Fluorescence Pattern Research Articles

Nuclear mTORC1 Live-Cell Sensor nTORSEL Reports Differential Nuclear mTORC1 Activity in Cell Lines

The mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is activated on the surface of lysosomes and phosphorylates substrates at various subcellular locations, including the lysosome, cytosol, and nucleus. However, the signaling and biological functions of nuclear mTORC1 (nmTORC1) are not well understood, primarily due to limited tools for monitoring mTORC1 activity in the nucleus. In this study, we developed a genetically encoded nmTORC1 sensor, termed nTORSEL, based on the phosphorylation of the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4EBP1) by mTORC1 within the nucleus. nTORSEL, like its predecessor TORSEL, exhibits a fluorescent punctate pattern in the nucleus through multivalent protein–protein interactions between oligomerized 4EBP1 and eIF4E when nmTORC1 activity is low. We validated nTORSEL using biochemical analyses and imaging techniques across representative cell lines with varying levels of nmTORC1 activity. Notably, nTORSEL specifically detects physiological, pharmacological, and genetic inhibition of nmTORC1 in mouse embryonic fibroblast (MEF) cells but not in HEK293T cells. Therefore, nTORSEL is an effective tool for investigating nuclear mTORC1 signaling in cell lines.

Luminescence Patterns on Multimillimeter Single-Crystal MAPbBr3 Microplatelets via Thermal Ablation Effects.

The single crystal distinguishes itself as the most outstanding representative of excellent optoelectronic performance among perovskite semiconductors due to its exceptional charge carrier properties, extraordinary optophysics, precise structural geometries, and superior material stabilities. However, it exhibits mediocre performance in light emission, which can be attributed to its excessive carrier diffusion lengths and extremely low recombination rates. To address this challenge, this study puts forward a controllable high-temperature annealing treatment strategy concentrating on multimillimeter-scale MAPbBr3 micrometer-thick platelet single crystals (MPSCs). Through the utilization of thermal ablation effects to convert the surface monocrystalline lattices into polycrystalline perovskite nanocrystals (PNCs) with a remarkably increased specific surface area, the fluorescence intensity arising from the rapid recombination of free carriers on the PNC surface is enhanced by 320 times compared to the pristine MPSC surface. These PNCs produced through surface annealing, with the characteristic of loose adhesion to surfaces, can be mechanically wiped away and regenerated in situ via reannealing the residual MAPbBr3 MPSCs, guaranteeing high intensity, durability, and standard geometric fluorescence. Moreover, by regulating the surface distributions of thermal ablation effects and carrying out embossing annealing treatment or laser direct writing, we can design and fabricate relatively complex, high-contrast (255×), and clearly resolved fluorescence patterns on MPSC surfaces.

Differentiating the Aβ42 aggregation states via intrinsic tyrosine fluorescence spectrum

Aggregation of amyloid β-peptide (Aβ) into β-sheet-rich fibrils is central to the development of Alzheimer’s disease, with Aβ42 more prone to aggregation over Aβ40. Using the intrinsic tyrosine fluorescence spectrum, we show that Aβ42 exhibits a biphasic fluorescence pattern featuring a broad band and a narrow one, distinct from Aβ40 and dissolved tyrosine. Molecular dynamics simulations highlighted the differences in tyrosine’s rotamer populations and dynamics between dissolved and aggregated amyloids. Fibrillar Aβ42 shows slower, more uniform tyrosine rotations, corresponding to the narrower fluorescence band. This approach offers a rapid means to differentiate Aβ42 aggregates, benefiting Aβ-related research.

Multilevel Stimuli-Responsive Smart "Sandwich" Label with Physical Unclonable Functions Bionic Wrinkles and Space-Selective Fluorescence Patterns.

With the increasing popularity of the internet, it brings convenience to lives while also increases security risks. Physical Unclonable Functions (PUFs) can generate random, unclonable, and unique identifiers using their inherent physical characteristics, which have broad prospects in anti-counterfeiting. Herein, inspired by the irregular tree bark fissures and random skin wrinkles found in nature, a method for creating complex micro-wrinkles with unclonable random patterns is proposed by simply stretching hydrogels. The random texture information contained in the micro-wrinkles is digitized into binary codes using an adaptive threshold algorithm. Additionally, a novel "sandwich" label with a multilevel intelligent anti-counterfeiting system is proposed. The first-level involves photoluminescence encryption with adjustable luminescence within visible light range and modulated luminescence at different excitation wavelengths; the second-level includes strain-related mechanical encryption, and the third-level consists of highly random and unclonable micro-wrinkles. The certification difficulty increases as the anti-counterfeiting grade increases, thereby enhancing label security. Furthermore, space-selective doping of rare earth metal-organic framework (RE-MOF) fluorescent materials in hydrogels is achieved through the use of screen-printing technology. The concept of novel multilevel smart anti-counterfeiting PUF labels will further enhance current levels of counterfeiting prevention.

Boosting FRET Efficiency of Chromophores with Aggregation-Caused Quenching by a Crystallization-Induced Precise Co-assembly Strategy.

Förster resonance energy transfer (FRET) plays a critical role in organic optoelectronic materials. However, developing facile and effective strategies to achieve high-efficiency energy harvesting of chromophores with aggregation-caused quenching (ACQ) remains an appealing yet challenging task, that has not yet been explored. Herein, a subtly strategy, crystallization-induced precise co-assembly (CIPCA) involving a molecular "lightening agent," to effectively improve FRET efficiency of ACQ chromophores is developed. Bis(phenylethynyl)anthracene (BPA) and bis(phenylethynyl)naphthacene (BPN) with significant ACQ effect are chosen as representative FRET donor and acceptor, respectively, and weakly-fluorescent octafluoronaphthalene (OFN) acted as the "lightening agent." Thanks to precise co-assembly with OFN, the PLQY of solid BPA is enhanced by 107%, and the BPN powder can be unprecedentedly lighted. More importantly, through such powerful CIPCA, the monotonous and weak emission for BPA@BPN can be remarkably regulated to colorful and much brighter ones with FRET efficiency improvement of as high as 180-270%. An in-depth understanding of FRET regulation is elucidated through a precise correlation of the supramolecular structures and properties. Such achievements allow to successfully fabricate distinct multi-stimuli-responsive fluorescent patterns and highly-emissive colorful flowers with high flexibility. This research provides an efficient strategy to improve the FRET efficiency of ACQ pairs.

Influence of Carbon-Nitride Dot-Emitting Species and Evolution on Fluorescence-Based Sensing and Differentiation.

Carbon dots have attracted widespread interest for sensing applications based on their low cost, ease of synthesis, and robust optical properties. We investigate structure-function evolution on multiemitter fluorescence patterns for model carbon-nitride dots (CNDs) and their implications on trace-level sensing. Hydrothermally synthesized CNDs with different reaction times were used to determine how specific functionalities and their corresponding fluorescence signatures respond upon the addition of trace-level analytes. Archetype explosives molecules were chosen as a testbed due to similarities in substituent groups or inductive properties (i.e., electron withdrawing), and solution-based assays were performed using ratiometric fluorescence excitation-emission mapping (EEM). Analyte-specific quenching and enhancement responses were observed in EEM landscapes that varied with the CND reaction time. We then used self-organizing map models to examine EEM feature clustering with specific analytes. The results reveal that interactions between carbon-nitride frameworks and molecular-like species dictate response characteristics that may be harnessed to tailor sensor development for specific applications.

Valorization of nopal wastes to produce quantum dots: optimizing synthesis and exploring in smart textile applications

Quantum carbon dots (QCDs) were efficiently synthesized from post-extraction residues generated during nopal fabric production using a hydrothermal treatment. These QCDs were applied to nopal fabrics, enhancing their UV solar radiation absorption. The synthesized QCDs exhibited fluorescence emissions in the 200–300 nm range. An eco-friendly dispersion was created by incorporating QCDs into TiO2 for use in smart textiles, which underlines our commitment to maintaining a sustainable process. Bright and fluorescent patterns were successfully applied to commercial and nopal fabrics using a spray printing technique. Additionally, the QCDs demonstrated pH-sensitive color changes, paving the way for practical applications. This work represents an initial step towards a circular economy by utilizing residues from nopal fabric production to synthesize quantum dots, which may be employed in smart textiles applications with UV absorption capabilities.

Interference patterns of two-photon excited fluorescence by spatial beam shaping

We report on two-photon excited fluorescence interference patterns produced by spatial laser beam shaping. Thereto, a focused spatially modulated laser beam component is overlapped with an unfocused Gaussian beam component, and both are directed on a cuvette filled with dye. The phase retardance between the two common path laser components is precisely adjusted by a 2D liquid crystal modulator. With this method it is possible to generate various fluorescence patterns which exhibit sharp features and a high contrast due to constructive and destructive interference of a two-photon excitation process. The developed spatial shaping interference technique with two-photon excitations has a high potential for optical and biophotonic imaging applications.

Genetically encoded epigenetic sensors for visualization of H3K9me3, H3K9ac and H3K4me1 histone modifications in living cells

Post-translational modifications of histones play a crucial role in chromatin structure maintenance and epigenetic regulation. The LiveMIEL (Live-cell Microscopic Imaging of Epigenetic Landscape) method represents a promising approach for tracking histone modifications. It involves visualization of epigenetic modifications using genetically encoded fluorescent sensors and further analysis of the obtained intranuclear patterns by multiparametric image analysis. In this study, we designed three new red fluorescent sensors—MPP8-Red, AF9-Red and DPF3-Red—for live-cell visualization of patterns of H3K9me3, H3K8ac and H3K4me1, respectively. The observed fluorescent patterns were visually distinguishable, and LiveMIEL analysis clearly classified them into three corresponding groups. We propose that these sensors can be used for live-cell dynamic analysis of changes in organization of three epigenetic types of chromatin.

The tyrosine kinase inhibitor Nintedanib induces lysosomal dysfunctionality: Role of protonation-dependent crystallization processes

Nintedanib (NIN), a multi-tyrosine kinase inhibitor clinically approved for idiopathic pulmonary fibrosis and lung cancer, is characterized by protonation-dependent lysosomotropic behavior and appearance of lysosome-specific fluorescence emission properties. Here we investigate whether spontaneous formation of a so far unknown NIN matter within the acidic cell compartment is underlying these unexpected emissive properties and investigate the consequences on lysosome functionality. Lysosomes of cells treated with NIN, but not non-protonatable NIN derivatives, exhibited lysosome-associated birefringence signals co-localizing with the NIN-derived fluorescence emission. Sensitivity of both parameters towards vATPase inhibitors confirmed pH-dependent, spontaneous adoption of novel crystalline NIN structures in lysosomes. Accordingly, NIN crystallization from buffer solutions resulted in formation of multiple crystal polymorphs with pH-dependent fluorescence properties. Cell-free crystals grown at lysosomal-like pH conditions resembled NIN-treated cell lysosomes concerning fluorescence pattern, photobleaching dynamics, and Raman spectra. However, differences in birefringence intensity and FAIM-determined anisotropy, as well as predominant association with (intra)lysosomal membrane structures, suggested formation of a semi-solid NIN crystalline matter in acidic lysosomes. Despite comparable target kinase inhibition, NIN, but not its non-protonatable derivatives, impaired lysosomal functionality, mediated massive cell vacuolization, enhanced autophagy, deregulated lipid metabolism, and induced atypical phospholipidosis. Moreover, NIN exerted distinct phototoxicity, strictly dependent on lysosomal microcrystallization events. The spontaneous formation of NIN crystalline structures was also observable in the gut mucosa of orally NIN-treated mice. Summarizing, the here-described kinase inhibition-independent impact of NIN on lysosomal functionality mediates several of its cell biological activities and might contribute to NIN adverse effects.

Enhanced electro-optical properties of polymer dispersed liquid crystals doped with functional nanofibers for applications of efficient smart windows and advanced anti-counterfeiting

The development of polymer-dispersed liquid crystal (PDLC) capable of infrared smart tuning and displaying distinct information under ultraviolet, visible, and infrared light presents significant challenges. These challenges include the tuning of electro-optical properties and IR reflectivity, the preparation of complex patterns, and the display of different display states under various stimuli. In this work, nanofibers doped with alumina-zinc oxide nanoparticles (AZO NPs) and loaded with cesium tungstate nanoparticles (Cs0.33WO3 NPs) into PDLC not only enhanced their electro-optical properties but also added infrared shielding. Temperature regulation tests demonstrate that PDLC films can intelligently modulate infrared light reflection, offering superior shading and temperature regulation capabilities. PDLC smart windows integrated with nanofibers achieve a temperature coordination function, maintaining a temperature differential of 12.1 °C compared to standard PDLC. This significant enhancement underscores their potential for applications in thermally regulated smart windows. In addition, a full-wavelength anti-counterfeiting mode for PDLC was developed using a screen-printing strategy to display different information under various wavelengths of light. Noteworthily, the anti-counterfeiting system exhibits a bright fluorescent pattern under UV light, a scattering pattern under visible light, and an infrared-modulated pattern when viewed with an infrared detector. Furthermore, an advanced dynamic anti-counterfeiting mode with time response was created using nanofibers loaded with different concentrations of Cs0.33WO3 NPs. This advanced anti-counterfeiting material can be used as multimodal and dynamic anti-counterfeiting label, which greatly facilitates the development of novel anti-counterfeiting and optical sensing materials.

The Staged Photocatalytic Reactor in the Removal of Acetaminophen: Aspects of Adsorption and Photocatalysis.

The efficiency of a staged photocatalytic reactor prototype was evaluated on a semi-pilot scale with the removal of acetaminophen, for which anatase particles were synthesized by Sol-Gel and impregnated on rectangular plates of clay. X-ray diffraction and Energy Dispersive X-ray Fluorescence patterns show that the final composite is made up of Al2O3 (14 %), SiO2 (41 %), CaO (3 %) TiO2 (34 %), and Fe2O3 (7 %). The impregnation method favors the dispersion of Anatase on the surface of the adsorbent. TiO2-Anatase/Clay, classified as a macro-porous solid with H3-type hysteresis loops by N2 physisorption. Adsorption processes are improved when using TiO2-Anatase/Clay compared to using TiO2-Anatase. The external mass transfer has a greater influence on the removal rate. The dimensionless parameters of the Biot number indicate there are no limitations due to the diffusive effect on the interior of the particle. The evaluation of the kinetic data under the Langmuir-Hinshelwood equation shows a decrease in efficiency as the initial concentration increases. The acetaminophen molecule shows destabilization in the structure of the aromatic ring with a visible decrease in the signals of this functional group evaluated by High-Performance Liquid Chromatography and Raman Spectroscopy.

Induced micellization of reverse poloxamine as a supramolecular platform for saccharide differentiation

The development of methods for detection and differentiation of chemically similar saccharide molecules is a challenging task. This study describes a simple approach for saccharide differentiation based on saccharide induced micelle formation of a reverse poloxamine surfactant, T90R4. The differential response is derived from the different kosmotropic effect of each type of saccharide, which is not only dependent on the number of hydroxyl groups present in the saccharides, but is also sensitive to their stereochemistry. This results in different propensities of the saccharides for inducing micellization of T90R4, which in turn is reflected by variations in the emission spectrum of a solubilised fluorescence probe, 7-hydroxy-4-methylcoumarin (7H4MC). The effect of temperature on the emission spectrum of 7H4MC is also found to be uniquely dependent on the nature of the saccharides. These effects work in tandem to generate a supramolecular platform for fluorescence pattern based recognition of multiple saccharides.

Multi‐Mode Elastic Full‐Color Fluorescent Patterns for Multi‐Visual Encryption

AbstractThe development of multi‐mode fluorescent patterns has attracted widespread attention, particularly for applications in high‐density information storage and highly secure encryption. However, the fabrication process of multi‐mode fluorescent patterns generally involves tedious and expensive steps. Specifically, in the fabrication technology of elastic full‐color fluorescent patterns, there is still a lack of ideal patterning preparation technology that can overcome the solvent orthogonality problem associated with multi‐color fabrication while ensuring both high elasticity and luminescence efficiency of the pattern. Herein, the multi‐mode elastic fluorescent patterns are successfully fabricated by multiple transferring of the quantum dots (QDs) from the carrier substrates to the elastomeric PDMS substrate via femtosecond laser‐induced forward transfer (FsLIFT) technology. Due to its solvent‐free nature, absence of masking requirements, and no need for annealing during FsLIFT processing, the multiple transferred QD films achieve independent patterns and does not interfere with each other, providing a strong promise for the fabrication of multi‐mode elastic full‐color patterns. Based on these stretchable fluorescent patterns, multi‐visual encryption techniques are successfully demonstrated. The proposed FsLIFT technology offers an efficient and straightforward strategy for fabricating multi‐mode fluorescent patterns with potential applications in advanced commercial security information encryption and dynamic camouflage.

Programmable Aggregation of Self-Assembled DNA Constructs.

Biomolecular aggregates ensure the optimum concentration and proximity required for biochemical processes to take place. Synthetic aggregating systems are becoming increasingly essential to study/mimic dynamic condensates in nature. Herein the ratiometric DNA aggregation of self-assembled DNA constructs using lanthanide salts is reported. In addition, the aggregation is shown to be reversed by the addition of specific lanthanide-binding ligands. The aggregate formation is confirmed by dynamic light scatteringexperiment, electrophoretic mobility shift assay, and field emission scanning electron microscope. This programmed DNA aggregation and its reversion are applied to evaluating the lanthanide-DNA and lanthanide-ligand binding constants, respectively. To achieve this, Forster resonance energy transfer (FRET)pair dyes at the 3' or 5' end of the DNA strands are strategically placed that generate unique fluorescence patterns upon interaction with the DNA constructs and different triggers such as lanthanides/ligands/monovalent cations, thus enabling the tracking of various states of binding. It also demonstrates a "fast method" to form and stabilize G-quadruplex (GQ) using lanthanides which complements the existing slow formation of GQs with Na+/K+ ions. The formation of GQ by lanthanides is corroborated by FRET, circular dichroism (CD), and enzyme linked immunosorbent assay (ELISA) experiments. These DNA constructs, formed by lanthanides, have shown resistance to cleavage by DNase I, and distinctive binding to Protoporphyrin dyes and Thioflavin T.

Surface engineering and concentration-dependent emission activated flexible tunable fluorescence from lignin-based N-doped carbon dots

Achieving the flexible and tunable fluorescence of lignin-based N-doped carbon dots (LCDs) remains a win–win yet challenging strategy. In this study, combining the surface engineering and concentration regulation strategy enabled the flexible adjustment of LCDs fluorescence emission from blue to yellow regions (345 to 560 nm). The surface electron-donor –OH groups not only enhanced the photoluminescence quantum yield (PLQY) to 14.75 % but activated the spectral shift of reduced LCDs (RLCDs). These arose from the reinforced π-electron conjugation and generated new –OH-related extrinsic defects with elevating concentration. Conversely, the electron-withdrawing CO groups participated in the blue-to-green fluorescence modulation. Still, they drastically reduced the PLQY of oxidized LCDs (OLCDs) to 2.13 %, due to the larger particle size and more non-radiative transfer induced by CO groups. Based on these, CDs/PVA films and printed fluorescent patterns with multicolor fluorescence were realized for anti-counterfeiting, and the RLCDs@Al2O3 hybrid facilitated the precise fluorescence fingerprint tracking and recognition. This study provided a facile strategy for enriching and developing novel LCDs with flexible fluorescence for their advanced applications.

Expanding the transgene expression toolbox of the malaria vector Anopheles stephensi.

Anopheles stephensi Liston, 1901 (Diptera: culicidae) is a competent vector of Plasmodium falciparum (Haemosporida: plasmodiidae) malaria, and its expansion in the African continent is of concern due to its viability in urban settings and resistance to insecticides. To enhance its genetic tractability, we determined the utility of a ~2 kb An. stephensi lipophorin (lp) promoter fragment in driving transgene expression. Lipophorin genes are involved in lipid transport in insects, and an orthologous promoter in An. gambiae (AGAP001826) was previously demonstrated to successfully express a transgene. In the present study, we qualitatively characterised the expression of a ZsYellow fluorescent marker protein, expressed by An. stephensi lp promoter fragment. Our study indicated that the lp promoter fragment was effective, generating a distinct expression pattern in comparison to the commonly utilised 3xP3 promoter. The lp:ZsYellow fluorescence was largely visible in early instar larvae and appeared more intense in later instar larvae, pupae and adults, becoming especially conspicuous in adult females after a blood meal. Different isolines showed some variation in expression pattern and intensity. Aside from general transgene expression, as the lp promoter produces a suitable fluorescent protein marker expression pattern, it may facilitate genotypic screening and aid the development of more complex genetic biocontrol systems, such as multi-component gene drives. This study represents an expansion of the An. stephensi genetic toolbox, an important endeavour to increase the speed of An. stephensi research and reach public health milestones in combating malaria.

Dynamic fluorescent patterns inspired by cuttlefish for anti-counterfeiting applications

Dynamic fluorescent patterns that convert external stimulation into intuitive color changes have drawn significant interest in various fields. However, the complex synthesis of materials with multiple emissions and technical barriers in the flexible fabrication of full-color fluorescent patterns hinder their applicability in diverse scenarios. Here, we present a bioinspired dynamic fluorescent pattern fabricated by femtosecond laser-induced forward transfer (FsLIFT), inspired by cuttlefish. The elastomeric substrate can contract and expand as muscle fibers of cuttlefish, and the patterned red, green, and blue quantum dot (QD) films can be regarded as pigment cells, achieving bionic preparation of function and morphology similar to cuttlefish. Notably, the fluorescent patterns can be modulated by manipulating strains and excitations. Using this bioinspired strategy, we successfully produced an advanced fluorescent anti-counterfeiting label via FsLIFT, which can only be recognized under specific strain and excitation light conditions. These dynamic fluorescent patterns show broad application potential in anti-counterfeiting, wearable devices, and information encryption.

Application of Ultraviolet-Enhanced Fluorescence Dermoscopy in Basal Cell Carcinoma.

Basal cell carcinoma (BCC) is the most common non-melanoma skin cancer. The aim of the current study was to analyze the ultraviolet-enhanced fluorescence dermoscopy (UVFD) characteristics of BCCs. BCCs were evaluated under polarized dermoscopy (PD) and UVFD. The findings in PD were described using predefined parameters for dermoscopic evaluation in dermato-oncology. UVFD characteristics were determined based on personal observations, and included interrupted follicle pattern, absence of pink-orange or blue-green fluorescence, well-demarcated borders, and dark silhouettes. In total, 163 BCCs were analyzed. Under UVFD, the interrupted follicle pattern (p < 0.001), absence of pink-orange fluorescence (p = 0.005) and well-demarcated borders (p = 0.031) were more frequently noted in BCCs < 5 mm than in bigger tumors. Lesions on the face showed clearly defined borders (p = 0.031) and interrupted follicle pattern (p < 0.001) more frequently than tumors located beyond the face. Nodular BCCs displayed interrupted follicle pattern (p = 0.001) and absence of pink-orange fluorescence (p < 0.001) more commonly than superficial subtypes. Non-pigmented BCCs more frequently showed lack of blue-green fluorescence (p = 0.007) and interrupted follicle pattern (p = 0.018) compared to pigmented variants. UVFD may be a valuable, complementary to PD, tool in the diagnosis of BCC, particularly in small tumors, lesions located on the face and nodular or non-pigmented subtypes.

Demixing fluorescence time traces transmitted by multimode fibers

Optical methods based on thin multimode fibers (MMFs) are promising tools for measuring neuronal activity in deep brain regions of freely moving mice thanks to their small diameter. However, current methods are limited: while fiber photometry provides only ensemble activity, imaging techniques using of long multimode fibers are very sensitive to bending and have not been applied to unrestrained rodents yet. Here, we demonstrate the fundamentals of a new approach using a short MMF coupled to a miniscope. In proof-of-principle in vitro experiments, we disentangled spatio-temporal fluorescence signals from multiple fluorescent sources transmitted by a thin (200 µm) and short (8 mm) MMF, using a general unconstrained non-negative matrix factorization algorithm directly on the raw video data. Furthermore, we show that low-cost open-source miniscopes have sufficient sensitivity to image the same fluorescence patterns seen in our proof-of-principle experiment, suggesting a new avenue for novel minimally invasive deep brain studies using multimode fibers in freely behaving mice.