High-sensitivity Fluorescence Research Articles

Responsive integration performed by laser-induced Er3+ structural doping and graphene nanoplatelets

Laser-induced preparation method has the advantages of high efficiency, high yield, no ionic diffusion restriction and competitive side reaction, which can prepare diversified composite materials based on small-scale and distributed characteristics. Besides, integrating multiple performance and conducting inductive feedback are helpful for the reliability and calibration of laser-induced materials in the precision field. Herein, responsive integration is designed based on graphene nanoplatelets/ZnSe:Er3+ composite with optical, electrical, thermal and visual properties for real-time feedback. Er3+ ions doped ZnSe is obtained by fast laser-induced method, combined with two-dimensional graphene nanoplatelets and assembled into temperature-sensitive polyimide and cellulose paper, realizing the characteristics of photoelectric conversion and photothermal conversion. Besides, red fluorescence guiding perceptual behavior can also be produced under the radiation of NIR laser. Based on the proposed composite, bionic flower is constructed, and exhibits good motion features in blooming. Meanwhile, large curvature bending, high thermal sensitivity and strong fluorescence emission are also revealed. Such performance combination design provides a convenient approach for multi signals cross feedback in soft actuator, which strengthens the monitoring and guidance of motion perceptions, and can be potentially applied in diversified fields.

Size photometry and fluorescence imaging of immobilized immersed extracellular vesicles.

Immunofluorescence analysis of individual extracellular vesicles (EVs) in common fluorescence microscopes is gaining popularity due to its accessibility and high fluorescence sensitivity; however, EV number and size are only measurable using fluorescent stains requiring extensive sample manipulations. Here we introduce highly sensitive label-free EV size photometry (SP) based on interferometric scattering (iSCAT) imaging of immersed EVs immobilized on a glass coverslip. We implement SP on a common inverted epifluorescence microscope with LED illumination and a simple 50:50 beamsplitter, permitting seamless integration of SP with fluorescence imaging (SPFI). We present a high-throughput SPFI workflow recording >10,000 EVs in 7min over ten 88×88µm2 fields of view, pre- and post-incubation imaging to suppress background, along with automated image alignment, aberration correction, spot detection and EV sizing. We achieve an EV sizing range from 37 to ∼220nm in diameter with a dual 440 and 740nm SP illumination scheme, and suggest that this range can be extended by more advanced image analysis or additional hardware customization. We benchmark SP to flow cytometry using calibrated silica nanoparticles and demonstrate superior, label-free sensitivity. We showcase SPFI's potential for EV analysis by experimentally distinguishing surface and volumetric EV dyes, observing the deformation of EVs adsorbed to a surface, and by uncovering distinct subpopulations in <100nm-in-diameter EVs with fluorescently tagged membrane proteins.

Single 5-nm quantum dot detection via microtoroid optical resonator photothermal microscopy

Label-free detection techniques for single particles and molecules play an important role in basic science, disease diagnostics, and nanomaterial investigations. While fluorescence-based methods are tools for single molecule detection and imaging, they are limited by available molecular probes and photoblinking and photobleaching. Photothermal microscopy has emerged as a label-free imaging technique capable of detecting individual nanoabsorbers with high sensitivity. Whispering gallery mode (WGM) microresonators can confine light in a small volume for enhanced light-matter interaction and thus are a promising ultra-sensitive photothermal microscopy platform. Previously, microtoroid optical resonators were combined with photothermal microscopy to detect 250 nm long gold nanorods and 100 nm long polymers. Here, we combine microtoroids with photothermal microscopy to spatially detect single 5 nm diameter quantum dots (QDs) with a signal-to-noise ratio exceeding 104. Photothermal images were generated by point-by-point scanning of the pump laser. Single particle detection was confirmed for 18 nm QDs by high sensitivity fluorescence imaging and for 5 nm QDs via comparison with theory. Our system demonstrates the capability to detect a minimum heat dissipation of 0.75 pW. To achieve this, we integrated our microtoroid based photothermal microscopy setup with a low amplitude modulated pump laser and utilized the proportional-integral-derivative controller output as the photothermal signal source to reduce noise and enhance signal stability. The heat dissipation of these QDs is below that from single dye molecules. We anticipate that our work will have application in a wide variety of fields, including the biological sciences, nanotechnology, materials science, chemistry, and medicine.

Preparation and application of wormlike micelles generated from a rosin-based aggregation-induced emission surfactant through coordinating aggregation

Fluorescent nanomaterials have drawn considerable attentions due to their high fluorescence sensitivity, excellent signal stability and tunable luminescent properties. A bio-based aggregation-induced emission (AIE) surfactant (Sodium 4-(tetraphenylstyrene)maleate, MPA-TPE-Na) was prepared from rosin, which showed typical AIE characteristic, obvious solvatochromism and a large Stokes shift (>100 nm). AIE viscoelastic solutions were further prepared by complexing MPA-TPE-Na and cetyltrimethylammonium bromide (CTAB) through coordinating aggregation and their microstructure, fluorescence properties and viscoelasticities were investigated. The aggregates in the viscoelastic solutions were wormlike micelles. As the concentration of MPA-TPE-Na increased, the fluorescence intensity of the viscoelastic solutions increased, and their viscosity increased at first and then decreased. The fluorescence emission intensity of the viscoelastic solutions was barely affected by the change in pH. But reducing the pH could increase its viscosity. This system can specifically recognize Fe3+ without any organic solvent and has anti-interference ability. The present work provided a bio-based fluorescent nanomaterial and verified the feasibility of preparing aqueous AIE nanomaterials by modifying AIE groups with rosin skeleton.

New 3-D Fluorescence Spectral Indices for Multiple Pigment Inversions of Plant Leaves via 3-D Fluorescence Spectra

High-sensitivity fluorescence monitoring has been widely used in agriculture and environmental science. However, the active fluorescence detection information of leaf segments mainly focuses on total chlorophyll, and the fluorescence information of chlorophyll a, chlorophyll b, and some other pigments has not been explored. This only considers the fluorescence spectrum characteristics at a single wavelength or the fluorescence integral from a range of wavelength regions and does not completely consider the linkage relation between the excitation, emission, and interference information. In this paper, the three-dimensional fluorescence spectrum, containing the excitation and emission fluorescence spectra, and the corresponding multiple pigment characteristics from the upgraded LOPEX_ZJU database were collected. The linkages of excitation and emission of the three-dimensional fluorescence spectra of these pigments were analyzed for the newly built multiple pigment 3-D fluorescence spectral indices (3-D FSIs), including those of chlorophyll a, chlorophyll b, carotenoid, anthocyanin, and flavonoid 3-D FSIs. Then, these pigment inversion models were established and validated. The results show that the 3-D FSIs performances for the photosynthetic pigment content inversion (including chlorophyll a and b, and carotenoids) were much better than those for the photo-protective pigments (including anthocyanins and flavonoids) from the 3-D fluorescence spectra of these plant leaves. Here, the 3-D fluorescence normalization index (FNI ((F430,690-F430,763)/(F430,690+F430,763))) for the chlorophyll a inversion model has a high accuracy, the RMSE is 2.96 μg/cm2, and the 3-D fluorescence reciprocal difference index (FRI (F650,704/F650,668) for the chlorophyll b model has an encouraging RMSE (2.01 μg/cm2). The RMSE of the 3-D fluorescence ratio index (FRI (F500,748/F500,717)) for the carotenoid inversion is 3.77 μg/cm2 RMSE. Only FRI (F370,615/F370,438) was selected for the modeling and validating evaluation of the leaf Flas content inversion, but the evaluation metrics were not good, with an RMSE (151.13 μg/cm2). For Ants, although there was a 3-D FSI (FRDI (1/F540,679-1/F540,557)), and its evaluation metrics, with an RMSE (2.8 μg/cm2), were at or over 0.05 level, the validating evaluation metric VC (98.3577%) was not encouraging. These results showed that fluorescence, as a nondestructive and efficient detection method, could determine the contents of chlorophyll a, chlorophyll b, and carotenoid in plant leaves, providing a new method to detect plant information. It can also provide a potential chance for the fluorescence images of fine photo-protective pigments, especially chlorophyll a and b, using the special active fluorescence excitation light source and a few fluorescence imaging channels from the optimal FSIs.

Single Side-Chain-Modulatory of Hemicyanine for Optimized Fluorescence and Photoacoustic Dual-Modality Imaging of H2S In Vivo.

Near-infrared fluorescence (NIRF)/photoacoustic (PA) dual-modality imaging integrated high-sensitivity fluorescence imaging with deep-penetration PA imaging has been recognized as a reliable tool for disease detection and diagnosis. However, it remains an immense challenge for a molecule probe to achieve the optimal NIRF and PA imaging by adjusting the energy allocation between radiative transition and nonradiative transition. Herein, a simple but effective strategy is reported to engineer a NIRF/PA dual-modality probe (Cl-HDN3) based on the near-infrared hemicyanine scaffold to optimize the energy allocation between radiative and nonradiative transition. Upon activation by H2S, the Cl-HDN3 shows a 3.6-fold enhancement in the PA signal and a 4.3-fold enhancement in the fluorescence signal. To achieve the sensitive and selective detection of H2S in vivo, the Cl-HDN3 is encapsulated within an amphiphilic lipid (DSPE-PEG2000) to form the Cl-HDN3-LP, which can successfully map the changes of H2S in a tumor-bearing mouse model with the NIRF/PA dual-modality imaging. This work presents a promising strategy for optimizing fluorescence and PA effects in a molecule probe, which may be extended to the NIRF/PA dual-modality imaging of other disease-relevant biomarkers.

Polyaniline-Coated Flower-Like Titanium Dioxide Magnetic Fluorescent Imprinted Nanoparticles for the Determination of Transferrin in Human Serum

As a typical glycoprotein and potential disease biomarker, transferrin (TrF) plays an important role in binding and transporting iron ions and regulating homeostasis in vivo. However, conventional serum TrF detection technologies often need complex sample pretreatments and long analysis times. Hence, fluorescent magnetic molecular imprinted polymers (FMMIPs) were prepared for the determination of TrF in human serum, which combined the high sensitivity of fluorescence and the specificity of molecularly imprinted polymers. This material is a labor-saving, easy-to-use, and noninvasive biomarker, which provides the determination of serum TrF with high selectivity. Moreover, the preparation and fluorescent properties of FMMIPs were systematically investigated. Under the optimized conditions, the prepared FMMIPs had a good adsorption and fast response with a low relative standard deviation (less than 5%) for TrF with an imprinting factor of 3.21 and detection limit of 0.107 μg/mL. This material provides direct, sensitive, and rapid protein determination for TrF in serum.

Aberrant topologies of bacterial membrane proteins revealed by high sensitivity fluorescence labelling

The cytoplasmic membrane compartmentalises the bacterial cell into cytoplasm and periplasm. Proteins located in this membrane have a defined topology that is established during their biogenesis. However, the accuracy of this fundamental biosynthetic process is unknown. We developed compartment-specific fluorescence labelling methods with up to single-molecule sensitivity. Application of these methods to the single and multi-spanning membrane proteins of the Tat protein transport system revealed rare topogenesis errors. This methodology also detected low level soluble protein mislocalization from the cytoplasm to the periplasm. This study shows that it is possible to uncover rare errors in protein localization by leveraging the high sensitivity of fluorescence methods.

New Rhodamine-based sensor for high-sensitivity fluorescence tracking of Cys and simultaneously colorimetric detection of H2S

Sulfhydryl-containing compounds including cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and hydrogen sulfide (H2S) are involved in many physiological processes. The development of single-molecule optical sensor for the distinguish detection of these bio-thiols is a critical and challenging effort. In this work, we designed a one-step synthesis of the Rhodamine-based sensor FR for specific fluorescent response of Cys and simultaneously colorimetric detection of H2S, in which the aldehyde and fluorine groups act as response sites. Sensor FR displays significant fluorescence enhancement at 565 nm toward Cys with high selectivity and low detection limits (49 nM) due to the low background fluorescent signal of the spirocyclic closed-state in Rhodamine structure. Meantime, after treatment of H2S, the color of the sensor changes significantly from colorless to blue-purple, which can be used as a visual colorimetric method to detect H2S. These response mechanisms were systematically characterized by 1H NMR and Mass spectrometry. Finally, sensor FR could be used to monitor exogenous and endogenous of intracellular Cys changes.

Sensitive fluorescent detection of phosmet and chlortetracycline in animal-derived food samples based on a water-stable Cd(II) chain-based zwitterionic metal-organic framework

The residues of pesticides and antibiotics have always been a major concern in agriculture and food safety. In order to provide a new method for the rapid detection of organophosphorus pesticides and antibiotics, a novel Cd(II) chain-based zwitterionic metal-organic framework MOF 1 with high sensitivity fluorescence sensing performance was successfully synthesized. A series of researches showed that the water- and pH-stable bifunctional MOF 1 has a great ability to detect phosmet (PSM) and chlortetracycline (CTC) in water through fluorescence quenching effect, with high detection sensitivity, low detection limits (0.0124 μM and 0.0131 μM), short response time (40 s) and reusability. Practical application results revealed that MOF 1 could detect PSM and CTC in milk, beef, chicken and egg samples, with satisfactory recoveries (95.2%–103.7%). As a novel fluorescence probe, MOF 1, is known the first case that can detect PSM in animal-derived samples, and the first dual-function material capable of detecting PSM and CTC. Mechanism studies displayed that competitive absorption and photoinduced electron transfer clearly authenticate the high quenching performance of the material.

Fluorescent probe for the detection of acetylcholinesterase inhibitors using high performance thin layer chromatography effect-directed assay in complex matrices

In recent years Bioautography has become an efficient bioassay for finding active compounds in complex matrices including extracts of; bacteria, plants or fungi. High Performance Thin Layer Chromatography (HPTLC) is a technique which allows effect-directive analysis (EDA) through the separation and identification of biologically active substances on a Thin Layer Chromatography (TLC) plate and can be run as a high throughput screening assay for enzymes. This paper presents a new bioautography method using a novel fluorescent probe derived from coumarin and its validation with Acetylcholinesterase (AChE) inhibition. This method combines the advantages of bioautography and the high sensitivity of fluorescence for detection. Combining these advantages, the limit of detection (LOD) and limit of quantification (LOQ) limits usually obtained with traditional chromophores has been reduced by three times. Therefore our new method has been applied on 14 mushroom extracts and we highlight a compound (variegatic acid) as a potentially new AChE inhibitor in X. Chrysenteron.

Europium- and Black Phosphorus-Functionalized Porphyrin as an l-Arginine Sensor and l-Arginine-Activated PDT/PTT Agent for Bacterial Treatment.

The attenuation of bacterial metabolism provides an adjunct to the treatment of bacterial infections. To develop a bacterial eradication agent, a bioactivatable material (BP@Eu-TCPP) was designed and synthesized by coordination and reduction of europium(III) with thin-layer black phosphorus (BP) and tetrakis (4-carboxyphenyl) porphyrin (TCPP). The existence of the P-Eu bond and Eu2+ 3d5/2 in X-ray photoelectron spectroscopy confirmed the successful synthesis of BP@Eu-TCPP. This material showed high fluorescence sensitivity to l-Arginine (l-Arg) and the main binding ratio of BP@Eu-TCPP to l-Arg was ca. 1:2 or 1:3, with the limit of detection of 4.0 μM. The material also showed good photothermal properties and stability, with a photothermal conversion efficiency of 37.3%. Although metal coordination has blocked the generation of 1O2, the addition of l-Arg to BP@Eu-TCPP can restore 1O2 generation upon red light-emitting diode (LED) light irradiation due to the formation of water-soluble Arg-TCPP species. Additionally, BP@Eu-TCPP was enabled to change the bacterial membrane and interfered with the bacterial iron absorption that effectively contributes to bacterial eradication. Such BP@Eu-TCPP is promised to be a novel material for the detection of l-Arg and l-Arg-activated photodynamic therapy.

Polyamidoamine Dendrimers Functionalized Water-Stable Metal-Organic Frameworks for Sensitive Fluorescent Detection of Heavy Metal Ions in Aqueous Solution.

In this work, polyamidoamine (PAMAM)-functionalized water-stable Al-based metal-organic frameworks (MIL-53(Al)-NH2) were proposed with enhanced fluorescence intensity, and used for the sensitive detection of heavy metal ions in aqueous solution. The size and morphology of MIL-53(Al)-NH2 were effectively optimized by regulating the component of the reaction solvents. PAMAM dendrimers were subsequently grafted onto the surface with glutaraldehyde as a cross-linking agent. It was found that the size and morphology of MIL-53(Al)-NH2 have great influence on their fluorescence properties, and PAMAM grafting could distinctly further improve their fluorescence intensity. With higher fluorescence intensity, the PAMAM-grafted MIL-53(Al)-NH2 showed good linearity (R2 = 0.9925-0.9990) and satisfactory sensitivity (LOD = 1.1-8.6 μmol) in heavy metal ions determination. Fluorescence enhancement and heavy metal ions detection mechanisms were discussed following the experimental results. Furthermore, analogous water-stable Materials of Institute Lavoisier (MIL) metal-organic frameworks such as MIL-53(Fe)-NH2 were also proved to have similar fluorescence enhancement performance after PAMAM modification, which demonstrates the universality of the method and the great application prospects in the design of PAMAM-functionalized high-sensitivity fluorescence sensors.

A Review on Recent Advances in Peptide‐based Fluorescence Probes and their Potential Applications

AbstractWith the development and maturity of classical solid‐state synthesis techniques, peptide synthesis has become more convenient. With the continuous progress of research in the field of life sciences, more and more peptides have been elucidated in terms of their structure and biological functions. Due to the excellent biocompatibility, specificity, selectivity, and low cytotoxicity, peptide‐based fluorescent probes have been widely explored and applied in chemical, biological, medical and other fields. This review summarized the characteristics of peptide‐based fluorescence probes in terms of long fluorescence lifetime, high stability, large Stokes shift, high fluorescence quantum yield, high selectivity and sensitivity. Several types of peptide‐based fluorescence probes, such as peptide basic fluorescence, embedded fluorescence molecules, coupled fluorescence groups, and self‐assembled fluorescence, were also described. Finally, we summarized the applications of peptide‐based fluorescent probes in biological imaging, disease detection, ion detection, and biomolecule detection. This review may provide a guarantee and inspiration for further development of the peptide‐based fluorescent probes in the field of biological applications.

Detection of oxytetracycline in milk using a novel carbon dots-based fluorescence probe via facile pyrolysis synthesis.

Amphiphilic blue-fluorescence carbon dots (B-CDs) were synthesized via pyrolysis method with citric acid and oleamine as precursors. B-CDs are monodispersed in ethanol, toluene, and ultrapure water with the average particle sizes of 3.33nm, 2.05nm, and 4.12nm, respectively. The maximum emission wavelength of the B-CDs excitation at 370nm is located at 459nm. The B-CDs have good optical properties with excellent photostability. The fluorescence quantum yield (QY) of the as-prepared CDs is as high as 30.17%. The fluorescence of B-CDs is quenched because of static quenching by oxytetracycline. A high selective and sensitive fluorescence probe for detecting oxytetracycline was constructed with a linear range of 1.52-27.60µg/mL and the detection limit of 0.33µg/mL. The B-CDs-based fluorescence probe can be applied to analyze oxytetracycline in milk; the recoveries and relative standard are satisfactory. Furthermore, the B-CDs were exploited for imaging of SH-SY5Y cells. The results demonstrate that as-synthesized CDs can serve as a cellular imaging reagent owing to remarkable bioimaging performance. This work provides a new strategy for the detection of oxytetracycline in food.

High-accuracy total reflection X-ray fluorescence analysis for determining trace elements using substrate cleaned by ammonia-hydrogen peroxide mixture

Total reflection X-ray fluorescence (TXRF) analysis is one of the useful techniques for determining trace elements. Owing to the high detection sensitivity of X-ray fluorescence in small residues, the hydrophobic substrates are generally used to form dot-type residue. However, when measuring low-Z elements in sample solutions with high concentrations of matrix elements, obtaining sufficient measurement sensitivity can be difficult. X-ray fluorescence is absorbed by the matrix elements, which means that the absorption effect increases in the thicker dried residue compared to the thinner dried residue. To avoid this absorption effect, we have proposed a thin film-type residue. In this study, the glass substrate was treated with an ammonia-hydrogen peroxide mixture (APM). The hydrogen peroxide decomposes organic components, and ammonia slightly etches glass materials. The APM-treated region was limited to a diameter of 6 mm by using a polytetrafluoroethylene mask placed on the glass substrate. As a result, the surface is refreshed to expose the hydroxyl group to make it superhydrophilic. The measured contact angle was 5°. By using the APM-treated superhydrophilic substrate to prepare dried residue, the net intensities in low Z elements were improved. The ratio of Al Kα intensity, calculated as net intensity in the APM-treated substrate divided by it in the hydrophobic substrate, was 2.29. The recovery of each element in the APM-treated substrate was almost 100%; however, recoveries of elements (Al, P, K, and Ca) in the hydrophobic substrate showed significant deviations from 100% (the recovery of Al was approximately 32%). In summary, we successfully improved both analytical sensitivity and accuracy in TXRF analysis by using the APM treated substrate.

Detection of enrofloxacin residues in dairy products based on their fluorescence quenching effect on AgInS2 QDs

Water-soluble AgInS2 (AIS) quantum dots (QDs) were successfully prepared through the one-pot water phase method with thioglycolic acid (TGA) as the stabilizing agent. Because enrofloxacin (ENR) effectively quenches the fluorescence of AIS QDs, a highly-sensitive fluorescence detection method is proposed to detect ENR residues in milk. Under optimal detection conditions, there was a good linear relationship between the relative fluorescence quenching amount (ΔF/F0) of AgInS2 with ENR and ENR concentration (C). The detection range was 0.3125–20.00 μg/mL, r = 0.9964, and the detection limit (LOD) was 0.024 μg/mL (n = 11). The average recovery of ENR in milk ranged from 95.43 to 114.28%. The method established in this study has advantages including a high sensitivity, a low detection limit, simple operation and a low cost. The fluorescence quenching mechanism of AIS QDs with ENR was discussed and the dynamic quenching mechanism of light-induced electron transfer was proposed.

Fluorescence and Colorimetric Analysis of African Swine Fever Virus Based on the RPA-Assisted CRISPR/Cas12a Strategy.

It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the trans-cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze para-nitrophenyl phosphate to generate para-nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.

A triphenylamine-based fluorescence probe for detection of hypochlorite in mitochondria

The level of HClO/ClO− in mitochondria is essential to keep the normal function of mitochondria. Therefore, it is meaningful to accurately and quickly monitor ClO− in mitochondria. In this work, a new triphenylamine-based fluorescence probe PDTPA was designed and synthesized, in which pyridinium salt and dicyano-vinyl group were introduced as mitochondria targeting site and reaction site for ClO−. The probe showed high sensitivity and fast fluorescence response (<10 s) in the detection of ClO−. Moreover, the probe PDTPA had good linearity in a wide concentration range of ClO− and its detection limit was calculated as 10.5 μM. Confocal fluorescence images demonstrated that the probe could target mitochondria and track the fluctuations of endogenous/exogenous ClO− levels in the mitochondria of living cells.

Folate Receptor‐Targeted NIR‐II Dual‐Model Nanoprobes for Multiscale Visualization of Macrophages in Rheumatoid Arthritis

AbstractRheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint inflammation. Macrophages accelerate the pathological process of RA through the continuous cell accumulation and secretion of cytokines. Therefore, multiscale visualization of macrophages is of great significance for the early diagnosis of RA. In this work, folate receptor‐targeted Cptnc‐4F nanoprobes, named FA‐CF‐NPs, are developed for dual‐model molecular imaging of macrophages in RA mouse models. FA‐CF‐NPs exhibit complementary imaging advantages with high fluorescence sensitivity and resolution in the second near‐infrared window (NIR‐II) and promising photoacoustic (PA) imaging contrast with deep tissue penetration. The high affinity of the folate ligand on the surface of FA‐CF‐NPs to macrophages with overexpressed folate receptors ensures the specific targeting function of the nanoprobes for imaging in vivo. FA‐CF‐NPs are employed to achieve NIR‐II fluorescence imaging to accurately localize macrophages in the joint, obtaining a high signal‐to‐background ratio (SBR) of 15.5. Meanwhile, PA imaging is also performed to observe macrophage distribution in the longitudinal section of the whole joint. Moreover, macrophage infiltration is detected in the joint of RA mice with the absence of RA pathologies such as synovitis and cartilage loss. The results provide an opportunity for in vivo macrophage tracking and early diagnosis of RA.