Biological Imaging Applications Research Articles

Comparison of Vinyldimethylaniline and Indolizine Donor Groups on Si‐Substituted Xanthene Core Shortwave Infrared Fluorophores

AbstractSmall organic molecules absorbing and emitting in the shortwave infrared (SWIR, 1000–2000 nm) region are desirable for biological imaging applications due to low auto‐fluorescence, reduce photon scattering, and good tissue penetration depth of photons which allows for in vivo imaging with high resolution and sensitivity. Si‐substituted xanthene‐based fluorophores with indolizine donors have demonstrated some of the longest wavelengths of absorption and emission from organic dyes. This work seeks to compare an indolizine heterocyclic nitrogen with dimethyl aniline nitrogen donors on otherwise identical Si‐substituted xanthene fluorophores via optical spectroscopy, computational chemistry and electrochemistry. Three donors are compared including an indolizine donor, a ubiquitous dimethyl aniline donor, and a vinyl dimethyl aniline group that keeps the number of π‐bonds consistent with indolizine. Significantly higher quantum yields and molar absorptivity are observed in these studies for a dimethylamine‐based donor relative to a simple indolizine donor absorbing and emitting at similar wavelengths (~1312 nm emission). Substantially longer wavelengths are obtainable by appending aniline‐based groups to the indolizine donor (~1700 nm) indicating longer wavelengths can be accessed with indolizine donors while stronger emitters can be accessed with anilines in place of indolizine.

Enhanced Infrared Vision by Nonlinear Up-Conversion in Nonlocal Metasurfaces.

The ability to detect and image short-wave infrared light has important applications in surveillance, autonomous navigation, and biological imaging. However, the current infrared imaging technologies often pose challenges due to large footprint, large thermal noise and inability to augment infrared and visible imaging. Here, infrared imaging is demonstrated by nonlinear up-conversion to the visible in an ultra-compact, high-quality-factor lithium niobate resonant metasurface. Images with high conversion efficiency and resolution quality are obtained despite the strong nonlocality of the metasurface. The possibility of edge-detection image processing augmented with direct up-conversion imaging for advanced night vision applications is furthershown.

Development of an miRFP680-Based Fluorescent Calcium Ion Biosensor Using End-Optimized Transposons.

The development of new or improved single fluorescent protein (FP)-based biosensors (SFPBs), particularly those with excitation and emission at near-infrared wavelengths, is important for the continued advancement of biological imaging applications. In an effort to accelerate the development of new SFPBs, we report modified transposons for the transposase-based creation of libraries of FPs randomly inserted into analyte binding domains, or vice versa. These modified transposons feature ends that are optimized to minimize the length of the linkers that connect the FP to the analyte binding domain. We rationalized that shorter linkers between the domains should result in more effective allosteric coupling between the analyte binding-dependent conformational change in the binding domain and the fluorescence modulation of the chromophore of the FP domain. As a proof of concept, we employed end-modified Mu transposons for the discovery of SFPB prototypes based on the insertion of two circularly permuted red FPs (mApple and FusionRed) into binding proteins for l-lactate and spermidine. Using an analogous approach, we discovered calcium ion (Ca2+)-specific SFPBs by random insertion of calmodulin (CaM)-RS20 into miRFP680, a particularly bright near-infrared (NIR) FP based on a biliverdin (BV)-binding fluorescent protein. Starting from an miRFP680-based Ca2+ biosensor prototype, we performed extensive directed evolution, including under BV-deficient conditions, to create highly optimized biosensors designated the NIR-GECO3 series. We have extensively characterized the NIR-GECO3 series and explored their utility for biological Ca2+ imaging. The methods described in this work will serve to accelerate SFPB development and open avenues for further exploration and optimization of SFPBs across a spectrum of biological applications.

LiveLattice: Real-time visualization of tilted light-sheet microscopy data using a memory-efficient transformation algorithm.

Light-sheet fluorescence microscopy (LSFM), a prominent fluorescence microscopy technique, offers enhanced temporal resolution for imaging biological samples in four dimensions (4D; x, y, z, time). Some of the most recent implementations, including inverted selective plane illumination microscopy (iSPIM) and lattice light-sheet microscopy (LLSM), rely on a tilting of the sample plane with respect to the light sheet of 30-45 degrees to ease sample preparation. Data from such tilted-sample-plane LSFMs require subsequent deskewing and rotation for proper visualization and analysis. Such transformations currently demand substantial memory allocation. This poses computational challenges, especially with large datasets. The consequence is long processing times compared to data acquisition times, which currently limits the ability for live-viewing the data as it is being captured by the microscope. To enable the fast preprocessing of large light-sheet microscopy datasets without significant hardware demand, we have developed WH-Transform, a novel GPU-accelerated memory-efficient algorithm that integrates deskewing and rotation into a single transformation, significantly reducing memory requirements and reducing the preprocessing run time by at least 10-fold for large image stacks. Benchmarked against conventional methods and existing software, our approach demonstrates linear scalability. Processing large 3D stacks of up to 15 GB is now possible within one minute using a single GPU with 24 GB of memory. Applied to 4D LLSM datasets of human hepatocytes, human lung organoid tissue, and human brain organoid tissue, our method outperforms alternatives, providing rapid, accurate preprocessing within seconds. Importantly, such processing speeds now allow visualization of the raw microscope data stream in real time, significantly improving the usability of LLSM in biology. In summary, this advancement holds transformative potential for light-sheet microscopy, enabling real-time, on-the-fly data processing, visualization, and analysis on standard workstations, thereby revolutionizing biological imaging applications for LLSM, SPIM and similar light microscopes.

Peptide-Directed Synthesis of Aggregation-Induced Emission Enhancement-Active Gold Nanoclusters for Single- and Two-Photon Imaging of Lysosome and Expressed αvβ3 Integrin Receptors.

This study explores the synthesis and characterization of aggregation-induced emission enhancement (AIEE)-active gold nanoclusters (AuNCs), focusing on their near-infrared luminescence properties and potential applications in biological imaging. These AIEE-active AuNCs were synthesized via the NaBH4-mediated reduction of HAuCl4 in the presence of peptides. We systematically investigated the influence of the peptide sequence on the optical features of the AuNCs, highlighting the role of glutamic acid in enhancing their quantum yield (QY). Among the synthesized peptide-stabilized AuNCs, EECEE-stabilized AuNCs exhibited the maximum QY and a pronounced AIEE effect at pH 5.0, making them suitable for the luminescence imaging of intracellular lysosomes. The AIEE characteristic of the EECEE-stabilized AuNCs was demonstrated through examinations using transmission electron microscopy, dynamic light scattering, zeta potential analysis, and single-particle imaging. The formation of the EECEE-stabilized AuNCs was confirmed by size-exclusion chromatography and mass spectrometry. Spectroscopic and electrochemical examinations uncover the formation process of EECEE-stabilized AuNCs, comprising EECEE-mediated reduction, NaBH4-induced nucleation, complex aggregation, and subsequent cluster growth. Furthermore, we demonstrated the utility of these AuNCs as luminescent probes for intracellular lysosomal imaging, leveraging their pH-responsive AIEE behavior. Additionally, cyclic arginylglycylaspartic acid (RGD)-modified AIEE dots, derived from cyclic RGD-linked peptide-induced aggregation of EECEE-stabilized AuNCs, were developed for single- and two-photon luminescence imaging of αvβ3 integrin receptor-positive cancer cells.

A novel “Turn-Off-On” fluorescent probe for specific sequential detection of Cu2+ and glyphosate and its application in biological imaging

As common pollutants, Cu2+ and glyphosate pose a serious threat to human health and the ecosystem. Herein, a fluorescent probe (E)-7-(diethylamino)-N'(4-(diethylamino)-2-hydroxybenzyl)-2-oxo-2H chromophore-3-carbazide (DDHC) was designed and synthesised for the sequential recognition of Cu2+ and glyphosate. DDHC has the advantages of a short synthesis path, easy-to-obtain raw materials, good anti-interference ability, and strong stability. The interaction of the DDHC-Cu2+ complexes with glyphosate allows the amino and carboxyl groups in glyphosate molecules to coordinate with Cu2+ strongly, competing for the Cu2+ in the DDHC-Cu2+ complexes and releasing the DDHC, leading to the recovery of fluorescence. The recognition was further validated through Job’s plot, HRMS, and DFT calculations. In addition, the successful recovery of Cu2+ and glyphosate in different environmental water samples fully demonstrates the practical application potential of DDHC. Especially, DDHC has low cytotoxicity and can enter zebrafish and HeLa cells, rapidly reacting with Cu2+ and glyphosate in the body, generating visible fluorescence quenching and recovery phenomena, achieving real-time visual monitoring of exogenous Cu2+ and glyphosate in zebrafish and HeLa cells. The targeting and dual selectivity of DDHC greatly enhance its potential application value in the field of detection, providing important theoretical support for studying the fate of multiple pollutants in the environment.

Debut of a responsive chemiluminescent probe for butyrylcholinesterase: Application in biological imaging and pesticide residue detection

Debut of a responsive chemiluminescent probe for butyrylcholinesterase: Application in biological imaging and pesticide residue detection

Aqueous synthesis of highly luminescent AgInSe2/ZnS core/shell nanocrystals

Ternary I-III-VI chalcopyrite-type nanocrystals (NCs) range among the most important alternative materials to Cd-based NCs. Within this materials family, AgInSe2 (AISe) presents a narrower bandgap than widely studied AgInS2 (AIS), making it more suitable for numerous applications. At present, it remains a long-standing challenge to directly synthesize high-quality AISe core and AISe/ZnS core/shell NCs in aqueous solution at atmospheric pressure. In this work, we describe their synthesis using glutathione and citric acid as dual stabilizers. First, to form AISe core NCs, the Se precursor is injected into a solution containing the Ag and In complexes at 96 °C for 20 min. In the second step, the AISe/ZnS core/shell structure is created by growing the ZnS shell on the AISe NCs surface at 90 °C for 60 min. The synthesized AISe and AISe/ZnS core/shell NCs are characterized using X-ray diffraction, transmission electron microscopy, and UV-Vis absorption and photoluminescence for optical spectroscopies. After the growth of the ZnS shell, AISe/ZnS core/shell NCs exhibit higher photostability and emit intense luminescence at a wavelength of 680 nm with an impressive quantum yield (QY) of 30 %, which represents a threefold higher than the AISe core NCs. These properties make the aqueous soluble AISe/ZnS core/shell NCs favorable candidates for lighting, displays, and biological imaging applications.

A novel triphenylamine-furan hydrazone-based sensing of Cu2+ ions and imaging in cancer cells

A novel fluorescent probe (TPA-TF) was designed and synthesized, incorporating triphenylamine-thiophene moieties coupled with furan and a hydrazone. First, the TPA-TF can be used as a sensor to detect Cu2+ in the environment/biosystem through an effective fluorescence quenching due to chelation-enhanced fluorescence quenching (CHEQ) and inhibiting the photoinduced electron transfer (PET) process. Various fluorescence spectra and density functional theory (DFT) computations were used to clarify the TPA-TF to Cu2+ detection characteristics. The Cu2+ sensor TPA-TF exhibited a fast response time of one minute. In this study, the linear range concentration for the Cu2+ ion detection was 0–1000 μM, with a broad pH working range (pH 2.7–7.4), and the detection limit of the sensor was 6.08 μM. With its broad applicability, the TPA-TF sensor was successfully used to detect and photograph Cu2+ in live cells, demonstrating its considerable potential for applications in biological imaging.

An innovative stepwise activated coupling reaction for fabricating functional fluorescence probes with potential photosensitive applications

Coupling bright organic emitters with biocomponents such as proteins and amino acids to fabricate biocompatible optical functional materials is receiving increasing attention. Previous reports of an efficient, typical reaction between maleic anhydride (MAH) and amino groups indicated that MAH derivatives have great potential for bioconjugation applications. However, the obstacle of complex synthesis methods and weak fluorescence to this kind of derivative structures based on MAH, have greatly limited their applications. Herein, an innovative strategy was developed based on stepwise activated coupling via a multi-step process. First, a series of donor–acceptor emitting frameworks was constructed by introducing different commercially available electron donors into the electron-withdrawing squaric acid core. Then, these frameworks were further activated to obtain different 3,4-diaryl MAH precursors for bioconjugation, which contributed to a novel light-triggered ring-enlargement effect. Importantly, the MAH precursors triggered catalyst-free coupling reactions at ambient temperature with organic small molecules and biological macromolecules containing primary amine groups. Different fluorescent precursors and abundant biological components were effectively combined, and a water-soluble, biocompatible photosensitizer with efficient photodynamic and photothermal therapy effects was obtained. This strategy is expected to have great potential for biological imaging and theragnostic applications.

Isolation of phase edges using off-axis q-plate filters.

Edge-enhanced microscopes with a q-plate have attracted more attention to enhance the edges of phase-amplitude objects in biological samples due to their capacity for all-directional edge enhancement, while differential interference-contrast microscopy enhances edges in only one-direction. However, the edge-enhanced microscopes cannot distinguish the edges of phase and amplitude objects, as both edges are equally enhanced. This study introduces a novel method for isolating the edge of a phase object from an amplitude object using an off-axis q-plate filter in a 4f system. Herein, we combined off-axis q-plates with four different displacements to isolate the phase object edge from the amplitude object. To demonstrate the proposed method, we conducted experiments using two distinct samples. The first sample comprised a phase test target surrounded by an aperture, and the second sample involved an overlap between the phase test target and a white hair with non-zero transmittance. In the samples, the isolated phase object edge is in good agreement with the theoretical expectations, and the amplitude object edge was reduced by approximately 93%. The proposed method is a novel and effective approach for isolating the edge of a phase object from an amplitude object and can be useful in various biological imaging applications.

Quasi-Chromophores Segregated by Single-Chain Nanoparticles of Fluorinated Zwitterionic Random Copolymers Showing Remarkably Enhanced Fluorescence Emission Capable of Fluorescent Cell Imaging.

Organic and polymer fluorescent nanomaterials are a frontier research focus. Here in this work, a series of fluorinated zwitterionic random copolymers end-attached with a quasi-chromophoric group of pyrene or tetraphenylethylene (TPE) are well synthesized via atom transfer radical polymerization with activators regenerated by electron transfer (ARGET ATRP). Those random copolymers with total degree of polymerization 100 or 200 are able to produce fluorescent single-chain nanoparticles (SCNPs) through intra-chain self-folding assembly with quite uniform diameters in the range of 10-20nm as characterized by dynamic light scattering and transmission electron microscopy. By virtue of the segregation or confinement effect, both SCNPs functionalized with pyrene or TPE group are capable of emitting fluorescence, with pyrene tethered SCNPs exhibiting stronger fluorescence emission reaching the highest quantum yield ≈20%. Moreover, such kind of fluorescent SCNPs manifest low cytotoxicity and good cell imaging performance for Hela cells. The creation of fluorescent SCNPs through covalently attached one quasi-chromophore to the end of one fluorinated zwitterionic random copolymer provides an alternative strategy for preparing polymeric luminescence nanomaterials, promisingly serving as a new type of fluorescent nanoprobes for biological imaging applications.

Super Broadband Emission Across NIR‐I and NIR‐II Under Blue Light Excitation of Cr3+, Ni2+ Co‐Doped Sr2GaTaO6 Phosphor Achieved by Two‐Site Occupation and Effective Energy Transfer

AbstractThe performance of the near‐infrared phosphor‐converted light‐emitting diodes (NIR pc‐LEDs) mainly depends on the NIR emitting phosphors used. Cr3+ doped materials can be excited by blue light chips, but their emission is located in the NIR‐I region (650–1000 nm). Ni2+ doped materials are mainly located in the NIR‐II region (1000–1700 nm), but they cannot be effectively excited by blue light chips. Herein, Cr3+, Ni2+ mono‐doped, and co‐doped Sr2GaTaO6 NIR emitting phosphors are prepared and investigated. Cr3+ and Ni2+ ions occupy two octahedral sites of Ga3+ and Ta5+. The co‐doping of Cr3+ ions has achieved two breakthroughs. One is to shift the optimal excitation wavelength from violet light to blue light due to the energy transfer (efficiency up to 70%) from Cr3+ to Ni2+. The other is to achieve the broadband and continuous emission across NIR‐I and NIR‐II regions (650–1700 nm, with a full width at half maximum (FWHM) of 410 nm (173 nm + 237 nm)). The prepared Sr2GaTaO6: 0.02Cr3+, 0.01Ni2+ phosphor is combined with a commercial 460 nm blue chip to realize its application in organic compounds identification, night vision, and biological imaging. This work points out a direction for the future development of efficient super broadband NIR‐emitting phosphors.

Biocatalytic Synthesis of Coumarin S-Glycosides: Towards Non-Cytotoxic Probes for Biomedical Imaging and Sensing.

This study unveils an innovative method for synthesizing coumarin S-glycosides, employing original biocatalysts able to graft diverse carbohydrate structures onto 7-mercapto-4-methyl-coumarin in one-pot reactions. The fluorescence properties of the generated thio-derivatives were assessed, providing valuable insights into their potential applications in biological imaging or sensing. In addition, the synthesized compounds exhibited no cytotoxicity across various human cell lines. This research presents a promising avenue for the development of coumarin S-glycosides, paving the way for their application in diverse biomedical research areas.

Dual-Emissive Iridium(III) Complexes and Their Applications in Biological Sensing and Imaging.

Phosphorescent iridium(III) complexes are widely recognized for their unique properties in the excited triplet state, making them crucial for various applications including biological sensing and imaging. Most of these complexes display single phosphorescence emission from the lowest-lying triplet state after undergoing highly efficient intersystem crossing (ISC) and ultrafast internal conversion (IC) processes. However, in cases where these excited-state processes are restricted, the less common phenomenon of dual emission has been observed. This dual emission phenomenon presents an opportunity for developing biological probes and imaging agents with multiple emission bands of different wavelengths. Compared to intensity-based biosensing, where the existence and concentration of an analyte are indicated by the brightness of the probe, the emission profile response involves modifications in emission color. This enables quantification by utilizing the intensity ratio of different wavelengths, which is self-calibrating and unaffected by the probe concentration and excitation laser power. Moreover, dual-emissive probes have the potential to demonstrate distinct responses to multiple analytes at separate wavelengths, providing orthogonal detection capabilities. In this concept, we focus on iridium(III) complexes displaying fluorescence-phosphorescence or phosphorescence-phosphorescence dual emission, along with their applications as biological probes for sensing and imaging.

Pyridine appended pyrimidine bis hydrazone: Zn2+/ATP detection, bioimaging and functional properties of its dinuclear Zn(II) complex

A pyridine functionalized pyrimidine-based system, H2P was successfully synthesized, characterized, and evaluated for its remarkable selective characteristics towards Zn2+ and ATP ions. The chemical sensing capabilities of H2P were demonstrated through absorption, fluorescence, and NMR spectroscopic techniques. The probe exhibited outstanding sensitivity when interacting with the ions, demonstrating relatively strong association constants and impressively low detection limits. The comprehensive binding mechanism of H2P with respect to Zn2+ and ATP ions was investigated using a combination of analytical methods, including Job's plot, NMR spectroscopy, mass spectrometry, and density functional theory (DFT) experiments. The interesting sensing ability of H2P for Zn2+/ATP ions was harnessed for live cell bioimaging and other diverse on-site detection purposes, including paper strips, cotton swabs, and applications involving mung bean sprouts. Further, the fluorescent probe demonstrated its effectiveness in detecting Zn2+ and ATP within live cells, indicating its significant potential in the realm of biological imaging applications. Moreover, the molecular configuration of the zinc complex (H2P–Zn2Cl4), derived from H2P, was elucidated using X-ray crystallography. This complex exhibited intriguing multifunctional attributes, encompassing its capability for detecting picric acid and for reversible acid/base sensing responses. The enhanced conducting behavior of the complex as well as its resistance properties were investigated by performing I–V characteristics and electrochemical impedance spectroscopic (EIS) experiments respectively.

Single microparticle characterization using multi-wavelength lens-free imaging

Holographic imaging captures an interference pattern, effectively encoding an object‘s properties such as size, shape and refractive index in the hologram. Lens-free holographic imaging offers a scalable solution with large field of view to analyze microparticles or cells in high-throughput biological imaging applications. We studied characterization of single particles based on their holographic fingerprint using multi-wavelength illumination based lens-free holography. Deciphering this information directly in the hologram domain with our multi-wavelength approach allows for reliable estimation of object refractive index along with its size without ambiguity. This work provides a path forward for lens-free imaging-based microparticle characterization that can prove useful in biological studies such as cell analysis and characterization.

Cloning and characterization of luciferase from an Asian firefly Pygoluciola qingyu and its comparison with other beetle luciferases.

The bioluminescence system of luminescent beetles has extensive applications in biological imaging, protein labeling and drug screening. To explore wild luciferases with excellent catalytic activity and thermal stability, we cloned the luciferase of Pygoluciola qingyu, one species living in areas of high temperature and with strong bioluminescence, by combining transcriptomic sequencing and reverse transcription polymerase chain reaction (RT-PCR). The total length of luciferase gene is 1638bp and the luciferase consists 544 amino acids. The recombinant P. qingyu luciferase was produced in vitro and its characteristics were compared with those of eight luciferases from China firefly species and two commercial luciferases. Compared with these luciferases, the P. qingyu luciferase shows the highest luminescence activity at room temperature (about 25-28℃) with similar KM value for D-luciferin and ATP to the Photinus pyralis luciferase. The P. qingyu luciferase activity was highest at 35℃ and can keep high activity at 30-40℃, which suggests the potential of P. qingyu luciferase for in vivo and cell application. Our results provide new insights into P. qingyu luciferase and give a new resource for the application of luciferases.

Microwave synthesis of chitosan-based carbon dots for Al3+ detection and biological application.

Yellow fluorescent carbon dots (Y-CDs) were prepared via microwave method using chitosan and o-phenylenediamine as the main raw materials. The obtained Y-CDs possesses good water solubility, excellent biocompatibility and luminous stability. During the microwave pyrolysis carbonization process, the surface of Y-CDs was modified with the functional groups such as amino and carboxyl, which can bind to Al3+ by forming complexes, further improving the selectivity and sensitivity of the Al3+ detection. And the fluorescence of Y-CDs was quenched by Al3+ by static quenching process. More importantly, Y-CDs as fluorescent sensor was further applied for the determination of Al3+ in the real water samples with high reliability and accuracy. In addition, Y-CDs present potential application in biological imaging. The cultivated zebrafish embryos with Y-CDs displayed clearly in vivo uptake and metabolic fluorescence images, further confirming its low toxicity and excellent biocompatibility.

Perspective and Prospects on persistent luminescent nanoparticles for biological imaging and tumor therapy.

Persistent luminescent nanoparticles (PLNPs) are photoluminescent materials that can still emit luminescence after the cessation of the excitation light source. In recent years, due to their unique optical properties, the PLNPs have attracted extensive attention in the biomedical field. Since the PLNPs effectively eliminate autofluorescence interference from biological tissues, many researchers have contributed a lot of work in the fields of biological imaging and tumor therapy. This article mainly introduces the synthesis methods of the PLNPs and their progress in the application of biological imaging and tumor therapy, as well as the challenges and development prospects.