Development of a system to sensitively and specifically visualize c-fos mRNA in living cells using bispyrene-modified RNA probes

In situ visualization of c-fos mRNA was shown both in fixed cells and in living cells using bispyrene-modified 2'-O-methyl RNA probes (OMUpy2) or phosphorothioate modified OMUpy2 (OMUpy2-S), which was RNA-specific with high sensitivity, and a system for time-lapse imaging of c-fos mRNA was successfully developed.     

MnO2‐Modified Persistent Luminescence Nanoparticles for Detection and Imaging of Glutathione in Living Cells and In Vivo

Persistent luminescence nanoparticles (PLNPs) hold great promise for the detection and imaging of biomolecules. Herein, we have demonstrated a novel nanoprobe, based on the manganese dioxide (MnO₂)‐modified PLNPs, that can detect and image glutathione in living cells and in vivo. The persistent luminescence of the PLNPs can be efficiently quenched by the MnO₂ nanosheets. In the presence of glutathione (GSH), MnO₂ was reduced to Mn²⁺ and the luminescence of PLNPs can be restored. The persistent luminescence property can allow detection and imaging without external excitation and avoid the background noise originating from the in situ excitation. This strategy can offer a promising platform for detection and imaging of reactive species in living cells or in vivo.     

A Telomerase-Assisted Strategy for Regeneration of DNA Nanomachines in Living Cells

DNA nanomachines have been engineered into diverse personalized devices for diagnostic imaging of biomarkers; however, the regeneration of DNA nanomachines in living cells remains challenging. Here, we report an ingenious DNA nanomachine that can implement telomerase (TE)-activated regeneration in living cells. Upon apurinic/apyrimidinic endonuclease 1 (APE1)-responsive initiation of the nanomachine, the walker of the nanomachine moves along tracks regenerated by TE, generating multiply amplified signals through which APE1 can be imaged in situ. Additionally, augmentation of the signal due to the regeneration of the nanomachines could reveal differential expression of TE in different cell lines. To the best of our knowledge, this is the first proof-of-concept demonstration of the use of biomarkers to assist in the regeneration of nanomachines in living cells. This study offers a new paradigm for the development of more applicable and efficient DNA nanomachines.     

Visualization and In Situ Ablation of Intracellular Bacterial Pathogens through Metabolic Labeling

Protected by the host cells, the hidden intracellular bacteria are typically difficult to kill by common antibiotics and cannot be visualized without complex cellular pretreatments. Herein, we successfully developed a bacteria-metabolizable dual-functional probe TPEPy-d -Ala, which is based on d -alanine and a photosensitizer with aggregation-induced emission for fluorescence turn-on imaging of intracellular bacteria in living host cells and photodynamic ablation in situ. Once metabolically incorporated into bacterial peptidoglycan, the intramolecular motions of TPEPy-d -Ala are inhibited, leading to an enhanced fluorescent signal, which allows the clear visualization of the intracellular bacteria. Moreover, TPEPy-d -Ala can effectively ablate the labeled intracellular bacteria in situ owing to covalent ligation to peptidoglycan, yielding a low intracellular minimum inhibitory concentration (MIC) of 20±0.5 μg mL⁻¹, much more efficient than that of a commonly used antibiotic, vancomycin.     

Disassembly‐Driven Fluorescence Turn‐on of Polymerized Micelles by Reductive Stimuli in Living Cells

Stimuli‐response nanoparticles have emerged as powerful tools for imaging and therapeutic applications. Ideally, they should be assembled from biodegradable materials featuring small size and cooperative response to biological stimuli that trigger particle disassembly and release of an active molecule that could be readily monitored in situ. A concept is developed that consists of organic nanoparticles, assembled from fluorescent amphiphiles and polymerized with a redox‐cleavable cross‐linker. We obtained 20 nm nanoparticles bearing self‐quenched Nile Red dye residues, which can disassemble in living cells into highly fluorescent molecular units owing to an external or internal reductive stimulus. The obtained results pave the way to new stimuli‐responsive nanomaterials for applications in background‐free imaging as well as in drug delivery, as the concept can be further extended to other active molecules including drugs and to cross‐linkers cleavable by other biological stimuli.     

An NIR Fluorescence Turn-on and MRl Bimodal Probe for Concurrent Real-time in vivo Sensing and Labeling of β-Galactosidase

To realize sensing and labeling biomarkers is quite challenging in terms of designing multimodal imaging probes. In this study, we developed a novel β-galactosidase (β-gal) activated bimodal imaging probe that combines near-infrared (NIR) fluorescence and magnetic resonance imaging (MRI) to enable real-time visualization of activity in living organisms. Upon β-gal activation, Gal-Cy-Gd-1 exhibits a remarkable 42-fold increase in NIR fluorescence intensity at 717 nm, allowing covalent labeling of adjacent target enzymes or proteins and avoiding molecular escape to promote probe accumulation at the tumor site. This fluorescence reaction enhances the longitudinal relaxivity by approximately 1.9 times, facilitating high-resolution MRI. The unique features of Gal-Cy-Gd-1 enable real-time and precise visualization of β-gal activity in live tumor cells and mice. The probe's utilization aids in identifying in situ ovarian tumors, offering valuable assistance in the precise removal of tumor tissue during surgical procedures in mice. The fusion of NIR fluorescence and MRI activation through self-immobilizing target enzymes or proteins provides a robust approach for visualizing β-gal activity. Moreover, this approach sets the groundwork for developing other activatable bimodal probes, allowing real-time in vivo imaging of enzyme activity and localization.     

Label-Free Electrochemiluminescence Imaging of Single-Cell Adhesions by Using Bipolar Nanoelectrode Array

A label-free and fast approach for positive electrochemiluminescence (ECL) imaging of single cells by bipolar nanoelectrode array is proposed. The reduction of oxygen at a platinized gold nanoelectrode array in a closed bipolar electrochemical system is coupled with an oxidative ECL process at the anodic side. For elevating the ECL imaging contrast of single cells, a driving voltage of −2.0 V is applied to in situ generate oxygen confined beneath cells that is subsequently used for ECL imaging at 1.1 V. High oxygen concentration in the confined space resulting from steric hindrance generates prominent oxygen reduction current at the cathodic side and higher ECL intensity at the anodic side, allowing positive ECL imaging of the cells adhesion region with excellent contrast. Cell morphology and adhesion strength can be successfully imaged with high image acquisition rate. This approach opens a new avenue for label-free imaging of single cells.     

Functionalized europium nanorods for in vitro imaging

Emissive europium hydroxide nanorods (ENR) (20 nm x 500 nm) functionalized by a surface coating of chromophore-containing organically modified silicate (ORMOSIL) layer, have been synthesized and characterized by high-resolution transmission electron microscopy (TEM). Low-temperature photophysical characterization of the functionalized nanorods (FENR) demonstrated a strong red 5D0 luminescence both in solid and in suspended solutions. Potentials of this nanorod material for live cell imaging have also been explored. Both the bare and functionalized nanorods are able to enter living human cells with no discernible cytotoxicity. Chromophore-to-Eu3+ energy-transfer in the functionalized nanorods enables staining of the cytoplasm of living human cells. This is confirmed by costaining with fluorescent dextran. The red chromophore-sensitized luminescence from the internalized nanorods in live human lung carcinoma cells (A549) can be observed by confocal microscopy 2 h after loading and reaches maximal emission after 24 h.     

Visualization and In Situ Ablation of Intracellular Bacterial Pathogens through Metabolic Labeling

Protected by the host cells, the hidden intracellular bacteria are typically difficult to kill by common antibiotics and cannot be visualized without complex cellular pretreatments. Herein, we successfully developed a bacteria‐metabolizable dual‐functional probe TPEPy‐d ‐Ala, which is based on d ‐alanine and a photosensitizer with aggregation‐induced emission for fluorescence turn‐on imaging of intracellular bacteria in living host cells and photodynamic ablation in situ. Once metabolically incorporated into bacterial peptidoglycan, the intramolecular motions of TPEPy‐d ‐Ala are inhibited, leading to an enhanced fluorescent signal, which allows the clear visualization of the intracellular bacteria. Moreover, TPEPy‐d ‐Ala can effectively ablate the labeled intracellular bacteria in situ owing to covalent ligation to peptidoglycan, yielding a low intracellular minimum inhibitory concentration (MIC) of 20±0.5 μg mL^?1, much more efficient than that of a commonly used antibiotic, vancomycin.     

Confocal/TEM overlay microscopy: a simple method for correlating confocal and electron microscopy of cells expressing GFP/YFP fusion proteins.

Genetic manipulation allows simultaneous expression of green fluorescent protein (GFP) and its derivatives with a wide variety of cellular proteins in a variety of living systems. Epifluorescent and confocal laser scanning microscopy (confocal) localization of GFP constructs within living tissue and cell cultures has become routine, but correlation of light microscopy and high resolution transmission electron microscopy (TEM) on components within identical cells has been problematic. In this study, we describe an approach that specifically localizes the position of GFP/yellow fluorescent protein (YFP) constructs within the same cultured cell imaged in the confocal and transmission electron microscopes. We present a simplified method for delivering cell cultures expressing fluorescent fusion proteins into LR White embedding media, which allows excellent GFP/YFP detection and also high-resolution imaging in the TEM. Confocal images from 0.5-microm-thick sections are overlaid atop TEM images of the same cells collected from the next serial ultrathin section. The overlay is achieved in Adobe Photoshop by making the confocal image somewhat transparent, then carefully aligning features within the confocal image over the same features visible in the TEM image. The method requires no specialized specimen preparation equipment; specimens are taken from live cultures to embedding within 8 h, and confocal transmission overlay microscopy can be completed within a few hours.     

基于共轭聚合物的纳米粒子用于肿瘤的近红外二区荧光成像及光热治疗

为提高生物组织荧光成像质量以及对肿瘤的高效光热治疗,设计合成了一种新型的窄带隙共轭聚合物(BDT-TTQ),并通过纳米沉积的方式将聚合物制备成水溶性纳米粒子(BDT-TTQ NPs).该共轭聚合物纳米粒子在1000~1200 nm近红外二区范围具有较好的吸收,在1064 nm的激发光下能实现1200~1400 nm的近红外二区荧光成像. BDT-TTQ NPs纳米粒子粒径分布较窄,形貌呈规则的球形且分散均匀,具有好的生物相容性.该纳米粒子既可以在体外实现较高的近红外二区荧光成像穿透深度,又可以实现对小鼠活体血管的高清晰度的近红外二区荧光成像.此外,BDT-TTQ NPs纳米粒子在1064 nm激光下展现出优异的光热转换效率,具有较高的光毒性,对体外的肿瘤细胞以及小鼠的异质瘤具有高的光热杀伤能力.     

Carbon nanotube electrostatic biprism: principle of operation and proof of concept.

During in situ transmission electron microscopy (TEM) field emission experiments, carbon nanotubes are observed to strongly diffract the imaging TEM electron beam. We demonstrate that this effect is identical to that of a standard electrostatic biprism. We also demonstrate that the nanotube biprism can be used to capture electron-holographic information.     

Near‐Infrared Fluorescent Probe with New Recognition Moiety for Specific Detection of Tyrosinase Activity: Design,Synthesis, and Application in Living Cells and Zebrafish

Fluorescence imaging of tyrosinase (a cancer biomarker) in living organisms is of great importance for biological studies. However, selective detection of tyrosinase remains a great challenge because current fluorescent probes that contain the 4‐hydroxyphenyl moiety show similar fluorescence responses to both tyrosinase and some reactive oxygen species (ROS), thereby suffering from ROS interference. Herein, a new tyrosinase‐recognition 3‐hydroxybenzyloxy moiety, which exhibits distinct fluorescence responses for tyrosinase and ROS, is proposed. Using the recognition moiety, we develop a near‐infrared fluorescence probe for tyrosinase activity, which effectively eliminates the interference from ROS. The high specificity of the probe was demonstrated by imaging and detecting endogenous tyrosinase activity in live cells and zebrafish and further validated by an enzyme‐linked immunosorbent assay. The probe is expected to be useful for the accurate detection of tyrosinase in complex biosystems.     

Near‐Infrared Fluorescent Probe with New Recognition Moiety for Specific Detection of Tyrosinase Activity: Design,Synthesis, and Application in Living Cells and Zebrafish

Fluorescence imaging of tyrosinase (a cancer biomarker) in living organisms is of great importance for biological studies. However, selective detection of tyrosinase remains a great challenge because current fluorescent probes that contain the 4‐hydroxyphenyl moiety show similar fluorescence responses to both tyrosinase and some reactive oxygen species (ROS), thereby suffering from ROS interference. Herein, a new tyrosinase‐recognition 3‐hydroxybenzyloxy moiety, which exhibits distinct fluorescence responses for tyrosinase and ROS, is proposed. Using the recognition moiety, we develop a near‐infrared fluorescence probe for tyrosinase activity, which effectively eliminates the interference from ROS. The high specificity of the probe was demonstrated by imaging and detecting endogenous tyrosinase activity in live cells and zebrafish and further validated by an enzyme‐linked immunosorbent assay. The probe is expected to be useful for the accurate detection of tyrosinase in complex biosystems.     

Monitoring H2O2 on the Surface of Single Cells with Liquid Crystal Elastomer Microspheres

Live‐imaging of signaling molecules released from living cells is a fundamental challenge in life sciences. Herein, we synthesized liquid crystal elastomer microspheres functionalized with horse‐radish peroxidase (LCEM‐HRP), which can be immobilized directly on the cell membrane to monitor real‐time release of H₂O₂ at the single‐cell level. LCEM‐HRP could report H₂O₂ through a concentric‐to‐radial (C‐R) transfiguration, which is due to the deprotonation of LCEM‐HRP and the break of inter or intra‐chain hydrogen bonding in LCEM‐HRP caused by HRP‐catalyzed reduction of H₂O₂. The level of transfiguration of LCEM‐HRP revealed the different amounts of H₂O₂ released from cells. The estimated detection sensitivity was ≈2.2×10^?7 μm for 10 min of detection time. The cell lines and cell–cell heterogeneity was explored from different configurations. LCEM‐HRP presents a new approach for in situ real‐time imaging of H₂O₂ release from living cells and can be the basis for seeking more advanced chemical probes for imaging of various signaling molecules in the cellular microenvironment.     

A unique approach to development of near-infrared fluorescent sensors for in vivo imaging

Near-infrared (NIR) fluorescent sensors have emerged as promising molecular tools for imaging biomolecules in living systems. However, NIR fluorescent sensors are very challenging to be developed. Herein, we describe the discovery of a new class of NIR fluorescent dyes represented by 1a/1c/1e, which are superior to the traditional 7-hydroxycoumarin and fluorescein with both absorption and emission in the NIR region while retaining an optically tunable hydroxyl group. Quantum chemical calculations with the B3LYP exchange functional employing 6-31G(d) basis sets provide insights into the optical property distinctions between 1a/1c/1e and their alkoxy derivatives. The unique optical properties of the new type of fluorescent dyes can be exploited as a useful strategy for development of NIR fluorescent sensors. Employing this strategy, two different types of NIR fluorescent sensors, NIR-H(2)O(2) and NIR-thiol, for H(2)O(2) and thiols, respectively, were constructed. These novel sensors respond to H(2)O(2) or thiols with a large turn-on NIR fluorescence signal upon excitation in the NIR region. Furthermore, NIR-H(2)O(2) and NIR-thiol are capable of imaging endogenously produced H(2)O(2) and thiols, respectively, not only in living cells but also in living mice, demonstrating the value of the new NIR fluorescent sensor design strategy. The new type of NIR dyes presented herein may open up new opportunities for the development of NIR fluorescent sensors based on the hydroxyl functionalized reactive sites for biological imaging applications in living animals.     

Encapsulation and dispersion of carbon black by an in situ controlling radical polymerization of AA/BA/St with DPE as a control agent

In this paper, a new strategy to encapsulate and disperse carbon black by an in situ controlling free radical polymerization of 1,1-Diphenylenthyene (DPE) method was developed. Firstly, a living amphipathic precursor polymer of P (AA-BA) containing DPE unit was synthesized. This precursor could be grafted or absorbed on the surface of small carbon black particles to prevent further aggregation of carbon black. And the DPE unit in the living amphipathic precursor could initiate following monomer to form polymer shell via in situ polymerization. Carbon black/polymer core-shell composite particles with 69.6 wt.% polymer shell were prepared. The encapsulated carbon black had a small particle size and high performance on dispersibility and stability. Encapsulation mechanism of this method was confirmed by analyses of TEM, UV–vis, ¹H NMR, ¹³C NMR, TGA, and other instruments.     

DNA tetrahedron-based split aptamer probes for reliable imaging of ATP in living cells

Accurate detection and imaging of adenosine triphosphate(ATP) expression levels in living cells is of great value for understanding cell metabolism, physiological activities, and pathologic mechanisms. Here, we developed a DNA tetrahedron-based split aptamer probe(TD probe) for ratiometric fluorescence imaging of ATP in living cells. The TD probe is constructed by hybridizing two split ATP aptamer probes(Apt-a and Apt-b) to a DNA tetrahedron assembled by four DNA oligonucleotides(T1, T2, T3 and ...     

Reduction of tellurite and deesterification of fluorescein diacetate are not well correlated with the viability of mycobacteria

Both Mycobacterium leprae and M. lepraemurium (MLM) were capable of reducing tellurium as tellurite ion (Te⁴⁺) to elemental tellurium (Te), seen by electron microscopy as fine crystals within the bacterial cells. There appeared to be close correspondence between the capacity to reduce tellurite, bright green fluorescence after staining with fluorescein diacetate (FDA) and the ability of M. smegmatis to multiply in culture. Likewise, there appeared to be correspondence between tellurite reduction and fluorescence after FDA staining for MLM subjected to prolonged storage in the cold or to heating at 70°C. However, correspondence with tellurite-reduction or fluorescence after FDA staining was not observed when death of MLM occured     

基于苯并吡喃腈-香豆素体系的近红外比率型荧光探针用于检测He La细胞中的过氧亚硝酸盐(英文)

过氧亚硝酸根(ONOO-)是生物体内的一种重要的活性氧,与人体内的各种生理活动息息相关.利用苯并吡喃腈-香豆素体系,合成了一种用于ONOO-检测的近红外比率型荧光探针(E)-7-(二乙胺基)-3-(2-(苯并吡喃腈)乙烯基)-香豆素(DCCM).该探针在ONOO-存在下表现出强烈的响应,颜色从紫色变为浅粉色,最大发射峰蓝移217nm,荧光颜色从淡紫色变为蓝色,能够直观地对溶液中的ONOO-进行监测. DCCM可以灵敏地检测ONOO^-,最低检测限为6.0×10^-7 mol·L^-1,该探针被成功用于HeLa细胞中的ONOO-成像检测.