Biological Imaging Applications Research Articles

Site-specific albumin tagging with chloride-containing near-infrared cyanine dyes: molecular engineering, mechanism, and imaging applications.

Near-infrared dyes, particularly cyanine dyes, have shown great potential in biomedical imaging due to their deep tissue penetration, high resolution, and minimal tissue autofluorescence/scattering. These dyes can be adjusted in terms of absorption and emission wavelengths by modifying their chemical structures. The current issues with cyanine dyes include aggregation-induced quenching, poor photostability, and short in vivo circulation time. Encapsulating cyanine dyes with albumin, whether exogenous or endogenous, has been proven to be an effective strategy for improving their brightness and pharmacokinetics. In detail, the chloride-containing (Cl-containing) cyanine dyes have been found to selectively bind to albumin to achieve site-specific albumin tagging, resulting in enhanced optical properties and improved biosafety. This feature article provides an overview of the progress in the covalent binding of Cl-containing cyanine dyes with albumin, including molecular engineering methods, binding sites, and the selective binding mechanism. The improved optical properties of cyanine dyes and albumin complexes have led to cutting-edge applications in biological imaging, such as tumor imaging (diagnostics) and imaging-guided surgery.

Advances in high spatiotemporal resolution fluorescence microscopic imaging technique based on point scanning

Laser point-scanning fluorescence microscopy serves as an indispensable tool in the life science research, owing to its merits of excellent resolution, high sensitivity, remarkable specificity, three-dimensional optical-sectioning capability, and dynamic imaging. However, conventional laser point-scanning fluorescence microscopy confronts a series of challenges in the rapidly evolving field of life sciences, because of the limitations imposed by optical diffraction and point scanning detection. Over the past two decades, substantial advancements have been made in super-resolution fluorescence microscopic imaging techniques. Researchers have developed various high spatial and temporal resolution point-scanning microtechniques, which hold great significance for biological optical imaging and other relevant applications. Regrettably, there are still few review articles covering the recent progress of this field. It is essential to provide a comprehensive review of laser point-scanning fluorescence microscopic techniques for their future developments and trends. In this article, the basic principles and recent advances in different point-scanning fluorescence microscopy imaging techniques are introduced from the perspectives of temporal resolution and spatial resolution, and the progress and applications of high spatio-temporal resolution microscopic imaging techniques based on point-scanning mode are summarized. Finally, the development trends and challenges of high spatio-temporal resolution point scanning fluorescence microscopic imaging technique are discussed.

Small-molecule fluorescent probes for bioactive species in inflammatory disease: arthritis, pneumonia and hepatitis.

Inflammation as an adaptive response underlies a wide variety of physiological and pathological processes. The progression of inflammation is closely intertwined with various bioactive molecules. To dissect the biological mechanisms and physiopathological functions of these molecules, exploitation of versatile detection mean is of great importance. Fluorescence imaging technique has been widely employed to track bioactive species in living systems. As a result, many small-molecule fluorescent probes for bioactive species in inflammatory disease have been developed. However, this interesting and frontier topic hasn't been systematically categorized. Therefore, in this review, we have generalized the construction strategies and biological imaging applications of small-molecule fluorescent probes for various bioactive species, including reactive oxygen/nitrogen/sulfur species, enzyme, mainly in arthritis, pneumonia and hepatitis. Moreover, the future challenges in constructing novel fluorescent probes for inflammatory disease are also present. This review will facilitate the comprehension of superior fluorescent probes for active molecules associated with inflammation.

Detection of Cobalamin and In Vitro Cell Imaging Based on Nitrogen-Doped Yellow Fluorescent Carbon Dots with Nano Architectonics.

As novel fluorescent nanomaterials, carbon dots have attracted increasing research attention because of their simple synthesis, robust fluorescence, low toxicity, and easy functionalisation. Previous research was focused on preparing carbon dots from biomass and chemical materials; however, most of these carbon dots exhibited blue fluorescence. Moreover, the fluorescence quantum yield was generally low, significantly limiting their application in biological imaging. To broaden the application scope of carbon dots, this study prepared long-wavelength emitting nano-carbon dots that exhibited increased quantum yield. Novel N-doped yellow fluorescent nano-carbon dots (Y-CDs) were synthesised via a hydrothermal method using L-tartaric acid and urea as the precursors. The Y-CDs had a high quantum yield (15.9%) and demonstrated photostability at various pHs, temperatures, and ionic strengths. The Y-CDs could detect cobalamin effectively and selectively, showing a linear relationship between fluorescence intensity and cobalamin concentration. The related coefficient was 0.997, and the detection limit was 2.101 μmol/L. In addition, the Y-CDs were successfully used as an imaging probe for MDA-MB-231 cells. Therefore, the Y-CDs developed in this study can be used for cobalamin detection and cell imaging.

Rational design of a negative photochromic spiropyran-containing fluorescent polymeric nanoprobe for sulfur dioxide derivative ratiometric detection and cell imaging.

It is highly desirable to develop high-performance ratiometric fluorescent probes for SO2 derivative detection and realize their application in biological imaging. In this study, we report the rational design of a novel negative photochromic spiropyran derivative, spiro[azahomoadamantane-pyran] (MAHD-SP), with notable orange fluorescence in its stable ring-opened state without UV regulation. The unsaturated double bond of MAHD-SP underwent the Michael addition reaction of the SO2 derivative, making the fluorescence quenching of MAHD-SP obvious. Then, MAHD-SP, a fluorescent conjugated polymer PFO and a polymeric surfactant PEO113-b-PS49 were used to construct a ratiometric fluorescent polymeric nanoprobe (RFPN) via a coprecipitation method. The probe exhibited high sensitivity and selectivity for the ratiometric detection of SO2 derivatives in pure aqueous solutions. Moreover, the good biocompatibility of RFPN can be used to visualize exogenous and endogenous SO2 derivative generation in living cells.

Ratiometric Sensing of Intracellular pH Based on Dual Emissive Carbon Dots.

Accurate monitoring of intracellular pH in living cells is critical for developing a better understanding of cellular activities. In the current study, label-free carbon dots (p-CDs), which were fabricated using a straightforward one-pot solvothermal treatment of p-phenylenediamine and urea, were employed to create a new ratiometric pH nanosensor. Under single-wavelength excitation (λex = 500nm), the p-CDs gave dual emission bands at 525 and 623nm. The fluorescent intensity ratio (I525/I623) was linearly related to pH over the range 4.0 to 8.8 in buffer solutions, indicating that the ratiometric fluorescence nanoprobe may be useful for pH sensing. In pH measurements, the p-CDs also demonstrated outstanding selectivity, reversibility, and photostability. Owing to the advantages outlined above, the nanoprobe was used to monitor the pH of HeLa cells effectively. The label-free CD-based ratiometric nanoprobe features comparatively easy manufacturing and longer excitation and emission wavelengths than the majority of previously reported CD-based ratiometric pH sensors, which is ultimately beneficial for applications in biological imaging.

Enhancing red upconversion emission in NaErF4@NaYF4 core-shell nanoparticles by introducing Yb3+ ions as energy trapping centers

Rare-earth-doped red upconversion nanocrystals (UCNPs) have shown excellent prospects for application in biological imaging, labeling and other fields. In this work, NaErF4@NaYF4 core-shell (CS) UCNPs were successfully constructed to enhance red emission by introducing Yb3+ ions as energy trapping centers under different near-infrared excitation wavelengths. The experimental results showed that the red upconversion emission intensity of NaErF4@ NaYF4 CS UCNPs doped with 15%Yb3+ ions in the core is nearly 202 times higher than that of NaErF4 UCNPs under 980 nm excitation wavelength, with a red/green emission intensity ratio of 18.7. When Yb3+ ions were introduced into the core and shell of NaErF4@NaYF4 CS UCNPs, NaErF4:15%Yb3+@NaYF4:10%Yb3+ CS UCNPs showed the best red emission due to the energy network characteristics of the Yb3+ ions. Furthermore, the enhancement mechanism of red emission in NaErF4@NaYF4 CS UCNPs with different Yb3+ ion doping concentrations were discussed, based on the spectral characteristics and lifetime. It was found that doping of Yb3+ ions in NaErF4@ NaYF4 CS UCNPs can significantly improve the red emission intensity of Er3+ ions through controlling energy migration (EM) and energy back transfer (EBT) processes, which has considerable potential for applications in bioimaging and anti-counterfeiting.

NaYF4 upconversion crystals with red light emission by low Er3+ concentration doping

In the work, a range of Er3+-sensitized NaYF4 upconversion (UC) particles doped with Tm3+ and Ho3+ are successfully prepared by hydrothermal process. The UC crystals appear two green emission peaks at 529 and 541 nm and a remarkable red emission peak at 655 nm under 980 and 1532 nm irradiations. Here, Tm3+ and Ho3+ serve as trapping centers, which confine the excitation energy efficiently to promote the red UC emission and improve the red to green ratio. UC mechanisms in the Er3+/Tm3+ co-doped and Er3+/Ho3+ co-doped NaYF4 crystals are discussed at length based on the UC luminescence spectroscopy. Unlike the traditional sensitization with high Er3+ concentration doping to obtain red light, the red UC luminescence is acquired by low Er3+ concentration doping, which can effectively avoid concentration quenching caused by high Er3+ concentration doping. Furthermore, NaYF4:Er3+, Tm3+ crystals exhibit stronger red UC emission compared with NaYF4:Er3+, Ho3+ counterparts. Er3+-sensitized NaYF4 crystals doped with Tm3+ and Ho3+ ions generate pure red light more efficaciously under 1532 nm excitation than under 980 nm excitation. The phosphors emitting brilliant red UC emission have the potential applications in solid state lasers and biological imaging.

Solvent-free and dispersant-free synthesis of narrow full width at half maximum carbon dots via sole precursor of p-phenylenediamine

Narrow band gap emissive carbon dots (CDs), especially those with full width at half maximum (FWHM) below 40 nm, is extremely important for biological imaging application due to higher color purity and better resolution. The synthesis of narrow FWHM emissive CDs usually requires the use of specific-structured molecular precursors with a large π conjugated domain. In comparison, the most common precursors for CDs, for instance p-phenylenediamine, are usually believed unsuitable for preparing CDs with FWHM emission below 40 nm. In this work, we challenged this stereotype by using p-phenylenediamine precursor to synthesize CDs with a small FWHM of only 29 nm. Instead of using traditional solvothermal method, a facile solvent-free method was developed to synthesize CDs with the powder of p-phenylenediamine in the autoclave under a reaction temperature of 200 °C. After dispersed in solution, CDs show a red emission around 615 nm with FWHM of 29 nm and a photoluminescence quantum yield (PLQY) of 12.8%. Through experiments and theoretical calculation, we found that the FWHM emission is positive correlated to the content of amino groups on the surface of CDs. In comparison to traditional solvothermal method, the current solvent-free method can promote the synthesis of CDs with less amino groups on the surface, reducing fluorescence center types, and hence realizing smaller FWHM emission. In addition, as-prepared CDs were used for biological imaging, and realized a rapid no-wash cell imaging.

Geometrical Parameter Effect on Plasmonic Scattering of Bimetallic Three-Layered Nanoshells.

Enhanced scattering from local surface plasmon resonance by light has attracted much attention due to its special applications in sensor, cell, and biological imaging . Here, we investigate the ratio of scattering to absorption in bimetallic three-layered nanoshells with different geometrical parameters using quasi-static theory. We show that the ratio of scattering to absorption strongly depends on the inner radius, shell thickness, middle dielectric function, and surrounding medium function. To gain insight into the effect of such geometrical parameters on the plasmonic scattering, we also provide a comparison between silver-dielectric-gold nanoshells and gold-dielectric-silver nanoshells. This work provides an alternative approach to analyze the optical properties of bimetallic three-layered nanoshells with potential applications in sensors and photo-detectors.

Coordinate Anchoring of Mixed Luminophores in Two Isostructural Hybrid Layers to Achieve Tunable Room-Temperature Phosphorescence.

Room-temperature phosphorescence (RTP) materials have widespread applications in biological imaging, anticounterfeiting, and optoelectronic devices. Because of the predesignability of metal-organic complexes (MOCs), the RTP materials based on MOC systems have received huge attention from researchers. The coordinate anchoring of luminophores to enhance the rigidity of organic molecules and restrict the nonradiative transition offers opportunities for generating MOC materials with captivating RTP performance. Hitherto, most of the MOC-based RTP materials feature a single luminophore ligand. The development of new MOC systems with RTP functionality is still challenging. Herein, we use the mixed-ligand synthetic strategy to produce isostructural MOCs, [Zn(TIMB)(X2-TPA)]·H2O (1, X = Cl; 2, X = Br; TIMB = 1,3,5-tris(2-methyl-1H-imidazol-1-yl)benzene; H2-X2-TPA = 2,5-dichloroterephthalic and 2,5-dibromoterephthalic acid), and modulate the RTP properties of resultant products via the synergy of coordinate anchoring and substitution synthesis. 1 and 2 feature similar coordination layers composed of neutral TIMB and anionic X2-TPA2- ligands, which provide a good structural model to tune the RTP performances of final products via substitution synthesis. Different from the reported RTP materials based on MOC systems, our study provides a general way to build and modulate MOC-based RTP materials with the assistance of coordinate anchoring and substitution synthesis strategies.

Luminescent Carbon Dots from Wet Olive Pomace: Structural Insights, Photophysical Properties and Cytotoxicity.

Carbon nanomaterials endowed with significant luminescence have been synthesized for the first time from an abundant, highly localized waste, the wet pomace (WP), a semi-solid by-product of industrial olive oil production. Synthetic efforts were undertaken to outshine the photoluminescence (PL) of carbon nanoparticles through a systematic search of the best reaction conditions to convert the waste biomass, mainly consisting in holocellulose, lignin and proteins, into carbon dots (CDs) by hydrothermal carbonization processes. Blue-emitting CDs with high fluorescence quantum yields were obtained. Using a comprehensive set of spectroscopic tools (FTIR, Raman, XPS, and 1H/13C NMR) in combination with steady-state and time-resolved fluorescence spectroscopy, a rational depiction of WP-CDs structures and their PL properties was reached. WP-CDs show the up-conversion of PL capabilities and negligible cytotoxicity against two mammalian cell lines (L929 and HeLa). Both properties are excellent indicators for their prospective application in biological imaging, biosensing, and dynamic therapies driven by light.

Dye-modified core/shell NaGdF4:Yb,Er@NaYF4 nanoparticles for the detection and irritation assessment of NO2−

Upconversion nanoparticles (UCNPs) have great potential applications in biological imaging, medical treatment and optical sensing. However, the development of an inorganic-organic hybrid fluorescent probe to accurately detect noxious ions and scientifically assess irritation in vivo remains a challenge. Herein, we demonstrate a fluorescent nanoprobe for detecting nitrites (NO2−) and assessing the irritation in jellyfish cell. The nanoprobe was assembled by linking neutral red to the surface of polyethylene glycol (PEG) modified NaGdF4:Yb,Er@NaYF4 core/shell nanoparticles. The nanoprobe could efficiently capture the NO2− in jellyfish and simultaneously visualize the process. It's known that the intensity ratio of green to red emission (IRGRE) of neutral red modified upconversion nanoparticles increases linearly with the amount of NO2− increasing. Moreover, the irritation of NO2− was assessed in jellyfish cells using neutral red modified fluorescent nanoprobe based on cell imaging and spectrum signals, that NO2− has irrecoverable injury to the cells. This work based on dye-modified upconversion luminescence nanoprobe is expected to shed further light on the biological analysis and detection of ions.

Coherent modulation imaging using a physics-driven neural network.

Coherent modulation imaging (CMI) is a lessness diffraction imaging technique, which uses an iterative algorithm to reconstruct a complex field from a single intensity diffraction pattern. Deep learning as a powerful optimization method can be used to solve highly ill-conditioned problems, including complex field phase retrieval. In this study, a physics-driven neural network for CMI is developed, termed CMINet, to reconstruct the complex-valued object from a single diffraction pattern. The developed approach optimizes the network's weights by a customized physical-model-based loss function, instead of using any ground truth of the reconstructed object for training beforehand. Simulation experiment results show that the developed CMINet has a high reconstruction quality with less noise and robustness to physical parameters. Besides, a trained CMINet can be used to reconstruct a dynamic process with a fast speed instead of iterations frame-by-frame. The biological experiment results show that CMINet can reconstruct high-quality amplitude and phase images with more sharp details, which is practical for biological imaging applications.

A novel Fe3+ fluorescent probe based on rhodamine derivatives and its application in biological imaging

In this paper, rhodamine B was used as the fluorophore to design and synthesize a probe RBPH for Fe3+ detection. The properties of the probe RBPH were investigated by UV–Vis absorption spectroscopy and fluorescence emission spectroscopy. The results showed that the probe had a detection limit of 75.9 nM and a binding constant of 0.608 × 104 M−1. Compared with the published rhodamine B class Fe3+ probes, the probe RBPH has the advantage of rapid response. Biological experiments show that the probe has good biocompatibility, low toxicity and good cell penetration ability. The probe RBPH can be successfully applied to the determination of Fe3+ ions in Hela cells and zebrafish, and a good fluorescence imaging effect can be seen by laser confocal microscopy.

Design and Synthesis of RhodIndolizine Dyes with Improved Stability and Shortwave Infrared Emission up to 1250 nm.

The design of shortwave infrared (SWIR) emissive small molecules with good stability in water remains an important challenge for fluorescence biological imaging applications. A series of four SWIR emissive rhodindolizine (RI) dyes were rationally designed and synthesized to probe the effects of nonconjugated substituents, conjugated donor groups, and nanoencapsulation in a water-soluble polymer on the stability and optical properties of the dyes. Steric protecting groups were added at the site of a significant LUMO presence to probe the effects on stability. Indolizine donor groups with added dimethylaniline groups were added to reduce the electrophilicity of the dyes toward nucleophiles such as water. All of the dyes were found to absorb (920-1096 nm peak values) and emit (1082-1256 nm peak values) within the SWIR region. Among xanthene-based emissive dyes, emission values >1200 nm are exceptional with 1256 nm peak emission being a longer emission than the recent record setting VIX-4 xanthene-based dye. Half-lives were improved from ∼5 to >24 h through the incorporation of either steric-based core protection groups or donors with increased donation strength. Importantly, the nanoencapsulation of the dyes in a water-soluble surfactant (Triton-X) allows for the use of these dyes in biological imaging applications.

Quantum-enhanced stimulated Brillouin scattering spectroscopy and imaging.

Brillouin microscopy is an emerging label-free imaging technique used to assess local viscoelastic properties. Quantum-enhanced stimulated Brillouin scattering is demonstrated using low power continuous-wave lasers at 795 nm. A signal-to-noise ratio enhancement of 3.4 dB is reported by using two-mode intensity-difference squeezed light generated with the four-wave mixing process in atomic rubidium vapor. The low optical power and the excitation wavelengths in the water transparency window have the potential to provide a powerful bio-imaging technique for probing mechanical properties of biological samples prone to phototoxicity and thermal effects. The performance enhancement affordable through the use of quantum light may pave the way for significantly improved sensitivity that cannot be achieved classically. The proposed method for utilizing squeezed light for enhanced stimulated Brillouin scattering can be easily adapted for both spectroscopic and imaging applications in biology.

Computing Metasurfaces Enabled Broad-Band Vectorial Differential Interference Contrast Microscopy

Differential interference contrast (DIC) microscopy provides the ability to visualize transparent phase samples such as living cells and tissues, which plays an essential role in applications in modern biology and medicine. However, existing DIC microscopy requires the introduction of complicated phase-contrast devices and is still limited to one-dimensional imaging. To mitigate these challenges, we propose a two-dimensional (2D) vectorial DIC microscopy based on computing metasurfaces to extract the 2D edge information for weakly scattering cells. Here, the Wollaston prism in a conventional DIC microscope is replaced by the computing metasurface, enabling our vectorial DIC microscope system to be miniaturized because of its compactness and multifunctionality. Imaging experiments with phase samples verify the capability to clearly observe a 2D vectorial DIC microscopy image with high contrast and its application in biological imaging. Particularly, because the designed computing metasurface is based on the Pancharatnam–Berry phase, our scheme may provide real-time, broad-band, and high-contrast phase imaging of living biomedical systems.

Strategy for Detecting Carbon Monoxide: Cu2+-Assisted Fluorescent Probe and Its Applications in Biological Imaging.

Herein, a novel strategy was proposed for identifying carbon monoxide (CO), which plays a crucial part in living systems. For the first time, we have managed to design, synthesize, and characterize successfully this new Cu2+-assisted fluorescent probe (DPHP) in detecting CO. Compared with the commonly adopted Pd0-mediated Tsuji-Trost reaction recognition method, such a new strategy did not engage costly palladium (II) salt and generated no leaving group, indicating a satisfactory anti-interference ability. The recognition mechanism was confirmed by IR, 1H NMR titration, HR-MS, cyclic voltammetry, X-ray photoelectron spectroscopy, electron paramagnetic resonance, and optical properties. Surprisingly, it was found that the new method achieved high selectivity and rapid identification of CO with a lower limit of detection (1.7 × 10-8 M). More intriguingly, it could recognize endogenous and exogenous CO in HeLa cells. The cytotoxicity of this new method was so low that it allowed the detection of CO in mice and zebrafish. Basically, our results trigger a novel viewpoint of rationally designing and synthesizing advanced materials for CO detection with unique features, impelling new research in detection chemistry.

Miniaturized Chemical Tags for Optical Imaging.

Recent advances in optical bioimaging have prompted the need for minimal chemical reporters that can retain the molecular recognition properties and activity profiles of biomolecules. As a result, several methodologies to reduce the size of fluorescent and Raman labels to a few atoms (e.g., single aryl fluorophores, Raman‐active triple bonds and isotopes) and embed them into building blocks (e.g., amino acids, nucleobases, sugars) to construct native‐like supramolecular structures have been described. The integration of small optical reporters into biomolecules has also led to smart molecular entities that were previously inaccessible in an expedite manner. In this article, we review recent chemical approaches to synthesize miniaturized optical tags as well as some of their multiple applications in biological imaging.