Multicolor Fluorescence Research Articles

Multi-Step Assembly of an RNA-Liposome Nanoparticle Formulation Revealed by Real-Time, Single-Particle Quantitative Imaging.

Self-assembly plays a critical role in nanoparticle-based applications. However, it remains challenging to monitor the self-assembly of multi-component nanomaterials at a single-particle level, in real-time, with high throughput, and in a model-independent manner. Here, multi-color fluorescence microscopy is applied to track the assembly of both liposomes and mRNA simultaneously in clinical mRNA-based cancer immunotherapy. Imaging reveals that the assembly occurs in discrete steps: initially, RNA adsorbs onto the liposomes; then, the RNA-coated liposomes cluster into heterogeneous structures ranging from sub-micrometer to tens of micrometers. The clustering process is consistent with a Smoluchowski model with a Brownian diffusion kernel. The transition between the two steps of assembly is determined by the orientation of RNA-mediated interactions. Given the facile application of this approach and the ubiquity of the components studied, the imaging and analysis in this work are readily applied to monitor multi-component assembly of diverse nanomaterials.

Chaperone-Mediated Heterotypic Phase Separation Prevents the Amyloid Formation of the Pathological Y145Stop Prion Protein Variant.

Biomolecular condensates formed via phase separation of proteins and nucleic acids are crucial for the spatiotemporal regulation of a diverse array of essential cellular functions and the maintenance of cellular homeostasis. However, aberrant liquid-to-solid phase transitions of such condensates are associated with several fatal human diseases. Such dynamic membraneless compartments can contain a range of molecular chaperones that can regulate the phase behavior of proteins involved in the formation of these biological condensates. Here, we show that a heat shock protein 40 (Hsp40), Ydj1, exhibits a holdase activity by potentiating the phase separation of a disease-associated stop codon mutant of the prion protein (Y145Stop) either by recruitment into Y145Stop condensates or via Y145Stop-Ydj1 two-component heterotypic phase separation that arrests the conformational conversion of Y145Stop into amyloid fibrils. Utilizing site-directed mutagenesis, multicolor fluorescence imaging, single-droplet steady-state and picosecond time-resolved fluorescence anisotropy, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy, we delineate the complex network of interactions that govern the heterotypic phase separation of Y145Stop and Ydj1. We also show that the properties of such heterotypic condensates can further be tuned by RNA that promotes the formation of multicomponent multiphasic protein-RNA condensates. Our vibrational Raman spectroscopy results in conjunction with atomic force microscopy imaging reveal that Ydj1 effectively redirects the self-assembly of Y145Stop towards a dynamically-arrested non-amyloidogenic pathway, preventing the formation of typical amyloid fibrils. Our findings underscore the importance of chaperone-mediated heterotypic phase separation in regulating aberrant phase transitions and amyloid formation associated with a wide range of deadly neurodegenerative diseases.

Multicolor solid-state fluorescence and multicolor afterglow carbon dots: preparation, luminescence regulation, and applications

We review multicolor solid-state fluorescence and multicolor afterglow carbon dots (CDs) in perspectives of synthesis methods, luminescence modulation mechanisms, and applications.

Determining ATG2 Localization During Autophagosome Formation in Mammalian Cells.

This chapter describes two imaging-based approaches for examining the localization of bridge-like lipid transfer proteins at membrane contact sites during native biological processes. These approaches use multi-color fluorescence imaging, enabling high spatial and temporal resolution and overcoming the limitations of biochemical methods. The first approach involves immunofluorescence in fixed cells, while the second utilizes time-lapse imaging in live cells. These methods are showcased through the example of ATG2, an essential autophagy-related protein, and demonstrate the ability to overcome technical difficulties such as large protein size, lack of high-quality antibodies, and imaging highly dynamic subcellular structures. These described methods provide a powerful tool for understanding protein function and biological processes and can be widely applied to various research questions in cell biology.

Characterization of a Natural Accession of Elymus sibiricus with In Situ Hybridization and Agronomic Evaluation.

Elymus sibiricus, valued for its perennial nature, broad adaptability, strong cold tolerance, and high economic value in forage production, plays a crucial role in combating grassland degradation, desertification, and salinization. Using morphological and cytogenetic methods, this study evaluated the cold tolerance, post-harvest regeneration capacity, and perennial characteristics of the E. sibiricus accession 20HSC-Z9 in the Harbin region of China from 2020 to 2023. This accession exhibited a germination rate of over 90% and a 100% green-up rate, with purple coleoptiles indicating its strong cold tolerance. Over the three growing seasons, 20HSC-Z9 maintained stable green-up and regeneration rates, confirming its perennial nature. Morphologically, 20HSC-Z9 had an average tiller count ranging from 56 to 74, similar to that of the control accession 20HSC-ES, and its plant height was significantly lower than that of 20HSC-IWG. Furthermore, 20HSC-Z9 produced over 100 grains per spike, with a seed setting rate exceeding 90%, and a thousand-grain weight comparable to that of 20HSC-IWG. The grain protein content of 20HSC-Z9 reached a maximum of 21.19%, greater than that of the control accessions (15.6% and 18.5%). Chromosome composition analysis, using sequential multicolor genomic in situ hybridization and multicolor fluorescence in situ hybridization, confirmed the StStHH genomic constitution of 20HSC-Z9 and revealed translocations between the St and H subgenome chromosomes. These results suggest that 20HSC-Z9 has significant potential as a new perennial forage grass germplasm for cold regions, suitable for further domestication and breeding efforts.

Genetic Characteristics of the Rat Fibroblast Cell Line Rat-1.

The Rat-1 cell line was established as a subclone of the parental rat fibroblastoid line F2408, derived from Fisher 344 rat embryos. Rat-1 cells are widely used in various research fields, especially in cancer biology, to study the effects of oncogenes on cell proliferation. They are also crucial for investigating signal transduction pathways and play a key role in drug testing and pharmacological studies due to their rapid proliferation. Therefore, Rat-1 cells are an indispensable research tool. While some cytogenetic information on their basic chromosomal features is available, detailed genomic analyses, such as karyotype analysis, short tandem repeat (STR) profiling, and whole-genome sequencing, have not been thoroughly conducted. As a result, the genetic stability and potential variations in Rat-1 cells over extended culture periods are poorly understood. This lack of comprehensive genetic characterization can limit the interpretation of experimental results and requires caution when generalizing findings from studies using this cell line. In this study, we describe the genetic characterization of the Rat-1 cell line. We established a karyotype, performed multicolor fluorescence in situ hybridization (mFISH), identified chromosomal losses and gains, and defined an STR profile for Rat-1 with 31 species-specific markers. Interestingly, the chromosomal imbalances found in Rat-1 cells resemble those found in human epithelioid sarcoma or liposarcoma. Additionally, we analyzed the transcriptome of Rat-1 cells through mRNA sequencing (mRNA-Seq) using next-generation sequencing (NGS). Finally, typical features of these fibroblastic cells were determined using electron microscopy, Western blotting, and fluorescent phalloidin conjugates.

Efficacy of multi-color near-infrared fluorescence with indocyanine green: A new imaging strategy and its early experience in laparoscopic cholecystectomy.

Near-infrared fluorescence imaging via using intravenous indocyanine green (ICG) has a wide range of applications in multiple surgical scenarios. In laparoscopic cholecystectomy (LC), it facilitates intraoperative identification of the biliary system and reduces the risk of bile duct injury. However, the usual single color fluorescence imaging (SCFI) has limitations in manifesting the fluorescence signal of the target structure when its intensity is relatively low. Moreover, surgeons often experience visual fatigue. We hypothesized that a novel imaging strategy, named multi-color fluorescence imaging (MCFI), could potentially address these issues by decreasing hepatic and background fluorescence pollution and improving biliary visualization. To investigate the novel imaging strategy MCFI in LC. This was a single-center retrospective study conducted at Peking Union Medical College Hospital, Beijing, China. Patients who underwent LC from June 2022 to March 2023 by the same surgical team were enrolled. Demographic features, clinical and surgical information were collected. The clarity, visual comfort, and effectiveness of different imaging strategies were subjectively evaluated by surgeons. A total of 155 patients were included, 60 patients were in the non-ICG group in which only bright light illuminance without ICG was applied, 60 patients were in the SCFI group, and 35 patients were in the MCFI group. No statistically significant differences were found in demographics or clinical history. Post-surgical complications were minimal in all 3 groups with no significant differences observed. MCFI improved the clarity of imaging and visual comfort. Clarity of imaging and visual comfort were improved with MCFI. MCFI improves biliary visualization and reduces liver fluorescence contamination, which supports its routine use in LC. MCFI may also be a better choice than SCFI in other clinical settings.

General and Versatile Nanoarchitectonics for Amino Acid-Based Glasses via Co-Assembly of Organic Counterions.

Amino acid-based biomolecular glasses represent an emerging material to meet the demand for sustainable development. However, most amino acids are difficult to vitrify due to their strong crystallization tendency, limiting further advancements of this field. In this study, we demonstrate that the introduction of counterions effectively suppresses crystallization, as hydrogen bonds within the system stabilize the disordered structures. Based on this, we propose a counterion co-assembly strategy to synthesize a wide range of amino acid-based glasses. This strategy enables the facile fabrication of glass with customizable shapes, high mechanical rigidity, and tunable multicolor fluorescence, ranging from blue to red depending on the excitation wavelength. Furthermore, this strategy allows the integration and enhancement of counterion properties within the glass matrix. Through the co-assembly of phosphorescent counterions, we synthesized a series of long-persistent luminescent glasses with significantly extended afterglow lifetimes. This work presents an effective approach for the synthesis of amino acid-based glasses and provides insights into the development of supramolecular glasses with tailored functionalities.

General and Versatile Nanoarchitectonics for Amino Acid‐Based Glasses via Co‐Assembly of Organic Counterions

Amino acid‐based biomolecular glasses represent an emerging material to meet the demand for sustainable development. However, most amino acids are difficult to vitrify due to their strong crystallization tendency, limiting further advancements of this field. In this study, we demonstrate that the introduction of counterions effectively suppresses crystallization, as hydrogen bonds within the system stabilize the disordered structures. Based on this, we propose a counterion co‐assembly strategy to synthesize a wide range of amino acid‐based glasses. This strategy enables the facile fabrication of glass with customizable shapes, high mechanical rigidity, and tunable multicolor fluorescence, ranging from blue to red depending on the excitation wavelength. Furthermore, this strategy allows the integration and enhancement of counterion properties within the glass matrix. Through the co‐assembly of phosphorescent counterions, we synthesized a series of long‐persistent luminescent glasses with significantly extended afterglow lifetimes. This work presents an effective approach for the synthesis of amino acid‐based glasses and provides insights into the development of supramolecular glasses with tailored functionalities.

Eu‐Doped Polyurethane with Efficient Multicolor Fluorescence, Self‐Healing, Stimuli‐Responsiveness and Its Diverse Applications

AbstractMulticolor lanthanide‐doped polymers have found versatile applications in data encryption, sensing, and smart display. However, multiple components, sophisticated design, and complex preparation procedures are often required. In contrast, their intrinsic and multicolor fluorescence emission is been effectively used. Herein, Eu‐doped polyurethane (ETP@PU), integrating tunable multicolor emission, self‐healing, and multi‐responsiveness, is prepared through a facile route via step‐growth polymerization of PU using commercial Eu‐ligand (Eu(TTA)3Phen, ETP) as a fluorophore. FTIR and XPS analysis demonstrate the successful coordination of Eu3+ with carbamate groups of PU, which not only enhances the mechanical properties of PU but also bestows unique luminescent properties. With increasing ETP content, the intrinsic fluorescence of PU declines, while that of Eu displays a discernible enhancement, resulting in the tunable multicolor emission. The highest quantum yield of Eu‐related emission reaches 71.3% due to the robust coordination and efficient energy transfer between Eu3+ and PU. Moreover, the ETP@PU exhibits excellent self‐healing capacity and multi‐responsiveness, and their potential applications are also investigated in the field of light‐emitting diodes, data encryption, and self‐healing process monitoring. This work provides a facile and efficient strategy to develop multicolor lanthanide‐doped polymers promising for various applications.

Evolution of Supramolecular Coordination Assemblies Visually Monitored by Time‐Dependent Multicolor Fluorescence

AbstractSupramolecular coordination assemblies play an important role in both material science and biological systems. Despite significant efforts in their development, most existing examples are thermodynamically controlled, which lack the adaptability and autonomy essential for the creation of advanced materials. In this work, we have developed non‐equilibrium coordination assembly systems that can evolve over time through a combination of thermodynamic and kinetic controls. The introduction of zinc ions resulted in the formation of metastable fiber assemblies in a kinetically trapped state, which autonomously converted to thermodynamically stable nanosheets over time. This evolution process can be regulated by external stimuli and visually monitored by the time‐dependent multicolor fluorescence. The construction strategy was versatile across other various ions such as Ca2+, Mg2+, and Al3+, offering inspiring insights for the design of complex systems that operated both in and out of their thermodynamic equilibrium.

Evolution of Supramolecular Coordination Assemblies Visually Monitored by Time-Dependent Multicolor Fluorescence.

Supramolecular coordination assemblies play an important role in both material science and biological systems. Despite significant efforts in their development, most existing examples are thermodynamically controlled, which lack the adaptability and autonomy essential for the creation of advanced materials. In this work, we have developed non-equilibrium coordination assembly systems that can evolve over time through a combination of thermodynamic and kinetic controls. The introduction of zinc ions resulted in the formation of metastable fiber assemblies in a kinetically trapped state, which autonomously converted to thermodynamically stable nanosheets over time. This evolution process can be regulated by external stimuli and visually monitored by the time-dependent multicolor fluorescence. The construction strategy was versatile across other various ions such as Ca2+, Mg2+, and Al3+, offering inspiring insights for the design of complex systems that operated both in and out of their thermodynamic equilibrium.

Multicamera simultaneous total internal reflection and interference reflection microscopy.

Interference Reflection Microscopy (IRM) is an optical technique that relies on the interference between the reflected light from an incident beam as it passes through materials of different refractive indices. This technique has been successfully used to image microtubules, biologically important biofilaments with a diameter of 25nm. However, it is often desirable to image both the microtubule and microtubule interacting proteins simultaneously. Here we present a simple modification to a standard multicolour total internal reflection fluorescence (TIRF) microscope that enables simultaneous high-speed IRM and single molecule TIRF imaging. Our design utilises a camera for each channel (IRM and TIRF) allowing independent optimisation of camera parameters for the two different modalities. We illustrate its application by imaging unlabelled microtubules and GFP-labelled end-binding protein EB1, which forms comets on the tips of polymerising microtubules. Our design is easily implemented, and with minimal cost, making it accessible to any laboratory with an existing fluorescence microscope.

Integration of Eu-based metal-organic frameworks and carbon dots for multicolor visual intelligent detection of phosphate

Phosphate (Pi) has an important influence on the water environment and physiological processes. Therefore, developing fluorescent probe for quantitative detection of Pi is crucial for water environment monitoring and human health assessment. This work designed a dual-emission ratio nano-fluorescent probe GCDs/Eu-BDC based on europium-based metal-organic frameworks (Eu-MOFs) and blue carbon dots (GCDs) for multicolor fluorescence detection of Pi. The GCDs/Eu-BDC realized multicolor fluorescence detection of Pi based on the red-to-blue fluorescence change. The probe has high selectivity and a detection limit of 70 nM in the range of 0–45 μM. GCDs/Eu-BDC can be used to detect Pi in environmental water samples and serum samples, proving the feasibility of quantitative analysis of Pi in real samples. In addition, a portable paper-based sensor was prepared in this work. Combined with the chromaticity analysis App in smartphones, the intelligent real-time detection of Pi can be realized, which has certain practical application potential.

Traffic signal-inspired fluorescence lateral flow immunoassay utilizing self-assembled AIENP@Ni/EC for simultaneous multi-pesticide residue detection

The presence of multiple pesticide residues in fruits and vegetables poses a serious threat to human health. Conventional gold nanoparticles (AuNPs) used in multiplex lateral flow immunoassays (M−LFIA) often exhibit poor sensitivity and potential for misidentification. This study introduces a traffic signal-inspired fluorescent LFIA (T-FLFIA) designed for the simultaneous quantitative detection of chlorothalonil (CTN), paclobutrazol (PBZ), and fipronil (FIP) in cowpea and apple samples. Utilizing the hydrophobic interactions of aggregation-induced emission luminogens (AIEgens) and the coordination of epicatechin (EC) with Ni2+, we developed a novel nanostructure-aggregation-induced emission nanoparticle (AIENP) coated with a metal-polyphenol network of Ni/EC (AIENP@Ni/EC) via a self-assembly method and employed it as fluorescence probe in T-FLFIA. Three distinct colors of AIENP@Ni/EC featuring green, yellow, and red fluorescence were successfully synthesized by coating various AIEgens. This multicolor fluorescence capability facilitates more precise and expedited discrimination among multiple detection targets. High-performance AIE fluorescence probes were fabricated by directly coupling AIENP@Ni/EC with different antibodies through electrostatic interactions, leveraging the exceptional biocompatibility of Ni/EC. The T-FLFIA exhibited sensitive detection of CTN, PBZ, and FIP at concentrations as low as 0.038 ng/mL, 0.025 ng/mL, and 0.046 ng/mL, respectively, with high qualitative accuracy (92.5 %) within a rapid timeframe (5 s). This exceptional performance can be attributed to its outstanding optical properties and distinguishable colors. This approach demonstrated enhanced sensitivity and accuracy, reduced consumption of antibodies and immunoprobes, and a broader detection range compared to single-color AuNPs-M−LFIA. This study introduces a novel, practical, and cost-effective tool for rapid screening of diverse pesticide residues in field settings.

Development and characterization of a novel wheat-rye T2DS·2DL-2RL translocation line with high stripe rust resistance

Rye (Secale cereale L.), a close relative of common wheat, represents a valuable genetic resource for enhancing the disease resistance of common wheat. Introducing novel rye-derived genes into wheat can potentially improve disease resistance. In this study, we successfully developed a novel wheat-rye derivative line LCR4 through hybridization between hexaploid triticale line Currency and common wheat cultivar Jimai 22 (JM22). We confirmed that LCR4 was a T2DS·2DL-2RL translocation line via comprehensive molecular cytogenetic analyses, including genomic in situ hybridization, multi-color fluorescence in situ hybridization, molecular marker analysis, and wheat SNP-arrays genotyping. Notably, upon inoculation with Puccinia striiformis f. sp. tritici (Pst) race V26 at the seedling stage and mixed Pst races at the adult stage, LCR4 exhibited robust resistance against stripe rust infection at both stages. Subsequent genetic analysis further elucidated that the translocated 2RL chromosome segment is responsible for this resistance. Consequently, LCR4 harboring elite agronomic traits can be effectively employed in breeding programs against stripe rust.

Solid-state solvatochromism of oxazolidine in multi-functionalized copolymer nanoparticles: Development of advanced materials with multi-color fluorescence

Investigating the solvatochromism of stimuli-chromic compounds such as oxazolidine in polymer matrices with various polarities or functionalities is a unique approach to developing advanced materials for anticounterfeiting, sensor, optoelectronic, and displayers. In a novel strategy, multi-functionalized copolymer nanoparticles were synthesized by copolymerization of methyl methacrylate (MMA) with various functional comonomers in emulsion media for post-polymerization modification with oxazolidine and investigation of its solvatochromism in both colloidal solution and solid phase. The particle size and morphology of nanoparticles were influenced significantly by the polarity of functional groups, and the morphology evolution from sphere to anisotropic shapes was observed by increasing the polarity of functional groups. Investigation of oxazolidine solvatochromism in colloidal solution and solid polymer powders indicated different mechanisms for solvatochromism, in which the polarity of media is the main effective parameter in colloidal solution and the polarity of functional groups in the polymer structure is the main effective parameter in solid polymer phase. The solvatochromism of oxazolidine was confirmed by observed red shift and blue shift in UV–Vis and fluorescence spectra, in which the intensity of absorbance and emission peaks was changed as a function of the polarity of functional groups. Solvatochromic photoluminescent polymer nanoparticles were used for several advanced applications such as solid anticounterfeiting inks to information encryption, dual-mode visualization of latent fingerprints, and also development of organic light-emitting diodes (OLEDs). For the first time, the results indicate a significant effect of polymer chain local polarity induced by functional groups on the tuning of optical properties of the oxazolidine by the solid-state solvatochromic phenomenon. Solvatochromism of oxazolidine in functionalized polymer nanoparticles is an interesting approach to developing novel intelligent materials that have advanced applications in different fields such as anticounterfeiting and information encryption, optoelectronic, polarity sensor, and visualization of latent fingerprints.

Cr3+-Activated Deep Trap Electron-Trapping Biphasic Mixture of Zn (Al,Ga)2O4 and Zn2GeO4 for Multilevel Information Storage by Trap Multiplexing and Wavelength Multiplexing.

Double wavelength emitting phosphors with hypersensitive thermal response are important for optical/thermal sensors and multiplexing optical data storage technology. However, it is a daunting challenge to develop multicolor electron-trapping materials with superheat sensitivity and anomalous quenching luminescence characteristics for information storage. Here, based on the selection of host lattice and the control of doping ion types, we have designed Mn2+- and/or Cr3+-activated persistent materials with excellent fluorescence properties. Among these phosphors, Mn,Cr co-activated biphasic mixture exhibits not only sensitive anomalous thermosensitive fluorescence properties but also multicolor and multimode fluorescence emission characteristics derived from Mn and Cr simultaneously. The corresponding multimode fluorescence mechanism is proposed based on afterglow energy transfer and deepening trap effect. Especially, based on the excellent fluorescence performances, these phosphors as information storage medium demonstrate multilevel information storage by a combination of the trap multiplexing and wavelength multiplexing technologies. The study here provides not only the ideas for designing and synthesizing new electronic materials but also a material foundation for the fourth generation of optical information storage and encryption.

Development and Characterization of Two Wheat-Rye Introgression Lines with Resistance to Stripe Rust and Powdery Mildew.

Rye (Secale cereale L.) genes, which contribute to the tertiary gene pool of wheat, include multiple disease resistance genes useful for the genetic improvement of wheat. Introgression lines are the most valuable materials for wheat breeding because of their small alien segments and limited or lack of linkage drag. In the present study, wheat-rye derivative lines SN21627-2 and SN21627-6 were produced via distant hybridization. A genomic in situ hybridization analysis revealed that SN21627-2 and SN21627-6 lack alien segments, while a multi-color fluorescence in situ hybridization analysis detected structural changes in both introgression lines. At the seedling and adult plant stages, SN21627-2 and SN21627-6 were highly resistant to stripe rust and powdery mildew. Primers for 86 PCR-based landmark unique gene markers and 345 rye-specific SLAF markers were used to amplify SN21627-2 and SN21627-6 genomic DNA. Eight markers specific to rye chromosome 2R were detected in both introgression lines, implying these lines carry chromosome 2R segments with genes conferring stripe rust and powdery mildew resistance. Therefore, SN21627-2 and SN21627-6 are resistant to more than one major wheat disease, making them promising bridging parents for breeding disease-resistant wheat lines.

Wet-spinning fabrication of stretchable multicolor fluorescent fibers augmented with dual-state emissive dyes

Flexible luminous fibers have attracted wide attention in the field of functional and smart textiles. In this work, stretchable multicolor fluorescent fibers were successfully prepared by wet-spinning using thermoplastic polyurethane (TPU) and the boron ketoimine fluorophores (TPA-BKI dyes). Due to the donor (D)-acceptor (A) push-pull electronic system and highly twisted triphenylamine (TPA) skeleton, TPA-BKI dyes exhibited bright emission in both solution and solid states. Meanwhile, the polymer chains further improved the emission efficiency of TPA-BKI dyes in TPU matrix by avoiding close contact between dye molecules, as well as preventing intramolecular rotations in the excited states. Wet-spinning can be used for large-scale and continuous preparation of fluorescent fibers. The TPA-BKI/TPU fibers showed excellent flexibility, high mechanical strength, and bright multicolor fluorescent emission (green, yellow, and red, respectively) when the dye content was as low as 0.1 wt%. Overall, the feasibility of large-scale preparation of fluorescent fibers and their excellent properties, such as flexibility, multicolor fluorescence, and mechanical stability, make them suitable for potential applications, including clothing displays, fashion designs, security, and anti-counterfeiting.