereas the accumulation, dynamics, and retention of IR780 in biological tissue, especially in tumor is still under-explored. Diffuse fluorescence tomography (DFT) can be used for localization and quantification of the three-dimensional distribution of NIR fluorophores. Herein, a homemade DFT imaging system combined with tumor-targeted IR780 was utilized for cancer imaging and pharmacokinetic evaluation. The aim of this study is to comprehensively assess the biochemical and pharmacokinetic characteristics of IR780 with the aid of DFT imaging. The optimal IR780 concentration (20 μg/mL) was achieved first. Subsequently, the good biocompatibility and cellar uptake of IR780 was demonstrated through the mouse acute toxic test and cell assay. In vivo, DFT imaging effectively identified various subcutaneous tumors and revealed the long-term retention of IR780 in tumors and rapid metabolism in the liver. Ex vivo imaging indicated IR780 was mainly concentrated in tumor and lung with significantly different from the distribution in other organs. DFT imaging allowed sensitive tumor detection and pharmacokinetic rates analysis. Simultaneously, the kinetics of IR780 in tumors and liver provided more valuable information for application and development of IR780.

Nanocarrier systems loaded with IR780, iron oxide nanoparticles and chlorambucil for cancer theragnostics.

Theragnostics has become a popular term nowadays, since it enables both diagnosis and therapy at the same time while only using one carrier platform. Therefore, formulating a nanocarrier system that could serve as theragnostic agent by using simple techniques would be an advantage during production. In this project, we aimed to develop a nanocarrier that can be loaded with the chemotherapeutic medication chlorambucil and magnetic resonance imaging agents (e.g., iron oxide nanoparticles and near-infrared fluorophore IR780) for theragnostics. Poly(lactic-co-glycolic acid) was combined with the aforementioned ingredients to generate poly(vinyl alcohol)-based nanoparticles (NPs) using the single emulsion technique. Then the NPs were coated with F127 and F127-folate by simple incubation for five days. The nanoparticles have the hydrodynamic size of approx. 250 nm with negative charge. Similar to chlorambucil and IR780, iron oxide loadings were observed for all three kinds of NPs. The release of chlorambucil was quicker at pH 5.4 than at pH 7.4 at 37 °C. The F127@NPs and F127-folate@NPs demonstrated much greater cell uptake and toxicity up to 72 h after incubation. Our in vitro results of F127@NPs and F127-folate@NPs have demonstrated the ability of these systems to serve as medication and imaging agent carriers for cancer treatment and diagnostics, respectively.

Five near-infrared-emissive graphene quantum dots for multiplex bioimaging

Due to high tissue penetration depth and low autofluorescence backgrounds, near-infrared (NIR) fluorescence imaging has recently become an advantageous diagnostic technique used in a variety of fields. However, most of the NIR fluorophores do not have therapeutic delivery capabilities, exhibit low photostabilities, and raise toxicity concerns. To address these issues, we developed and tested five types of biocompatible graphene quantum dots (GQDs) exhibiting spectrally-separated fluorescence in the NIR range of 928–1053 nm with NIR excitation. Their optical properties in the NIR are attributed to either rare-earth metal dopants (Ho-NGQDs, Yb-NGQDs, Nd-NGQDs) or defect-states (nitrogen doped GQDS (NGQDs), reduced graphene oxides) as verified by Hartree-Fock calculations. Moderate up to 1.34% quantum yields of these GQDs are well-compensated by their remarkable >4 h photostability. At the biocompatible concentrations of up to 0.5–2 mg ml−1 GQDs successfully internalize into HEK-293 cells and enable in vitro imaging in the visible and NIR. Tested all together in HEK-293 cells five GQD types enable simultaneous multiplex imaging in the NIR-I and NIR-II shown for the first time in this work for GQD platforms. Substantial photostability, spectrally-separated NIR emission, and high biocompatibility of five GQD types developed here suggest their promising potential in multianalyte testing and multiwavelength bioimaging of combination therapies.

Precise and safe pulmonary segmentectomy enabled by visualizing cancer margins with dual-channel near-infrared fluorescence.

Segmentectomy is a type of limited resection surgery indicated for patients with very early-stage lung cancer or compromised function because it can improve quality of life with minimal removal of normal tissue. For segmentectomy, an accurate detection of the tumor with simultaneous identification of the lung intersegment plane is critical. However, it is not easy to identify both during surgery. Here, the authors report dual-channel image-guided lung cancer surgery using renally clearable and physiochemically stable targeted fluorophores to visualize the tumor and intersegmental plane distinctly with different colors; cRGD-ZW800 (800nm channel) targets tumors specifically, and ZW700 (700nm channel) simultaneously helps discriminate segmental planes. The near-infrared (NIR) fluorophores with 700nm and with 800nm channels were developed and evaluated the feasibility of dual-channel fluorescence imaging of lung tumors and intersegmental lines simultaneously in mouse, rabbit, and canine animal models. Expression levels of integrin αvβ3, which is targeted by cRGD-ZW800-PEG, were retrospectively studied in the lung tissue of 61 patients who underwent lung cancer surgery. cRGD-ZW800-PEG has clinically useful optical properties and outperforms the FDA-approved NIR fluorophore indocyanine green and serum unstable cRGD-ZW800-1 in multiple animal models of lung cancer. Combined with the blood-pooling agent ZW700-1C, cRGD-ZW800-PEG permits dual-channel NIR fluorescence imaging for intraoperative identification of lung segment lines and tumor margins with different colors simultaneously and accurately. This dual-channel image-guided surgery enables complete tumor resection with adequate negative margins that can reduce the recurrence rate and increase the survival rate of lung cancer patients.

Bifunctional Tumor-Targeted Bioprobe for Phothotheranosis.

Background: Near-infrared (NIR) phototheranostics provide promising noninvasive imaging and treatment for head and neck squamous cell carcinoma (HNSCC), capitalizing on its adjacency to skin or mucosal surfaces. Activated by laser irradiation, targeted NIR fluorophores can selectively eradicate cancer cells, harnessing the power of synergistic photodynamic therapy and photothermal therapy. However, there is a paucity of NIR bioprobes showing tumor-specific targeting and effective phototheranosis without hurting surrounding healthy tissues. Methods: We engineered a tumor-specific bifunctional NIR bioprobe designed to precisely target HNSCC and induce phototheranosis using bioconjugation of a cyclic arginine-glycine-aspartic acid (cRGD) motif and zwitterionic polymethine NIR fluorophore. The cytotoxic effects of cRGD-ZW800-PEG were measured by assessing heat and reactive oxygen species (ROS) generation upon an 808-nm laser irradiation. We then determined the invivo efficacy of cRGD-ZW800-PEG in the FaDu xenograft mouse model of HNSCC, as well as its biodistribution and clearance, using a customized portable NIR imaging system. Results: Real-time NIR imaging revealed that intravenously administered cRGD-ZW800-PEG targeted tumors rapidly within 4 h postintravenous injection in tumor-bearing mice. Upon laser irradiation, cRGD-ZW800-PEG produced ROS and heat simultaneously and exhibited synergistic photothermal and photodynamic effects on the tumoral tissue without affecting the neighboring healthy tissues. Importantly, all unbound bioprobes were cleared through renal excretion. Conclusions: By harnessing phototheranosis in combination with tailored tumor selectivity, our targeted bioprobe ushers in a promising paradigm in cancer treatment. It promises safer and more efficacious therapeutic avenues against cancer, marking a substantial advancement in the field.