Intra-Operative Definition of Glioma Infiltrative Margins by Visualizing Immunosuppressive Tumor-Associated Macrophages.

Accurate delineation of glioma infiltrative margins remains a challenge due to the low density of cancer cells in these regions. Here, a hierarchical imaging strategy to define glioma margins by locating the immunosuppressive tumor-associated macrophages (TAMs) is proposed. A pH ratiometric fluorescent probe CP2-M that targets immunosuppressive TAMs by binding to mannose receptor (CD206) is developed, and it subsequently senses the acidic phagosomal lumen, resulting in a remarkable fluorescence enhancement. With assistance of CP2-M, glioma xenografts in mouse models with a tumor-to-background ratio exceeding 3.0 for up to 6 h are successfully visualized. Furthermore, by intra-operatively mapping the pH distribution of exposed tissue after craniotomy, the glioma allograft in rat models is precisely excised. The overall survival of rat models significantly surpasses that achieved using clinically employed fluorescent probes. This work presents a novel strategy for locating glioma margins, thereby improving surgical outcomes for tumors with infiltrative characteristics.

A Ratiometric SERS Probe for Imaging the Macrophage Phenotypes in Live Mice with Epilepsy and Brain Tumor.

Macrophage performs multiple functions such as pathogen phagocytosis, antigen presentation, and tissue remodeling by polarizing toward a spectrum of phenotypes. Dynamic imaging of macrophage phenotypes is critical for evaluating disease progression and the therapeutic response of drug candidates. However, current technologies cannot identify macrophage phenotypes in vivo. Herein, a surface-enhanced Raman scattering nanoprobe, AH1, which enables the accurate determination of physiological pH with high sensitivity and tissue penetration depth through ratiometric Raman signals is developed. Due to the phenotype-dependent metabolic reprogramming, AH1 can effectively identify macrophage subpopulations by measuring the acidity levels in phagosomes. After intravenous administration, AH1 not only visualizes the spatial distribution of macrophage phenotypes in brain tumors and epileptic regions of mouse models, but also reveals the repolarization of macrophages in brain lesions after drug intervention. This work provides a new tool for dynamically monitoring the disease-associated immune microenvironment and evaluating the efficacy of immune-therapeutics in vivo.

Rigid and Photostable Shortwave Infrared Dye Absorbing/Emitting beyond 1200 nm for High-Contrast Multiplexed Imaging.

The shortwave infrared (SWIR) spectral region beyond 1200 nm offers optimal tissue penetration depth and has broad potential in diagnosis, therapy, and surgery. Here, we devised a novel class of fluorochromic scaffold, i.e., a tetra-benzannulated xanthenoid (EC7). EC7 absorbs/emits maximally at 1204/1290 nm in CH2Cl2 and exhibits an unparalleled molar absorptivity of 3.91 × 105 cm-1 M-1 and high transparency to light at 400-900 nm. It also exhibited high resistance toward both photobleaching and symmetry breaking due to its unique structural rigidity. It is feasible for in vivo bioimaging and particularly suitable to couple with the shorter-wavelength analogues for high-contrast multiplexing. High-contrast dual-channel intraoperative imaging of the hepatobiliary system and three-channel in vivo imaging of the intestine, the stomach, and the vasculature were showcased. EC7 is a benchmark fluorochrome for facile biomedical exploitation of the SWIR region beyond 1200 nm.

The pursuit of xanthenoid fluorophores with near-infrared-II emission for in vivo applications.

As fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) has gained increasing attention, it is inevitable that NIR-II fluorophores, the cornerstone of NIR-II imaging, have come to the middle of the stage. NIR-II xanthenoid fluorophores with good stability, high brightness, and fluorescence adjustability are becoming popular. We here reviewed the recent progress of xanthenoid fluorophores with NIR-II emission for in vivo applications. Especially, we focus on the strategies used for longer wavelength and fluorescence regulation to construct OFF-ON or ratiometric NIR-II fluorescent probes.

NIR-II Dyad-Doped Ratiometric Nanosensor with Enhanced Spectral Fidelity in Biological Media for In Vivo Biosensing.

Ratiometric fluorescence nanosensors provide quantitative biological information. However, spectral shift and distortion of ratiometric nanosensors in biological media often compromise sensing accuracy, limiting in vivo applications. Here, we develop a fluorescent dyad (aBOP-IR1110) in the second near-infrared (NIR-II) window by covalently linking an asymmetric aza-BODIPY with a ONOO--responsive meso-thiocyanine. The dyad encapsulated in the PEGylated nanomicelle largely improves spectral fidelity in serum culture by >9.4 times compared to that of its noncovalent counterpart. The increased molecular weights (>1480 Da) and hydrophobicity (LogP of 7.87-12.36) lock dyads inside the micelles, which act as the shield against the external environment. ONOO--altered intramolecular Förster resonance energy transfer (FRET) generates linear ratiometric response with better serum tolerance, enabling us to monitor the dynamics of oxidative stress in traumatic brain injury and evaluate therapeutic efficiency. The results show high correlation with in vitro triphenyltetrazolium chloride staining, suggesting the potential of NIR-II dyad-doped nanosensor for in vivo high-fidelity sensing applications.

The pursuit of polymethine fluorophores with NIR-II emission and high brightness for in vivo applications.

Polymethine cyanine dyes, as the most important class of organic near-infrared-II (NIR-II) fluorophores, recently received increasing attention due to their high molar extinction coefficients, intensive fluorescence brightness, and flexible wavelength tunability for fluorescent bioimaging applications. Very recently, remarkable advances have been made in the development of NIR-II polymethine fluorophores with improved optical performance, mainly including tunable fluorescence, improved brightness, improved water solubility and stability. In this review, we summarize the recent research advances in molecular tailoring design strategies of NIR-II polymethine fluorophores, and then emphasize the representative bioimaging and biosensing applications. The potential challenges and perspectives of NIR-II polymethine fluorophores in this emerging field are also discussed. This review may provide guidance and reference for further development of high-performance NIR-II polymethine fluorophores to boost their clinical translation in the future.

Polymethine Molecular Platform for Ratiometric Fluorescent Probes in the Second near-Infrared Window.

Visualizing biomolecules such as enzymes in the deep tissue of living organisms via molecular ratiometric fluorescent probes in the second near-infrared window (NIR-II) with a built-in self-calibration function can provide reliable information about relevant pathophysiological processes directly but so far is not feasible due to the lack of a fluorescence modulation strategy in the NIR-II window. Here we present a molecular platform Py-2 by integrating the rhodamine 6G scaffold and polymethine. The maximal emission wavelength of Py-2 was 1010 nm and blue-shifted to 945 nm when its secondary amine was acylated. Based on Py-2, two molecular ratiometric NIR-II fluorescent probes, nitroreductase-responsive Rap-N and ROS-responsive Rap-R, were constructed and successfully demonstrated in vitro and in vivo. Overall, this report presents a unique approach to developing ratiometric NIR-II molecular probes for in vivo biosensing.

ROS/RNS and Base Dual Activatable Merocyanine-Based NIR-II Fluorescent Molecular Probe for in vivo Biosensing.

Inflammation usually results in high-level reactive oxygen species (ROS) and reactive nitrogen species (RNS) not only in acidic tissue but also in alkaline tissue. However, noninvasively in vivo monitoring reactive species specifically within alkaline tissue remains a huge challenge. Here we introduce a dual activatable fluorescent probe PN910 located in the second near-infrared window (NIR-II, 900-1700 nm), which shows high selectivity toward H2 O2 and OONO- at pH beyond 7.4. Then we verified that PN910 could be used for the real-time, specific and accurate monitoring of cystitis and colitis for living animals. This report presents a unique approach to the development of dual activatable probe for in vivo biosensing.

NIR-II cell endocytosis-activated fluorescent probes for in vivo high-contrast bioimaging diagnostics.

Fluorescence probes have great potential to empower bioimaging, precision clinical diagnostics and surgery. However, current probes are limited to in vivo high-contrast diagnostics, due to the substantial background interference from tissue scattering and nonspecific activation in blood and normal tissues. Here, we developed a kind of cell endocytosis-activated fluorescence (CEAF) probe, which consists of a hydrophilic polymer unit and an acid pH-sensitive small-molecule fluorescent moiety that operates in the "tissue-transparent" second near-infrared (NIR-II) window. The CEAF probe stably presents in the form of quenched nanoaggregates in water and blood, and can be selectively activated and retained in lysosomes through cell endocytosis, driven by a synergetic mechanism of disaggregation and protonation. In vivo imaging of tumor and inflammation with a passive-targeting and affinity-tagged CEAF probe, respectively, yields highly specific signals with target-to-background ratios over 15 and prolonged observation time up to 35 hours, enabling positive implications for surgical, diagnostic and fundamental biomedical studies.

A Promising NIR-II Fluorescent Sensor for Peptide-Mediated Long-Term Monitoring of Kidney Dysfunction.

Kidney disease is usually "silent" at the early stage but can lead to severe kidney failure later on. The development of bioimaging probes with rapid distribution and long-term retention in the kidney is significant for the precise diagnosis of renal diseases. Here, a strategy for the peptide-mediated delivery and long-term accumulation (>48 h) of second near-infrared window (NIR-II) fluorophores into the kidney is demonstrated. It is shown that both the hepatic-cleared organic molecules and fast renal-cleared ultrasmall nanoparticles can be retained in the kidney after conjugation to the peptide with high polarity. Moreover, a ROS-responsive activatable bilateral NIR-II sensor was designed based on the kidney targeting peptide, which enables both in vivo long-term kidney monitoring and in vitro urine analysis. The capability of the peptide-based sensor to detect early kidney injury and report on kidney dysfunctional progression is particularly crucial for chemotherapy regimen optimization and timely renoprotective intervention during medication.

Unsymmetrical pentamethine cyanines for visualizing physiological acidities from the whole-animal to the cellular scale with pH-responsive deep-red fluorescence.

Acidity plays an important role in numerous physiological and pathological events. Non-invasively monitoring pH dynamics would be valuable for understanding pathological processes and optimizing therapeutic strategies. Although numerous near-infrared (NIR) fluorophores have been developed to monitor acidification in vivo, the experimental results are difficult to verify at the molecular or cellular level using a fluorescence microscope or flow cytometer due to the lack of lasers with excitation wavelengths in the NIR wavelength range. This work presents a sequential condensation strategy for obtaining unsymmetrical pentamethine cyanines with fine-tuned pK a values and improved yields. These deep-red fluorophores with pH responsiveness can not only be used to monitor acidification in live cells using confocal microscopic imaging and flow cytometry, but they can also be used to non-invasively identify infected tissue with a low pH value in live mouse models. In addition, the acidity in infected tissue slices was verified under a conventional confocal microscope. Overall, this work demonstrates a new synthetic method with improved yields for unsymmetrical pentamethine cyanines that can report acidity. These pH-responsive deep-red fluorophores not only provide new tools for accessing pH-associated physiological and pathological events, but they can also help in understanding in vivo imaging results at the molecular or cellular level due to their detectability by multiple imaging instruments.

Molecular Engineering of NIR-II Fluorophores for Improved Biomedical Detection.

The development of fluorophores for the second near-infrared window (NIR-II, 1000-1700 nm) represents an emerging, significant, and vibrant field in analytic chemistry, chemical biology, and biomedical engineering. The wavelength, brightness, and stability are three crucial factors that determine the performance of an NIR-II fluorophore. Up to now, significant progress has been made in the development of NIR-II fluorescence molecular probes, including the synthesis of D-A-D and D-π-A fluorophores with improved NIR-II fluorescence imaging performance and the construction of off-on probes and ratiometric probes via energy transfer or molecular structure modification. In this review, we summarize the most recent advances in molecular engineering design strategies of NIR-II fluorophores and probes, then highlight a selection of bioimaging and biosensing applications. We also provide perspectives on potential challenges and opportunities in this emerging field.

NIR-II pH Sensor with a FRET Adjustable Transition Point for In Situ Dynamic Tumor Microenvironment Visualization.

Monitoring the pH in tumor lesions provides abundant physiological information. However, currently developed pH sensors only achieve sensitive detection in the settled response region around the pH transition point (pHt ). To realize tumor pH monitoring with high sensitivity within a wider response region, reported here are serial pHt adjustable sensors (pTAS) that simply regulate the component ratio of second near-infrared (NIR-II) emission aza-BODIPY (NAB) donor and pH sensitive rhodamine-based pre-acceptor (NRh) in Förster resonance energy transfer system. Combining the pH response regions of pTAS, a twofold widened pH detection range (6.11-7.22) is obtained compared to the pHt settled sensor (6.38-6.94). With an adjustable pHt , in vivo tumor pH increase and decrease processes could be dynamically visualized through dual-channel ratiometric bioimaging within the NIR-II window, with a coefficient of variation under 1 % compared to the standard pH meter.

Engineering the Charge-Transfer State to Facilitate Spin-Orbit Charge Transfer Intersystem Crossing in Spirobis[anthracene]diones.

Spiro conjugation has been proposed to dictate the efficiency of charge transfer, which could directly affect the spin-orbit charge transfer intersystem crossing (SOCT-ISC) process. However, this process has yet to be exemplified. Herein, we prepared three spirobis[anthracene]diones, in which two benzophenone moieties are locked in close proximity and differentially functionalized to fine-tune the charge transfer state. Its feasibility for SOCT-ISC was theoretically predicted, then experimentally evaluated. Through fine-tuning the spiro conjugation coupling and varying the solvent dielectric constants, ISC rate constants were engineered to vary in a dynamic range of three orders of magnitude, from 7.8×108  s-1 to 1.0×1011  s-1 , which is the highest ISC rate reported for SOCT-ISC system to our knowledge. Our findings substantiate the key factors for effective SOCT-ISC and offer a new avenue for the rational design of heavy atom free triplet sensitizers.

J-Aggregates of Cyanine Dye for NIR-II in Vivo Dynamic Vascular Imaging beyond 1500 nm.

Light in the second near-infrared window, especially beyond 1500 nm, shows enhanced tissue transparency for high-resolution in vivo optical bioimaging due to decreased tissue scattering, absorption, and autofluorescence. Despite some inorganic luminescent nanoparticles have been developed to improve the bioimaging around 1500 nm, it is still a great challenge to synthesize organic molecules with the absorption and emission toward this region. Here, we present J-aggregates with 1360 nm absorption and 1370 nm emission formed by self-assembly of amphiphilic cyanine dye FD-1080 and 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Molecular dynamics simulations were further employed to illustrate the self-assembly process. Superior spatial resolution and high signal-to-background ratio of J-aggregates were demonstrated for noninvasive brain and hindlimb vasculature bioimaging beyond 1500 nm. The efficacy evaluation of the clinically used hypotensor is successfully achieved by high-resolution in vivo dynamic vascular imaging with J-aggregates.

A Threshold-Limited Fluorescence Probe for Viscosity.

Viscosity of body fluid is an established biomarker of pathological conditions. Abnormality of cellular viscosity occurs when cells are challenged with external stresses. Small molecule probes to assess the viscosity are sought after for both disease diagnostics and basic studies. Fluorescence based probes are particular attractive due to their potentials for convenient and high spatiotemporal resolution microscopic monitoring of biological samples. The dyes with a floppy push-pull backbone or dyes with a rotatable substituent exhibits a viscosity responsive fluorescence enhancement and therefore viable viscosity probes. The scaffold of the existing viscosity probes contains typically one such floppy site. Therefore, they typically linearly respond to log(viscosity). We argue that minor viscosity fluctuation could potentially be physiological as the biological system is dynamic. We wish to develop a type of conceptually-new, threshold-limited viscosity probes, to complement the existing probes. Such probes do not exhibit a fluorescence enhancement when challenged with minor and presumably physiological enhancement of viscosity. When the viscosity is higher than a certain threshold, their fluorescence turns on. We hypothesize that a dye with two far-apart floppy sites could potentially yield such a threshold-limited signal and designed VPZ2 and VPZ3. Through spectral titration, VPZ3 was found to yield the desired threshold-limited signal. VPZ3 was suitable for in vitro bioimaging of viscosity under one-photon or two-photon excitation. VPZ3 is potentially useful in many downstream applications. Future work includes fine-tune of the threshold to allow tailored limit for fluorescence turn-on to better meet the need of different applications. Besides the implications in the real-world applications, the design concept could also be translated to design of alternative substrates.

Stable, Wavelength-Tunable Fluorescent Dyes in the NIR-II Region for In†Vivo High-Contrast Bioimaging and Multiplexed Biosensing.

Small-molecule organic fluorophores, spectrally active in the 900-1700 nm region, with tunable wavelength and sensing properties are sought-after for in vivo optical imaging and biosensing. A panel of fluorescent dyes (CX) has been developed to meet this challenge. CX dyes exhibit the wavelength tunability of cyanine dyes and have a rigidified polymethine chain to guarantee their stability. They are chemo- and photo-stable in an aqueous environment and have tunable optical properties with maximal absorbing/emitting wavelength at 1089/1140 nm. They show great potential in high-contrast in vivo bioimaging and multicolor detection with negligible optical cross talk. Förster resonance energy transfer (FRET) between CX dyes was demonstrated in deep tissue, providing an approach for monitoring drug-induced hepatotoxicity by detection of OONO- . This report presents a series of NIR-II dyes with promising spectroscopic properties for high-contrast bioimaging and multiplexed biosensing.

Anti-quenching NIR-II molecular fluorophores for in vivo high-contrast imaging and pH sensing.

The contrast and sensitivity of in vivo fluorescence imaging has been revolutionized by molecular fluorophores operating in the second near-infrared window (NIR-II; 1000-1700 nm), but an ongoing challenge is the solvatochromism-caused quenching in aqueous solution for the long-wavelength absorbing fluorophores. Herein, we develop a series of anti-quenching pentamethine cyanine fluorophores that significantly overcome the severe solvatochromism, thus affording stable absorption/emission beyond 1000 nm with up to ~ 44-fold enhanced brightness and superior photostability in aqueous solution. These advantages allow for deep optical penetration (8 mm) as well as high-contrast and highly-stable lymphatic imaging superior to clinical-approved indocyanine green. Additionally, these fluorophores exhibit pH-responsive fluorescence, allowing for noninvasive ratiometric fluorescence imaging and quantification of gastric pH in vivo. The results demonstrate reliable accuracy in tissue as deep as 4 mm, comparable to standard pH electrode method. This work unlocks the potential of anti-quenching pentamethine cyanines for NIR-II biological applications.

Peroxynitrite Activatable NIR-II Fluorescent Molecular Probe for Drug-Induced Hepatotoxicity Monitoring.

Drug-induced hepatotoxicity represents an important challenge for safety in drug development. The production of peroxynitrite (ONOO-) is proposed as an early sign in the progression of drug-induced hepatotoxicity. Currently, reported ONOO- probes mainly emit in the visible range or the first NIR window, which have limited in vivo biosensing application due to the autofluorescence and photon scattering. Herein, we developed a peroxynitrite activatable second near-infrared window (NIR-II) molecular probe for drug-induced hepatotoxicity monitoring, based on the fusion of an NIR-II fluorescence turn-on benzothiopyrylium cyanines skeleton and the phenyl borate. In the presence of ONOO-, the probe IRBTP-B can turn on its NIR-II fluorescence by yielding its fluorophore IRBTP-O and display good linear response to ONOO-. Tissue phantom study confirmed reliable activated signals could be acquired at a penetration depth up to 5 mm. Using this probe, we disclose the upregulation of ONOO- in a preclinical drug-induced liver injury model and the remediation with N-acetyl cysteine (NAC) in vivo. We expect that this strategy will serve as a general method for the development of an activatable NIR-II probe based on the hydroxyl functionalized reactive sites by analyte-specific triggering.

A diversity-oriented rhodamine library for wide-spectrum bactericidal agents with low inducible resistance against resistant pathogens.

Antimicrobial resistance is a public health emergency and warrants coordinated global efforts. Challenge is that no alternative molecular platform has been identified for discovery of abundant antimicrobial hit compounds. Xanthene libraries have been screened for bioactive compounds. However, the potentially accessible chemistry space of xanthene dyes is limited by the existing xanthene synthesis. Herein we report a mild one-step synthesis, which permits late-stage introduction of a xanthene moiety onto i.e. natural products, pharmaceuticals, and bioactive compounds and construction of a focused library of rhodamine dyes exhibiting facile functional, topographical and stereochemical diversity. In vitro screening yields 37 analogs with mid-to-high bactericidal activity against WHO priority drug-resistant pathogens. These findings suggest that synthetic dye libraries exhibiting high structural diversity is a feasible chemical space combating antibacterial resistance, to complement the natural sources.