Fluorescence Imaging Applications Research Articles

Aptamer-guided luminous microsphere for anchoring and lightening Salmonella enterica serovar Typhimurium

Aptamers are recognition elements that are easy to synthesize and store, but their softness makes their stability and affinity subject to the influence of the environment. Herein, by taking advantage of the aptamer-specific ability to recognize targets and the fluorescence of nontoxic carbon dots (CDs), we constructed aptamer-guided luminous microspheres (~200 nm in size). These microspheres, with tetrahedral DNA (Td) as the skeleton, can actively recognize Salmonella enterica serovar Typhimurium (S. Typhimurium) and fluoresce after sensitively binding to the target. The fluorescence intensity emitted by the microspheres increased 3.05 times. The limitation of detection was 9 CFU/mL, with a detection range of 10–108 CFU/mL, and the recovery rate in qualified pasteurized milk and drinking water samples was 95.35–103.01%. Additionally, this presented fluorescence signal amplification strategy provides novel insights into the analysis of various food threat factors and other fluorescence imaging applications.

Liposome-Loaded Targeted Theranostic Fluorescent Nano-Probes for Diagnosis and Treatment of Cervix Carcinoma

Near-infrared fluorescence imaging, with its high sensitivity, non-invasiveness, and superior real-time feedback properties, has become a powerful skill for accurate diagnosis in the clinic. Nanoparticle-assisted chemotherapy is an effective cure for cancer. Specifically, the combination of near-infrared fluorescence imaging with chemotherapy represents a promising method for precise diagnosis and treatment of cervical cancer. To realize this approach, it is necessary to design and synthesize therapeutic nano-probes with detection abilities. In this work, an organic NIRF emissive heptamethine cyanine dye, IR783, was utilized and encapsulated in biocompatible drug-carrier liposomes). Then, the anticancer drug doxorubicin was loaded, to form LP-IR783-DOX nanoparticles. The LP-IR783-DOX nanoparticles had spherical shapes and were smoothly dispersed in aqueous solutions. Favorable absorption (a peak of 800 nm) and fluorescence (a peak of 896 nm) features were obtained from LP-IR783-DOX nanoparticles in the near-infrared region. Moreover, the specific detection abilities of nanoparticles were confirmed in different cell lines, and nanoparticles exhibited strong detection abilities in human cervix carcinoma cells in particular. To analyze the chemotherapeutic properties of LP-IR783-DOX nanoparticles, live HeLa cells were studied in detail, and the application of these NPs resulted in a chemotherapeutic efficiency of 56.75% based on fluorescein isothiocyanate staining and flow cytometry. The results indicate that nanoparticles have great potential for theranostic application of fluorescence imaging and chemotherapy in cases of cervical cancer.

A synergistic strategy to develop photostable and bright dyes with long Stokes shift for nanoscopy

The quality and application of super-resolution fluorescence imaging greatly lie in the dyes’ properties, including photostability, brightness, and Stokes shift. Here we report a synergistic strategy to simultaneously improve such properties of regular fluorophores. Introduction of quinoxaline motif with fine-tuned electron density to conventional rhodamines generates new dyes with vibration structure and inhibited twisted-intramolecular-charge-transfer (TICT) formation synchronously, thus increasing the brightness and photostability while enlarging Stokes shift. The new fluorophore YL578 exhibits around twofold greater brightness and Stokes shift than its parental fluorophore, Rhodamine B. Importantly, in Stimulated Emission Depletion (STED) microscopy, YL578 derived probe possesses a superior photostability and thus renders threefold more frames than carbopyronine based probes (CPY-Halo and 580CP-Halo), known as photostable fluorophores for STED imaging. Furthermore, the strategy is well generalized to offer a new class of bright and photostable fluorescent probes with long Stokes shift (up to 136 nm) for bioimaging and biosensing.

Synthesis and Photoluminescence Characterization of 3- MPA Capped CdZnTe Quantum Dots

3-MPA capped CdZnTe alloy quantum dots synthesized using Na2TeO3 as the tellurium source under open atmosphere conditions in aqueous medium. The optical and photoluminescence properties of the CdZnTe quantum dots characterized using UV-Visible absorption and Photoluminescence spectrum. An excitonic peak at 527 nm observed in the absorption spectrum. Tauc relation and Urbach relation were employed to find the bandgap energy and urbach energy of the CdZnTe quantum dot. The quantum dot particle size was estimated using the bandgap energy from Tauc plot employing tight binding approximation, which was 4.75 nm. A high intense emission peak at 567 nm obtained in the photoluminescence spectrum. The emission peaks correspond to yellow fluorescence. The CIE chromaticity diagram gives color coordinate at (0.41,0.56) with a color purity of 92%. The high intense yellow fluorescent CdZnTe quantum can placed as potential candidates for fluorescent probes, biolabeling and cell imaging applications.

Stable red nanoparticles loaded neutral luminescent radicals for fluorescence imaging

Although neutral luminescent radicals have arisen much attention recently, their applications in fluorescence imaging are rarely reported due to their insolubility in water and weak luminescence in polar solvents. Herein, we encapsulated a luminescent radical and its precursor into nanoparticles (NPs) by an amphiphilic polymer matrix, DSPE-PEG2000, to avoid aggregation-caused quenching and poor solubility in water to further achieve its application in cell-imaging. The obtained radical NPs exhibited strong red emission, excellent stability and remarkable biocompatibility under physiological conditions. The radical NPs were incubated with live HCT116 cells and showed good cellular uptake, thus making fluorescence imaging possible in vitro. The results confirm the feasibility of stable neutral luminescent radical as a promising candidate for the application in fluorescence imaging.

ATP-Triggered Intracellular In Situ Aggregation of a Gold-Nanoparticle-Equipped Triple-Helix Molecular Switch for Fluorescence Imaging and Photothermal Tumor Therapy.

Isotropic gold nanoparticles (AuNPs) can generate a plasma-plasma interaction when aggregating and can also produce ideal photothermal effects. Some studies have designed ATP-responsive nanodrug delivery systems by taking advantage of the differences between internal and external ATP in tumor cells, but few studies have focused on the photothermal effects of ATP-induced AuNP aggregation in tumors. Here, a triple-helix probe (THP) molecular switch and MUC1 aptamer-functionalized AuNPs were constructed for fluorescence imaging analysis and photothermal therapy (PTT). The MUC1 aptamer guides THP-AuNP targeting in tumor cells, followed by the high concentration of ATP inducing structural changes in triple-helix probes and causing the intracellular aggregation of AuNPs, which cannot escape from the tumor site, enabling tumor imaging while performing PTT. Therefore, the designed THP-AuNPs have promising applications in fluorescence imaging and PTT.

Combined TPEF and SHG Imaging for the Microstructural Characterization of Different Wood Species Used in Artworks

The morphological and chemical conformation of wood microstructures is characteristic of individual species and strongly influences the macromechanical properties of the material, as well as its sensitivity to deterioration factors. Noninvasive techniques enabling the visualization of wood microstructures, while simultaneously providing compositional information, can significantly facilitate the analysis of wooden artworks for conservation purposes. In this paper, we present the application of combined two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) imaging as a versatile diagnostic tool for the microcharacterization of three hardwood species never analyzed by this method. Multimodal mapping of the molecular constituents based on the detected nonlinear signals provides useful information for studying the biological and biochemical deterioration of wood, opening a new field of application for a well-established and widely used imaging technology.

A Facile and Highly Efficient Approach to Obtain a Fluorescent Chromogenic Porous Organic Polymer for Lymphatic Targeting Imaging.

Porous organic polymers have an open architecture, excellent stability, and tunable structural components, revealing great application potential in the field of fluorescence imaging, but this part of the research is still in its infancy. In this study, we aimed to tailor the physical and chemical characteristics of indocyanine green using sulfonic acid groups and conjugated fragments, and prepared amino-grafted porous polymers. The resulting material had excellent solvent and thermal stability, and possessed a relatively large pore structure with a size of 3.4 nm. Based on the synergistic effect of electrostatic bonding and π–π interactions, the fluorescent chromogenic agent, indocyanine green, was tightly incorporated into the pore cavity of POP solids through a one-step immersion method. Accordingly, the fluorescent chromogenic POP demonstrated excellent imaging capabilities in biological experiments. This preparation of fluorescent chromogenic porous organic polymer illustrates a promising application of POP-based solids in both fluorescence imaging and biomedicine applications.

Investigation on photoluminescence properties of MWCNTs@Gd2O3: RE3+ (RE = Eu, Tb, and Tm) hybrid nanocomposites

A facile synthesis of rare earth (RE3+) activated gadolinium oxide (Gd2O3) decorated on the surface of multiwall carbon nanotubes (MWCNT), i.e., MWCNTs@Gd2O3: RE3+ (RE = Eu, Tb, and Tm) have been carried out. The X-ray diffraction patterns and scanning electron microscope (SEM) micrographs of as-prepared nanocomposites confirm the homogeneous dispersion of the nanocomposites. The emission spectrum of MWCNTs@Gd2O3:Eu3+ nanocomposite exhibits the characteristic emission lines of Eu3+ at 590 nm and 610 nm with the radiation of 254 nm. MWCNTs@Gd2O3:Tb3+ nanocomposite displays an intense green emission at 547 nm, while MWCNTs@Gd2O3:Tm3+ nanocomposite shows a strong blue emission at 457 nm under ultra-violet (UV) excitation of 360 nm. The calculated values of emission lifetimes in the studied phosphors could attribute to more radiative relaxation. Henceforth, the results affirm that these spectral engineered luminescent materials may envision prospective applications in fluorescent imaging, bio-separation, fluorescent lamps, and display devices, etc.

Simultaneous Enhancement of the Long-Wavelength NIR-II Brightness and Photothermal Performance of Semiconducting Polymer Nanoparticles.

Theranostic agents with fluorescence in the second near-infrared (NIR-II) window, especially in its long-wavelength region, and NIR-II-excitable photothermal effect is promising but challenging in tumor diagnosis and therapy. Here, we report a simple but effective strategy to develop semiconducting polymer nanoparticles-based theranostic agents (PBQx NPs) and demonstrate their applications for long-wavelength NIR-II fluorescence imaging beyond 1400 nm and photothermal therapy (PTT) of tumors upon excitation at 1064 nm. Both experimental results and theory calculations show that the brightness and photothermal performance of PBQx NPs can be simultaneously improved by simply increasing the repeating unit number of semiconducting polymers. For example, PBQ45 NPs have 5-fold higher brightness than PBQ5 NPs and 6.7-fold higher photothermal effect (based on PCE × ε) than PBQ3 NPs, and exhibit promising applications in long-wavelength NIR-II fluorescence abdomen imaging, image-guided tumor resection, and image-guided PTT. This study demonstrates the effectiveness and importance of repeating unit numbers in regulating the theranostic performance, which has not received enough attention before.

Molecular fluorophores for in vivo bioimaging in the second near-infrared window.

This systematic review aims to summarize the current developments of fluorescence and chemi/bioluminescence imaging based on the molecular fluorophores for in vivo imaging in the second near-infrared window. By investigating most of the relevant references on the web of science and some journals, this review firstly begins with an overview of the background of fluorescence and chemi/bioluminescence imaging. Secondly, the chemical and optical properties of NIR-II dyes are discussed, such as water solubility, chemostability and photo-stability, and brightness. Thirdly, the bioimaging based on NIR-II fluorescence emission is outlined, including the in vivo imaging of polymethine dyes, donor - acceptor - donor (D - A - D) chromophores, and lanthanide complexes. Fourthly, we demonstrate the chemi/bioluminescence in vivo imaging in the second near-infrared window. Fifthly, the clinical application and translation of near-infrared fluorescence imaging are presented. Finally, the current challenges, feasible strategies and potential prospects of the fluorophores and in vivo bioimaging are discussed. Based on the above literature research on the applications of molecular fluorescent and chemi/bioluminescent probes in the second near-infrared window in recent years, this review weighs the advantages and disadvantages of fluorescence and chemi/bioluminescence imaging, and NIR-II fluorophores based on polymethine dyes, D - A - D chromophores, and lanthanide complexes. Besides, this review also provides a very important guidance for expanding the imaging applications of molecular fluorophores in the second near-infrared window.

Bacteria-targeted fluorescence imaging of extracted osteosynthesis devices for rapid visualization of fracture-related infections

PurposeFracture-related infection (FRI) is a serious complication in orthopedic trauma surgery worldwide. Especially, the distinction of infection from sterile inflammation and the detection of low-grade infection are highly challenging. The objective of the present study was to obtain proof-of-principle for the use of bacteria-targeted fluorescence imaging to detect FRI on extracted osteosynthesis devices as a step-up towards real-time image-guided trauma surgery.MethodsExtracted osteosynthesis devices from 13 patients, who needed revision surgery after fracture treatment, were incubated with a near-infrared fluorescent tracer composed of the antibiotic vancomycin and the fluorophore IRDye800CW (i.e., vanco-800CW). Subsequently, the devices were imaged, and vanco-800CW fluorescence signals were correlated to the results of microbiological culturing and to bacterial growth upon replica plating of the imaged devices on blood agar.ResultsImportantly, compared to culturing, the bacteria-targeted fluorescence imaging of extracted osteosynthesis devices with vanco-800CW allows for a prompt diagnosis of FRI, reducing the time-to-result from days to less than 30 min. Moreover, bacteria-targeted imaging can provide surgeons with real-time visual information on the presence and extent of infection.ConclusionHere, we present the first clinical application of fluorescence imaging for the detection of FRI. We conclude that imaging with vanco-800CW can provide early, accurate, and real-time visual diagnostic information on FRI in the clinical setting, even in the case of low-grade infections.Graphical abstract

Frontispiece: 19F MRI Probes for Multimodal Imaging

This review focuses on multimodal imaging encompassing 19F MRI (magnetic resonance imaging) as one of the modalities. Structure of the probes and example applications in optical fluorescence imaging (OFI), 1H MRI, chemical exchange saturation transfer (CEST), MRI, ultrasonography (USG), X-ray computed tomography (CT), single-photon emission tomography (SPECT), positron emission tomography (PET), and photoacoustic imaging (PAI) as well as other less popular techniques were discussed. For more information, see the Review by D. Janasik and T. Krawczyk on page 4–35.

Determination of surgical margins in laparoscopic parenchyma-sparing hepatectomy of neuroendocrine tumors liver metastases using indocyanine green fluorescence imaging.

Neuroendocrine tumors (NETs) are a group of heterogenous tumors originating from neuroendocrine system. Approximately, 40 percent will go through liver metastases, and liver-directed therapy was proved to improve the survival outcome. Parenchyma-sparing hepatectomy is advocated for the resection of NETs liver metastases while the possible relatively low negative margin rate is concerned. Indocyanine green (ICG) fluorescence imaging provides a real-time navigation on determination of surgical margins in colorectal cancer liver metastases. However, there was no previous study that reported the applications of ICG fluorescence imaging in NETs liver metastases. The present study aimed to evaluate the feasibility and security of using ICG fluorescence imaging to determine surgical margins of NETs liver metastases during operation. A retrospective two-arm cohort study was performed on 25 consecutive patients with NETs liver metastases who underwent laparoscopic parenchyma-sparing hepatectomy (LPSH). Patients were divided into two groups according to whether or not the ICG fluorescence imaging was used. Data on sociodemographic characteristics, laboratory parameters, pathology results, and surgical outcomes were collected. A total of 145 tumors pathologically diagnosed with NETs liver metastases were resected from 25 patients. The pathological results indicated negative margins in all tumors (102/102) in LPSH with ICG fluorescence imaging group. The negative margin rate was significantly higher in LPSH using the ICG fluorescence imaging (100% v.s 88.4%, p = 0.002). Surgical outcomes, including operation time, estimated blood loss, intraoperative transfusion rate, and postoperative morbidity, were comparable between LPSH with and without ICG fluorescence imaging groups. ICG fluorescence imaging showed the potential to identify tumor boundaries and determine surgical margins. This technique may serve as a valuable intraoperative navigation in patients with NETs liver metastases.

Excellent Multiphoton Excitation Fluorescence with Large Multiphoton Absorption Cross Sections of Arginine-Modified Gold Nanoclusters for Bioimaging.

Fluorescent gold nanoclusters (Au NCs) with excellent one-photon and multiphoton properties have been demonstrated as promising candidates in many application fields. However, small multiphoton absorption (MPA) cross sections and weak multiphoton excitation (MPE) fluorescence impede their practical applications under near-infrared (NIR) excitation for biological imaging. Here, we report the regulated one-photon and multiphoton properties and mechanisms of arginine-stabilized 6-aza-2-thiothymine Au NCs (Arg/ATT-Au NCs) and the applications for MPE fluorescence imaging. The introduction of arginine into the capping layer of ATT-Au NCs significantly modifies the electronic structure, the absorption cross sections, and the relaxation dynamics of the lowest excited state, drastically reducing the nonradiative relaxation, suppressing the blinking, and greatly enhancing the fluorescence. Besides the improved one-photon properties, Arg/ATT-Au NCs demonstrate remarkable MPE fluorescence with a large MPA cross section. The two-photon (λex = 850 nm), three-photon (λex = 1400 nm), and four-photon (λex = 1700 nm) absorption cross sections have been determined to be 6.1 × 10-47 cm4 s1 photon-1, 1.5 × 10-78 cm6 s2 photon-2, and 5.5 × 10-108 cm8 s3 photon-3, respectively, much higher than those of conventional organic compounds and previously reported Au NCs. Moreover, Arg/ATT-Au NCs have been successfully applied in two-photon and three-photon excitation fluorescence imaging of living cells with NIR excitation. The manifold advantages of small size, high quantum yield, suppressed blinking, good photostability and cytocompatibility, large MPA cross sections, and excellent MPE fluorescence imaging performances make fluorescent Arg/ATT-Au NCs a great candidate of imaging probes with vis-NIR excitation.

A nanoarchitecture of a gold cluster conjugated gold nanorod hybrid system and its application in fluorescence imaging and plasmonic photothermal therapy.

Engineering different nanomaterials into a single functional material can impart unique properties of the parental nanoparticles, especially in the field of bio imaging and therapy. Gold nanomaterials having different sizes, shapes and dimensionalities exhibit exceptional properties apart from their non-toxicity and hence are strong candidates in the biomedical field. Designing a hybrid nanomaterial of two gold nanostructures retaining the individual properties of the parental nanomaterials is challenging. Here, we demonstrate the synthesis of a hybrid nanomaterial (GQC@GNR), comprising an extremely small gold nanocluster and a representative of the asymmetric gold nanostructure, i.e., a gold nanorod, both having their own different exclusive optical properties like tuneable emission and NIR absorption characteristics, respectively. The hybrid system is designed to retain its emission and absorption in the NIR region to use it as an agent for simultaneous imaging and therapy. The formation of GQC@GNR and its architectonics heavily depend on the synthesis route and the parameters adopted which in turn have a direct influence on its properties. The architecture and its connection to the optical properties are explained using UV-Vis absorption and photoluminescence spectroscopy, zeta potential, transmission electron microscopy, etc. DFT-based computational modelling supports architectonics as explained by the experimental findings. The formation of the gold-gold hybrid system witnessed interesting science with a strong indication that materials with desired properties can be designed by appropriately modulating the architectonics of hybrid formation. Finally, folate conjugated GQC@GNR demonstrated its efficacy for targeted imaging and photothermal therapy in HeLa cells and tumor-bearing animal models. The detailed therapeutic efficacy of GQC@GNR is also explained based on Raman spectroscopy.

Management of fluorescent organic/inorganic nanohybrids for biomedical applications in the NIR-II region.

Biomedical fluorescence imaging in the second near-infrared (NIR-II, 100-1700 nm) window provides great potential for visualizing physiological and pathological processes, owing to the reduced tissue absorption, scattering, and autofluorescence. Various types of NIR-II probes have been reported in the past decade. Among them, NIR-II organic/inorganic nanohybrids have attracted widespread attention due to their unique properties by integrating the advantages of both organic and inorganic species. Versatile organic/inorganic nanohybrids provide the possibility of realizing a combination of functions, controllable size, and multiple optical features. This tutorial review summarizes the reported organic and inorganic species in nanohybrids, and their biomedical applications in NIR-II fluorescence and lifetime imaging. Finally, the challenges and outlook of organic/inorganic nanohybrids in biomedical applications are discussed.

Shortwave-infrared (SWIR) emitting annexin V for high-contrast fluorescence molecular imaging of tumor apoptosis in living mice.

Recently, shortwave infrared (SWIR) fluorescence imaging over 1000 nm has attracted much attention for in vivo optical imaging because of the higher signal to background ratios in the SWIR region. For the application of SWIR fluorescence imaging to biomedical fields, the development of SWIR fluorescent molecular probes with high biocompatibility is crucial. Although many researchers have designed a variety of SWIR emitting probes based on organic dyes, the synthesis of biocompatible SWIR fluorescent molecular imaging probes is still challenging. In this work we synthesized indocyanine green (ICG) and π-conjugation extended ICG (ICG-C11) labelled annexin V as SWIR fluorescent probes for tumor apoptosis. Annexin V is an endogenous protein with binding ability to phosphatidylserine (PS) which appears on the outer monolayer of apoptotic cell membranes. Although there are many types of visible and NIR fluorescent annexin V, there are no SWIR emitting fluorescent probes that can be used for high contrast fluorescence imaging of apoptosis in vivo. Herein, we report the synthesis and application of ICG and ICG-C11 conjugated annexin V for SWIR fluorescence imaging of tumor apoptosis. The presented fluorescent annexin V is the first SWIR emitting probe for in vivo optical imaging of tumor apoptosis. We demonstrate that SWIR emitting ICG- and ICG-C11 conjugated annexin V enable high-contrast fluorescence imaging of tumor apoptosis in living mice. We further demonstrate that ICG-C11-annexin V can be used for long-term (ca. two weeks) SWIR fluorescence imaging of tumor apoptosis. The SWIR fluorescent annexin V will greatly contribute not only to the study of tumor-apoptosis induced by anti-cancer drugs, but also to the study of apoptosis-related diseases in a living system.

A benzophenoxazine-based NIR fluorescent probe for the detection of hydrogen sulfide and imaging in living cells.

Hydrogen sulfide (H2S) plays a vital role in regulating many important physiological functions. However, H2S-containing industrial wastewater is inevitably pumped into the environment which seriously contaminates water supplies and foodstuffs. So it is crucial to develop a single fluorescent probe for H2S detection with high sensitivity and selectivity. Herein, a colorimetric and turn-on near infrared (NIR) fluorescent probe NRDNP based on benzophenoxazine was designed and synthesized by thiolysis of 2,4-dinitrophenyl (DNP) ether as the specific reaction site strategy to achieve highly specific H2S detection in living systems. The studies demonstrated that the probe NRDNP exhibited excellent sensing performance toward H2S with an about 80-fold NIR fluorescence enhancement, a rapid response within 10 min, excellent sensitivity with a detection limit of 19 nM and good selectivity. Furthermore, the NRDNP is an optical sensor which visually changes in terms of the fluorescence colour/intensity upon sensing gaseous H2S molecules. NRDNP has low cytotoxicity and has been successfully applied in the fluorescence imaging of H2S in living cells, suggesting that it would be an effective tool for H2S detection in living systems.

Plasmonic random laser enabled artefact-free wide-field fluorescence bioimaging: uncovering finer cellular features.

Narrow bandwidth, high brightness, and spectral tunability are the unique properties of lasers that make them extremely desirable for fluorescence imaging applications. However, due to the high spatial coherence, conventional lasers are often incompatible for wide-field fluorescence imaging. The presence of parasitic artefacts under coherent illumination causes uneven excitation of fluorophores, which has a critical impact on the reliability, resolution, and efficiency of fluorescence imaging. Here, we demonstrate artefact-free wide-field fluorescence imaging with a bright and low threshold silver nanorod based plasmonic random laser, offering the capability to image finer cellular features with sub-micrometer resolution even in highly diffusive biological samples. A spatial resolution of 454 nm and up to 23% enhancement in the image contrast in comparison to conventional laser illumination are attained. Based on the results presented in this paper, random lasers, with their laser-like properties and spatial incoherence are envisioned to be the next-generation sources for developing highly efficient wide-field fluorescence imaging systems having high spatial and temporal resolution for real-time, in vivo bioimaging.