Near-infrared Fluorescent Probe Research Articles

Application of a wash-free NIR-fluorescent probe in detecting the polarity of lipid droplets and identifying NAFLD

Application of a wash-free NIR-fluorescent probe in detecting the polarity of lipid droplets and identifying NAFLD

Nanoparticle-enhanced near-infrared fluorescence probes as a new frontier in cancer imaging

Recent advancements in nanotechnology have significantly enhanced the development of near-infrared fluorescence (NIRF) probes, positioning them as powerful tools in cancer imaging. This paper explores the unique advantages of nanoparticles incorporating NIR dyes, such as indocyanine green (ICG) and DiR, which exhibit deep tissue penetration and minimal background autofluorescence. The enhanced permeability and retention (EPR) effect facilitates selective accumulation in tumor tissues, enabling sophisticated imaging and precision-targeted drug delivery systems. This review highlights the remarkable potential of NIRF imaging techniques in molecular diagnostics, emphasizing their ability to differentiate malignant tissues at a molecular level. Additionally, we discuss various NIRF dye classifications, including cyanine and BODIPY-based probes, along with the development of multifunctional agents that enhance imaging specificity and therapeutic effectiveness. The integration of advanced targeting capabilities, including the use of antibodies and small molecules, further improves the precision of these imaging agents. While challenges remain regarding the pharmacokinetics and potential toxicity of nanoparticle-based probes, their capacity for real-time tumor tracking and the promise of multimodal imaging approaches underscore their transformative role in cancer diagnostics and treatment. By advancing the field of theranostics, nanoparticle-enhanced NIRF probes pave the way for personalized medicine and improved patient outcomes in oncology.

Near-infrared fluorescent probe for visualization of nitroxyl in the plant response to stress

BackgroundNitroxyl (HNO) is an emerging signaling molecule that plays a significant regulatory role in various aspects of plant biology, including stress responses and developmental processes. However, understanding the precise actions of HNO in plants has been challenging due to the absence of highly sensitive and real-time in situ monitoring tools. Consequently, it is crucial to develop effective and accurate detection methods for HNO. Establishing such methodologies will enable researchers to elucidate the functional roles of HNO in plant physiological processes, thereby advancing our knowledge of plant resilience and adaptation under environmental stressors. ResultHerein, we successfully constructed a near-infrared fluorescent probe, DCIF-HNO, based on the dicyanoisophorone platform as fluorophore and 2-(diphenylphosphino)benzoate as HNO recognition site for identifying HNO in plants. Probe DCIF-HNO exhibited rapid response, excellent selectivity, and high sensitivity to HNO in vitro spectroscopic tests, while also demonstrating low toxicity and biocompatibility. A rapid and portable smartphone sensing platform for HNO in actual samples was successfully constructed based on probe DCIF-HNO and color recognition application. Moreover, probe DCIF-HNO was successfully applied to plant cells and tissues, enabling real-time visualization and detection of HNO and revealing the complex network of HNO interactions during H2S/NO crosstalk in plants. Furthermore, the increase in HNO levels in plants response to high salt and Cr stress was observed using probe DCIF-HNO. Transcriptome sequencing and differential metabolites analysis were employed to gain insight into the mechanism of HNO production under Cr stress. SignificanceDue to the optical properties and high-resolution imaging capabilities of DCIF-HNO, this study offers a novel framework for elucidating the signaling role of HNO in plant stress responses. The precise visualization of HNO dynamics enhances our understanding of the complex molecular pathways involved in plant adaptation to abiotic stressors. This research not only advances plant physiology but also has significant implications for developing strategies to enhance agricultural resilience in challenging environmental conditions.

Accurate diagnosis of early-stage lung cancer by targeting lipid droplets with a two-photon near-infrared fluorescence probe

Clinically, solid nodules with adenocarcinoma (SN–ADC) exhibit faster growth and greater invasiveness than ground-glass nodules with adenocarcinoma (GGN-ADC). Metabolic reprogramming is a critical feature of tumor cells and may facilitate the rapid progression of early-stage lung cancer. Herein, we used laser microdissection to precisely isolate malignant regions from five SN–ADC and six GGN–ADC samples for proteomic analysis. Bioinformatic analysis, western blotting, and electron microscopy conducted in this study revealed a significant upregulation of lipid droplet-associated protein (PLIN2) and marked increase in lipid droplet (LD) accumulation in SN–ADC tissues. Consequently, a novel two-photon near-infrared fluorescence probe was developed to monitor the dynamic changes in LDs in lung cancer cells. Further, experimental validation confirmed the effectiveness of the LD probe (LD-probe) in monitoring the changes in LDs in tumor cells and distinguishing LD differences between tumor and normal tissues. In particular, the probe successfully detected significant differences in LD behavior between SN–ADC and GGN–ADC in early-stage lung cancer. Thus, this study developed a rapid and effective tool for the diagnosis of early-stage lung cancer. The proposed method offers deeper insight into the monitoring of the behavior of LDs in tumor cells, with potential clinical applications.

Selenophene-containing silicon-rhodamine: A novel near-infrared fluorescent probe for Hg2+ detection and its application in cell imaging

Monitoring of mercury ion (Hg2+) pollution is one of the eternal themes due to its notorious toxicity. Herein, we elaborately designed a novel fluorescent probe N’-((selenophen-2-yl)methylene)Si-rhodamine B hydrazide (Se-SiRH) by integrating 2-formylselenophene with Si-rhodamine B hydrazide. Se-SiRH exhibited an excellent near-infrared response towards Hg2+ in MeOH/PBS solution (1:1, v/v, pH = 7.0) and superior specificity towards Hg2+ than other metal ions. Meanwhile, striking sensitivity towards Hg2+ was found and the limit of detection in fluorescence measurement was calculated to be 2.1 × 10−9 mol·L−1. What’s more, Hg2+-binding mechanism was confirmed with Job-plot measurement, HR-MS and theoretical calculation. Importantly, the success in detecting Hg2+ in real water samples and visualizing Hg2+ in HepG2 cells validated its eye-catching application capability in environmental and biological research.

Near-Infrared Visualization of NAD(P)H Dynamics in Live Cells and Drosophila melanogaster Larvae Using a Coumarin-Based Pyridinium Fluorescent Probe.

A near-infrared fluorescent probe, A, was designed by substituting the carbonyl group of the coumarin dye's lactone with a 4-cyano-1-methylpyridinium methylene group and then attaching an electron-withdrawing NADH-sensing methylquinolinium acceptor via a vinyl bond linkage to the coumarin dye at the 4-position. The probe exhibits primary absorption maxima at 603, 428, and 361 nm, and fluoresces weakly at 703 nm. The addition of NAD(P)H results in a significant blue shift in the fluorescence peak from 703 to 670 nm, accompanied by a substantial increase in fluorescence intensity. This spectral shift is attributed to the transformation from an A-π-A-π-D configuration to a D-π-A-π-D pyridinium platform in probe AH, owing to the addition of a hydride from NADH to the electron-accepting quinolinium acceptor producing the electron-contributing 1-methyl-1,4-dihydroquinoline donor in probe AH. This conclusion is supported by theoretical calculations. The probe was utilized to investigate NAD(P)H dynamics under various conditions. In HeLa cells, treatment with glucose or maltose resulted in a substantial elevation in near-infrared emission intensity, suggesting increased NAD(P)H levels. Chemotherapeutic agents including cisplatin and fludarabine at concentrations of 5, 10, and 20 μM brought about a dose-dependent increase in emission intensity, reflecting heightened NAD(P)H levels due to drug-induced stress and cellular damage. In vivo experiments with hatched, starved Drosophila melanogaster larvae were also conducted. The results showed a clear relationship between emission intensity and the levels of NADH, glucose, and oxaliplatin, confirming that the probe can detect variations in NAD(P)H levels in a living organism. Our investigation also demonstrates that NAD(P)H levels are significantly elevated in the cystic kidneys of ADPKD mouse models and human patients, indicating substantial metabolic alterations associated with the disease. This near-infrared emissive probe offers a highly sensitive and specific method for monitoring NAD(P)H levels across cellular, tissue and whole-organism systems. The ability to detect NAD(P)H variations in reaction to varying stimuli, including nutrient availability and chemotherapeutic stress, underscores its potential as a valuable resource for biomedical research and therapeutic monitoring.

A Novel Methacryloyl-Masked NIR Fluorescent Probe for Detection of Cysteine and Its Application in Bioimaging.

Cysteine (Cys) detection is recognized as an essential element in this investigation due to the critical function of Cys in several physiological processes in living organisms. A new NIR fluorescent probe SNC-Cys has been synthesized by incorporating a five-membered malononitrile derivatized ring as an electron withdrawing group, and a methacryloyl group detection moiety for cysteine. After adding Cys, SNC-Cys shows an emission of 654 nm and further works as a "Turn-on" probe via ICT photomechanism. SNC-Cys has high sensitivity and selectivity for Cys (LOD = 0.46 µM) and can discriminate it from other closely related amino acids, molecules with structural similarity, and in some cases very close functional group likeness. Thus, these results allow the effective imaging of Cys in living A549 cells which indicates good cell permeability and high applicability in live cell imaging. This study anticipates that SNC-Cys could be an aid in the detection of Cys-relevant diseases.

An ultrasensitive lysosome-targeting NIR fluorescence probe for detection of hydroxyl radical during ferroptosis and cuproptosis

Hydroxyl radical (•OH) is one of the most destructive reactive oxygen species in biosystems, mainly produced by the participation of transition metals. Lysosomes, the major metabolic center of intracellular metals, may accelerate the generation of •OH by metal-participated reactions due to their acidic microenvironment, which may make lysosomes a major intracellular site for •OH production. In this work, an ultrasensitive NIR lysosome-targeting •OH fluorescence probe, Lyso-OH, was developed for in situ detection of •OH in lysosomes. The reaction of Lyso-OH and •OH leads to a large spectroscopic red-shift and a significant NIR fluorescence response at 655 nm. Such large changes in π-conjugation and spectroscopic properties can achieve an extremely low background fluorescence, which is helpful to obtain a high-contrast and ultrasensitive NIR fluorescence response, enabling Lyso-OH to detect the trace •OH produced by autoxidation of low-valent metals (e.g. Fe2+ and Cu+). Lyso-OH has good lysosome-targeting ability and has been used for the fluorescence imaging of two transition metal-dependent regulated cell death processes, ferroptosis and cuproptosis. The results revealed that both ferroptosis and cuproptosis were accompanied by lysosomal •OH level increase. Moreover, Lyso-OH was also capable of monitoring the •OH level fluctuations in mice models of inflammation and tumor. The good analytical performance of Lyso-OH may allow it to be widely used in more •OH-associated physiological and pathological processes.

Near-infrared fluorescent molecular probes with cetuximab in the in vivo fluorescence imaging for epithelial ovarian cancer

BackgroundNear-infrared fluorescence (NIRF) imaging is an excellent choice for image-guided surgery due to its simple operation and non-invasiveness. Developing tumor-specific fluorescent molecular probes is key to fluorescence imaging-guided surgery. EGFR (epidermal growth factor receptor) is closely related to the proliferation and growth of tumor cells and is highly expressed in epithelial ovarian cancer (EOC). The study aims to construct a NIR fluorescent molecular probe using cetuximab (an EGFR monoclonal antibody) and investigate its feasibility for targeting EOC in vivo through fluorescence imaging.MethodsWe determined the expression of EGFR in EOC. NIR fluorescent molecular probe with cetuximab (cetuximab-Cy7) was chemically engineered and identified. The subcutaneous xenografted tumor model of EOC was induced using SKOV3-Luc cell line with positive expression of EGFR. Cetuximab-Cy7 was used for in vivo fluorescence imaging, and phosphate-buffered saline, free Cy7 dye and mouse isotype immunoglobulin G-Cy7 were used as controls. NIRF imaging system was performed to study the distribution and targeting of the probes. Tumors were imaged in situ and ex vivo, and fluorescent intensity was quantified. Resected specimens were analyzed to confirm diagnosis, and immunohistochemical (IHC) staining was used to identify EGFR expression.ResultsEGFR expression was increased in EOC tissues than fallopian tube tissues. The high expression of EGFR was significantly correlated with well-differentiation, residual lesions ≤ 1 cm, no recurrence and increased survival. NIRF imaging showed that the cetuximab-Cy7 enabled detection of tumor lesions in EOC-bearing mice with the optimal dose of 30 µg. The suitable imaging time window may be 24–96 h post-injection. Ex vivo fluorescence imaging indicated that fluorescent signal was mainly detected in the tumor and the lung. IHC results confirmed that xenografts were EGFR positive.ConclusionCetuximab-Cy7 can specifically target the tumors of EOC xenografted nude mice. This research lays the foundation for future studies on EOC surgery navigation.

A Near-Infrared Fluorescent Probe for Live-Cell Imaging of RNA in the Cancer Cells.

Ribonucleic acid (RNA), a biomolecular compound polymerized by many riboside monomers, is one of the most essential substances in life. Monitoring RNA changes within organisms will play an important role in the diagnosis and treatment of major genetically related diseases. Herein, we synthesized a novel compound QOT-NA as near-infrared probe for imaging RNA in cancer cells. The synthesis method and photophysical properties of the probe were presented. The titration and selectivity experiments demonstrated that the probe detected RNA in vitro. Moreover, the RNA imaging experiments indicated that the near-infrared RNA probe QOT-NA can locate RNA within cancer cells.

Near-infrared xanthene fluorescence probe for frequency upconversion fluorescence detection of HSO3− in evaluation of drug-induced hepatotoxicity

Bisulfite (HSO3−) plays an important role in life activities. Abnormal content of HSO3− may cause cardiovascular, cancer and other diseases. Frequency upconverted luminescent (FUCL) probes are a class of anti-Stokes luminescent materials with long-wavelength excitation and short-wavelength emission properties, which offer advantages in a range of applications due to their higher sensitivity and photostability. In addition, FUCL imaging has the advantages of high tissue penetration depth and low photo-damage, thus, it is more suitable for fluorescence imaging. In this work, a FUCL probe based on an xanthene fluorophore was designed and synthesised to detect HSO3−. The Probe PT-1 could respond to HSO3− and had high selectivity and sensitivity. It also exhibited a quenched FUCL signal. The detection limit of FUCL concerning HSO3− was 43 nM (λex = 730 nm), which is half the detection limit achieved under traditional excitation (93 nM, λex = 643 nm). Cell fluorescence imaging showed that PT-1 could effectively target mitochondria and monitor endogenous/exogenous HSO3− in living cells. More importantly, PT-1 was successfully used to monitor the level of HSO3− in mice with drug-induced liver injury through FUCL imaging.

Rapid detection and imaging of methylglyoxal in plant tissues by the near-infrared fluorescent probe SWJT-2

Methylglyoxal (MG) can be produced via various pathways in plants. MG is toxic for plant cells at high levels, however it acts as a signaling molecule at low levels, just as H2O2 in plants. Therefore, MG detection is very important for investigating its roles in plant cells, especially in plants under environmental stresses. The near-infrared fluorescent probe SWJT-2 is a novel probe with high sensitivity for the rapid detection of MG in human HeLa cells, but at present it is not clear whether the probe can be used to determine MG levels in plant tissues. In this present research, we tried to apply the probe in plant research. Our results showed that 40 min treatment of SWJT-2 (80 μM) can be applied to the detection and imaging of MG levels in tobacco (Nicotiana benthamiana) tissues.

Synthesis and characterization of Rosebengal end-capped poly(o-chloroaniline)/Ag nanocomposite-based NIR fluorescent probe: a structure–property relationship study

Synthesis and characterization of Rosebengal end-capped poly(o-chloroaniline)/Ag nanocomposite-based NIR fluorescent probe: a structure–property relationship study

Rationally Designed G-Quadruplex Selective "Turn-On" NIR Fluorescent Probe with Large Stokes Shift for Nucleic Acid Research-Based Applications.

Guanine-rich DNA/RNA sequences can form Hoogsteen bonds to adopt noncanonical secondary structures called G-quadruplexes, and these have been associated with diverse cellular processes. There has been considerable research interest in the design of G4-interacting ligands for cellular probing of the G4 structure and understanding its associated biological function. Most of the fluorescent G4 ligands either do not have significant selectivity over other nucleic acid structures, have high Stokes shift, or are not in the near-infrared (NIR) region, which limits its cellular visualization. The current work involves the rational design and synthesis of NIR fluorescent probes comprising a (Z)-1-methyl-2-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium scaffold. Among the designed molecules, 4a exhibited far-red fluorescence (λmax = 680 nm) with large Stokes shift (∼182 nm) upon selective binding to human telomeric G-quadruplexes. The dye 4a does not disturb the conformation and stability of G-quadruplexes, thereby making it suitable for nucleic acid research based applications. Interestingly, 4a showed remarkable selectivity over single- and double-stranded structures in contrast to a commercially available quadruplex binding probe, Thiazole orange (TO). The molecular docking studies indicate that 4a binds at the groove region of the telomeric DNA G-quadruplex through π-π stacking interactions with the quinoline and amine-substituted phenyl ring and with the phosphate backbone through anion-π interactions with the benzothiazole ring. The designed molecule 4a has interesting photophysical properties, cell permeability, and biocompatibility with minimal cytotoxicity. Fluorescence imaging studies in live HeLa cells showed that probe 4a binds to the transient population of the DNA G-quadruplex in the nucleus and RNA quadruplexes in the cytoplasm. In brief, G-quadruplex NIR fluorescent probe 4a with a higher signal/noise ratio has significant potential for cellular imaging studies and thus opens avenues to decipher the biological pathways for better understanding of G-quadruplex biology.

A mitochondria-targeted nitric oxide probe with large Stokes shift for real-time imaging and evaluation of inflammatory bowel disease in situ

BackgroundInflammatory bowel disease (IBD) is a prevalent inflammatory disorder, and the abnormal expression of nitric oxide (NO) produced by biocatalysis of iNOS enzyme in mitochondria is directly associated with the occurrence and progression of IBD. Activatable fluorescent probes offer promising tools for early diagnosis of IBD, however, inadequate biodistribution and limited targeting properties of these probes in vivo severely impede accurate diagnosis of IBD and real-time evaluation of inflammatory levels in situ. Therefore, it is necessary to design a highly efficient fluorescent probe towards NO to overcome inadequate biodistribution and achieve accurate diagnosis and evaluation of IBD in situ. ResultsWe designed a highly efficient mitochondria-targeted “turn-on” NIR fluorescent probe Cy-OMe which has excellent targeting properties and imaging ability. The response mechanism is probe Cy-OMe rapidly undergoes N-nitrosation reaction resulting in “turn-on” NIR fluorescence signal when exposed to NO. Cy-OMe exhibits high sensitivity and specificity in detecting NO content in vitro, owing to its large Stokes shift. Furthermore, the probe Cy-OMe not only efficiently targets mitochondria but also enables precise assessment of fluctuations in endogenous NO concertation across various cell types. Importantly, by virtue of large Stokes shift and excellent mitochondrial targeting ability, Cy-OMe has the capability to specifically evaluate dynamic fluctuations of NO in lipopolysaccharide (LPS)‐stimulated IBD mouse models in situ and Cy-OMe was achieved high-contrast imaging and precision diagnosis of intestinal inflammation diseases. SignificanceCy-OMe can accurately assess fluctuations in NO levels and show high signal fidelity in the diseased intestine region, which has prospects in the non-invasive diagnosis of intestinal inflammation in vivo. At the same time, it is expected to serve as a potential diagnose platform for investigating the physiological processes underlying NO-related inflammatory diseases and promoting understanding of the pathological functions of NO across diverse inflammatory diseases.

A Cascade Activation Probe with Double-Enhanced Near-Infrared Imaging for Monitoring Peroxynitrite Fluctuations in Vivo.

Monitoring peroxynitrite (ONOO-) fluctuations is particularly important for assessing pathological progression and oxidative damage due to their crucial role in maintaining the redox balance of organisms. However, due to the lack of efficient tools for differentially monitoring ONOO- fluctuations at different concentration ranges in vivo, the precise detection of endogenous ONOO- fluctuations under pathological conditions in living systems remains challenging. Herein, we rationally designed a double-enhanced emission cascade activatable near-infrared (NIR) fluorescent probe (B-TCF) for the measurement of ONOO-, which consists of a borate ester response group and a malononitrile hemicyanine fluorophore. Especially, after sequential oxidative hydrolysis of the borate ester group and xanthene skeleton, B-TCF exhibited a sequentially double-enhanced NIR emission response at 776 and 625 nm for different ONOO- concentration ranges. Moreover, B-TCF revealed excellent and promising performance for ONOO- in terms of high selectivity, sensitivity, and reaction rate (k = 28.2 M-1 s-1). Motivated by the two-step emission signal enhancement and large wavelength shift in the NIR region, B-TCF enabled discriminative imaging of ONOO- with the low and high concentrations in living cells. Importantly, B-TCF was successfully applied for assessing the pathological progression of isoniazid and acetaminophen-induced liver damage in vivo by detecting the endogenous different ONOO- levels. Overall, this study not only demonstrates the first double-enhanced emission cascade activatable NIR fluorescent probe for measuring the dynamic variation of ONOO- in related diseases but also shows great potential as an effective molecular tool for evaluating the various stages of drug-induced liver damage.

Visualization of the protective role of estrogen against female liver fibrosis via an ER viscosity NIR fluorescent probe

Visualization of the protective role of estrogen against female liver fibrosis via an ER viscosity NIR fluorescent probe

Detection of carboxylesterases by an activatable NIR fluorescence probe with high selectivity in living systems

Carboxylesterases (CEs) have attracted increasingly attention in the physiological and pathological processes of many diseases such as diabetes, hepatocellular carcinoma (HCC) and drug metabolism. Selective detection of CEs activity is imperative to evaluate the pathophysiological process of CEs-related disease. Herein, a selective near-infrared (NIR) fluorescent probe (DCM-CE) with high selectivity is reported. DCM-CE was composed of dicyanoisophorone-based fluorophore and carbamate unit which is a highly selective identification group for CEs. DCM-CE exhibited good sensitivity for CEs detection at physiological pH and temperature. Furthermore, DCM-CE featured large Stokes shift (175 nm) and good at tracking the drug-induced trace change of CEs activity in HepG2 cells with little effect on cell viability. Moreover, DCM-CE was applied to monitor the activity of CEs in living mice. Taken all together, DCM-CE had extensive potential application value in detecting CEs for evaluating related diseases.

A novel near-infrared fluorescent probe for butyrylcholinesterase: Research for screening of natural anti-AD inhibitors

Elevated levels of butyrylcholinesterase (BuChE) have the potential to be predictive in the timely detection and diagnosis of Alzheimer's disease (AD). By inhibiting of BuChE activity can raise acetylcholine levels and intervene AD processes. Therefore, BuChE as an important factor in treatment AD has been given more and more attention in clinical studies. Given the facts above, in this study, for precise monitoring of BuChE level changes and screening for possible butyrylcholinesterase inhibitor (BuChEI) for AD diagnosis and therapy, a near-infrared (NIR) fluorescence probe (NFP-BuChE) was created. The probe exhibits excellent sensitivity and selectivity for BuChE. NFP-BuChE has been successfully applied to the detection of endogenous BuChE levels in live cells, and we successfully constructed a screening system for BuChEI on cells and a novel natural efficient BuChEI (matrine) was discovered and identified, which significantly reduced BuChE activity and thus alleviated AD symptoms. Most importantly, for the first time, we measured the changes of BuChE levels in zebrafish (0–4 days) after fertilization, various organs of zebrafish, and AD zebrafish modeled by different concentrations of AlCl3 by NFP-BuChE, and at the same time, we also validated the inhibitory effect of matrine on BuChE by NFP-BuChE in zebrafish. In addition, NFP-BuChE has also been successfully used to measure the changes of BuChE levels in the brains of AD mice. These findings imply that NFP-BuChE is a potentially useful molecular tool for screening possible natural BuChEI quickly and for monitoring changes in BuChE activity, and it is expected that more value will be explored in mice. In addition, matrine and its derivatives are promising options for future Alzheimer's disease treatments.

Advanced deep-tissue imaging and manipulation enabled by biliverdin reductase knockout.

We developed near-infrared (NIR) photoacoustic and fluorescence probes, as well as optogenetic tools from bacteriophytochromes, and enhanced their performance using biliverdin reductase-A knock-out model (Blvra-/-). Blvra-/- elevates endogenous heme-derived biliverdin chromophore for bacteriophytochrome-derived NIR constructs. Consequently, light-controlled transcription with IsPadC-based optogenetic tool improved up to 25-fold compared to wild-type cells, with 100-fold activation in Blvra-/- neurons. In vivo , light-induced insulin production in Blvra-/- reduced blood glucose in diabetes by ∼60%, indicating high potential for optogenetic therapy. Using 3D photoacoustic, ultrasound, and two-photon fluorescence imaging, we overcame depth limitations of recording NIR probes. We achieved simultaneous photoacoustic imaging of DrBphP in neurons and super-resolution ultrasound localization microscopy of blood vessels ∼7 mm deep in the brain, with intact scalp and skull. Two-photon microscopy provided cell-level resolution of miRFP720-expressing neurons ∼2.2 mm deep. Blvra-/- significantly enhances efficacy of biliverdin-dependent NIR systems, making it promising platform for interrogation and manipulation of biological processes.