Application In Live Cell Imaging Research Articles

A large-Stokes-shift fluorescent probe for Zn2+ based on AIE, and application in live cell imaging.

A fluorescence-enhanced sensor based on aggregation-induced emission (AIE) was synthesized using a di(2-picolyl)amine (DPA) group as a highly selective metal chelating agent for Zn2+. The combination of the probe and Zn2+ was achieved in an environment where the volume fraction of water was 90%, giving the probe good biocompatibility, and a large Stokes shift (100nm) occurred after Zn2+ was combined with the probe. The obvious color change makes the probe visible to the naked eye, and gives it a high signal-to-noise ratio, and high contrast, and minimizes self-absorption. Because of the high selectivity of the DPA group to Zn2+, the sensitivity of the probe to detect Zn2+ has been improved. The mechanism of the formation of complexes between the probe and Zn2+ was confirmed by nuclear magnetic resonance spectroscopy (NMR), high-resolution mass spectrometry (HRMS), and particle size distribution. Under the optimal experimental conditions, the linear fluorescence reaction of Zn2+ was good, between 0.2 and 18μM, and the detection limit was 1.3 × 10-7M. The low toxicity and excellent membrane permeability of the probe in living cells enable it to be efficiently applied for Zn2+ imaging in cells. Graphical abstract.

A selective purine-based fluorescent chemosensor for the “naked-eye” detection of zinc ions (Zn2+): applications in live cell imaging and test strips

In this work, a novel purine based probe PTAHN was successfully designed and synthesized. PTAHN displayed high selectivity towards Zn2+via turn-on fluorescence. What's more, PTAHN can be proficiently employed for imaging Zn2+ in living cells.

Development and Applications of Fluorogen/Light-Up RNA Aptamer Pairs for RNA Detection and More.

The central role of RNA in living systems made it highly desirable to have noninvasive and sensitive technologies allowing for imaging the synthesis and the location of these molecules in living cells. This need motivated the development of small pro-fluorescent molecules called "fluorogens" that become fluorescent upon binding to genetically encodable RNAs called "light-up aptamers." Yet, the development of these fluorogen/light-up RNA pairs is a long and thorough process starting with the careful design of the fluorogen and pursued by the selection of a specific and efficient synthetic aptamer. This chapter summarizes the main design and the selection strategies used up to now prior to introducing the main pairs. Then, the vast application potential of these molecules for live-cell RNA imaging and other applications is presented and discussed.

A Novel Fluorescent Probe Based on Perylene Derivative for Hg2+ Ions and Biological Thiols and its Application in Live Cell Imaging and Theoretical Calculations

AbstractThe selective and sensitive detection of biothiols; cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) in aqueous solutions is of considerable importance because of their pivotal roles in maintaining the reducing environment in the cells. This study describes a strategy for the determination of biothiols based on the PDI/Met‐Hg2+complex platform. We designed and fabricated methionine modified perylene diimide molecule as a selective sensing probe for Hg2+ ions in aqueous solutions (PDI/Met‐Hg2+). The complex between perylene bisimide derivative (PDI/Met) and Hg2+ was investigated and it demonstrated turn‐on fluorescence response for the detection of the biological thiols. Besides, PDI/Met displayed fluorescence quenching response in the presence of mercury ions and the emission intensity of PDI/Met‐Hg2+ was recovered after transferring biothiols (Cys, Hcy, and GSH). Thus, PDI/Met could be utilized as a fluorescent chemosensor for the sequential recognition of mercury ions and biological thiols.

A simple coumarin based “fluorescent On” probe for the selective detection of Al3+ along with its application in live cell imaging via AGS cell line

A new coumarin based fluorescent switch (HCBP) has been fabricated which displays high selective sensing towards Al3+ among other metal cations at physiological pH. On gradual addition of Al3+ specifically, HCBP shows a brilliant “turn-on” emission enhancement of about ∼13-fold with about 50 nm red shift at 481 nm in MeOH/H2O (1/1, v/v) solution. The fluorescent switch is proven to be a reversible probe by addition of EDTA gradually into the HCBP-Al3+ solution. Detection limit as well as Binding constant values have been calculated and found to be in the order of 10−9 M and 103 M-1 respectively. DFT and TDDFT studies are conducted with the probe to establish a similarity between theoretical and experimental outcomes. We can also use this new fluorescent switch as a biomarker kit as it has shown a brilliant potential in the application of live cell imaging using gastric cancer cell (AGS cell).

High-speed low-frequency chirped coherent anti-Stokes Raman scattering microscopy using an ultra-steep long-pass filter.

Coherent anti-Stokes Raman scattering (CARS) microscopy is becoming a more common tool in biomedical research. High-speed CARS microscopy has important applications in live cell imaging and in label-free pathology. However, only a few realizations exist of CARS imaging applied in the few terahertz spectral range (<300 cm-1), in which much is unknown to date. Although single-beam CARS microscopy proved to be robust in this low-frequency region, pixel-dwell time using presently available schemes is still relatively long, in the millisecond scale. Single-beam notch-shaped chirped-CARS (C-CARS) microscopy in the fingerprint region can be performed without using lock-in detection, yet it necessitates double-notch shaping, resulting in a relatively complex system. Here, we demonstrate that C-CARS in the low-frequency regime can be achieved using a sharp-edge, which is created by an ultra-steep long-pass filter (ULPF). Furthermore, we demonstrate that this variant of C-CARS spectroscopy can be performed without post-processing analyses. This is used to image collagen in a biological sample with a pixel dwell time of 200 microseconds. This sharp-edge C-CARS method may find important application in rapid low-frequency CARS imaging of live cells or for imaging of fast flowing objects such as in microfluidic channels.

A coumarin-based turn-on chemosensor for selective detection of Zn(II) and application in live cell imaging.

A 2-oxo-2H-chromene-3-carbohydrazide (CHB) was synthesized by the reaction of salicylaldehyde with diethyl malonate and hydrazine hydrate. The recognition behaviors of CHB to Zn2+ were investigated and the results showed that CHB exhibits well selectivity and sensitivity to Zn2+ with fast response in PBS (pH = 7.24, 60% DMF), the co-existed cations and anions could not interfere the recognition between CHB and Zn2+. Besides, the detection limit of CHB for Zn2+ was calculated to be 0.95 μM. Furthermore, DFT, EI-MS data and Job's plot were applied for determining the sensing mechanism of CHB with Zn2+ and the results showed that a type of 2:1 complex was formed between CHB and Zn2+ with the binding constant was 1.32 × 104 M-2. At last, probe CHB was successfully applied for the imaging of Zn2+ in living cells.

A quinoline-based selective ‘turn on’ chemosensor for zinc(II) via quad-core complex, and its application in live cell imaging

An efficient quinoline-based fluorescent chemosensor (QLNPY) was successfully developed for the detection of zinc ions (Zn2+). This novel chemosensor displayed higher sensitivity and selectivity toward Zn2+ over other competitive metal ions accompanying with obvious fluorescence enhancement. The QLNPY-Zn2+ complex can be further used as a new fluorescent “turn-off” sensor for pyrophosphate (PPi) and sulfur ion (S2−) via a Zn2+ displacement approach. The limits of detection were calculated to be 3.8 × 10−8 M for Zn2+, 3.7 × 10−7 M for PPi and 4.9 × 10−7 M for S2−. The binding mechanism of QLNPY and Zn2+ was investigated through NMR, HR-MS analysis and further studied by crystallographic analysis. Additionally, further application of QLNPY for sequential bioimaging of Zn2+ and PPi was studied in HepG2 cells, suggesting that the quinoline-based chemosensor possesses great potential applications for the detection of intracellular Zn2+ and PPi in vivo.

Analysis of the Diffusivity Change from Single-Molecule Trajectories on Living Cells.

With the wide application of live-cell single-molecule imaging and tracking of biomolecules at work, deriving diffusion state changes of individual molecules is of particular interest as these changes reflect molecular oligomerization or interaction with other cellular components and thus correlate with functional changes. We have developed a Rayleigh mixture distribution-based hidden Markov model method to analyze time-lapse diffusivity change of single molecules, especially membrane proteins, with unknown dynamic states in living cells. With this method, the diffusion parameters, including diffusion state number, state transition probability, diffusion coefficient, and state mixture ratio, can be extracted from the single-molecule diffusion trajectories accurately via easy computation. The validity of our method has been demonstrated with not only experiments on synthetic trajectories but also single-molecule fluorescence imaging data of two typical membrane receptors. Our method offers a new analytical tool for the investigation of molecular interaction kinetics at the single-molecule level.

A highly sensitive turn-on fluorescent chemosensor for recognition of Zn(II) ions and its application in live cells imaging

A novel and simple fluorescent chemosensor, DH, was synthesized. As designed, DH displayed excellent selectivity exhibiting a “turn-on” response for Zn2+ over other relevant metal ions based on PET mechanism in 100% aqueous media, and the binding constant of Zn2+ binding to DH was calculated to be 7.27 × 108 M−2. Importantly, DH exhibited high sensitivity as a Zn2+ fluorescent chemosensor with a limit of detection (LOD) of 19.25 nM, which is lower than WHO guidelines (76.0 μM). Moreover, the 2:1 stoichiometry between the chemosensor DH and Zn2+ was verified by Job's plot, fluorescent titration and high resolution mass spectroscopy. In addition, the recognition process of DH toward Zn2+ was chemically reversible and recyclable by adding Na2EDTA. Moreover, due to its good biocompatibility and low cytotoxicity, DH was successfully applied to the fluorescence imaging of Zn2+ and Na2EDTA in living HK2 cells.

Ratiometric Polymer Probe for Detection of Peroxynitrite and the Application for Live-Cell Imaging.

Peroxynitrite (ONOO−) is one of the sources of oxidation stress involved in many biological signaling pathways. The role of ONOO− being a double-edged sword in biological systems drives the development of effective detection tools. In this work, a boronate-based polymeric fluorescent probe PB-PVA was synthesized and the probe performance was evaluated. The probe exhibits ratiometric sensing of ONOO− in a range of 0–6 µM. There is good linear relationship between the probe fluorescence intensity ratio and ONOO− concentration. The probe also displays moderate selectivity towards ONOO− over other ROS. Moreover, it is water-soluble and possesses good biocompatibility which aids the imaging of ONOO− in living cells. These properties could make the probe a promising tool in in vitro study related to ONOO−.

Use of Drosophila S2 Cells for Live Imaging of Cell Division.

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:α-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as 'CID' gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

A novel fluorescent probe based on isocoumarin for Hg2+ and Fe3+ ions and its application in live-cell imaging.

Synthesis of the 2-amino-4-phenyl-6- (isocoumarin-3-yl) -3-cyanopyridine (APICP) containing both isocoumarin and pyridine ring in its structure was carried out, and this compound was characterized by ATR-FTIR, 1H NMR, and 13C NMR spectral techniques. A fluorescence sensor determining Hg2+ and Fe3+ ions in DMSO/HEPES buffer solution (9/1 v/v, 5 μM, pH 7.0) was developed using the synthesized compound, and the detection limits of the sensor with exquisite selectivity were calculated as 8.12 nM and 5.51 nM for Hg2+ and Fe3+ ions, respectively. Jobs plot method was used to determine the stoichiometry of APICP-Hg2+/Fe3+ complexes as 2:1 and FT-IR and ESI-MS methods confirmed the results. Besides, cell growth inhibitory potentials of the sensor over HepG2 cells and invivo fluorescent cell imaging experiments were conducted. Findings revealed the relatively low cytotoxic effects of the synthesized sensor (IC50: 0.541 ± 0.039 mM), and it could be utilized as an intracellular imaging agent for the determination of Fe3+ and Hg2+ ions in biological systems.

Isonicotinoylhydrazide modified 3-acetylcoumarin scaffold as an efficient chemical reversible sensor to detect Al3+ selectively and its application in live cells imaging

A turn-on photochromic sensor based on coumarin was designed and synthesized for selective and sensitive detection of Aluminium(III) (Al3+) in ethanol (C2H5OH)/water (H2O; 1:1, v/v) with the detection limit of 0.126 μM. The mechanism was proposed to form a kind of 1:1-type complex between probe C1 and Al3+ in which the binding constant was 3.11 × 107 M−1. In addition, the sensor was effectively used for the detection of Al3+ in living HEK293T cells.

Crystal structure and spectral studies of green fluorescent protein (GFP) chromophore analogue ethyl 2-[(4Z)-(6-hydroxy naphthalen-2-yl) methylene)-2-methyl-5-oxo-4,5-di hydro-1H-imidazol-1-yl] acetate

Synthetically modified green fluorescent protein chromophore derivative was prepared, its crystal structure and spectral properties were studied. Crystal data for C19H18N2O4: triclinic, space group P-1 (no. 2), a = 8.2506(17) Å, b = 11.934(2) Å, c = 17.461(4) Å, α = 102.89(3)°, β = 94.62(3)°, γ = 96.68(3)°, V = 1654.5(6) Å3, Z = 4, T = 173(2) K, μ(MoKα) = 0.096 mm-1, Dcalc = 1.358 g/cm3, 7227 reflections measured (4.722° ≤ 2Θ ≤ 53.996°), 7227 unique (Rint = 0.0453, Rsigma = 0.0662) which were used in all calculations. The final R1 was 0.0561 (I &gt; 2σ(I)) and wR2 was 0.1658 (all data). The single crystal structure showed, the benzylidine moiety adopts Z-conformation in solid state and the molecules were associated by various O−H···O and C−H···O non-covalent interactions. The UV absorption-emission spectral analysis indicated that a significant red shift of emission observed at alkaline pH indicating its utility for live cell imaging applications.

Highly Selective Fluorescent Probe for the Detection of Copper (II) and Its Application in Live Cell Imaging.

The development of fluorescent methods for the detection of metal ions is of great importance due to their diverse environmental and biological roles. Herein, a rhodamine 6G-based off-on fluorescent probe (L1) with a t-butyl pyrrole moiety as the recognition site was designed and synthesized. Photophysical studies show that L1 exhibits excellent sensitivity and selectivity towards Cu2+ to other metal ions in neutral acetonitrile aqueous media. Mechanism studies suggest that the recognition process may associate with a Cu2+ promoted hydrolysis reaction of L1. Furthermore, L1 has been successfully applied in fluorescence imaging of Cu2+ ion in living cells.

Selective and Sensitive Fluorescence Probe for Detection of Al3+ in Food Samples Based on Aggregation-Induced Emission and Its Application for Live Cell Imaging

A novel fluorescent probe L was designed for rapid detection of Al3+ with high selectivity in water-acetonitrile medium based on aggregation-induced emission (AIE). The recognition mechanism was investigated by fluorescence spectra, SEM, FI-IR, 1H NMR, and mass spectra. The detection limit of Al3+ was 5.3 × 10−8 M. The recoveries were within the range of 84.5–103.3% and the relative standard deviation (RSD) values were within the range of 1.7–6.4%. The AIE behavior could be attributed to the J-aggregates formation and the chelation-enhanced fluorescence (CHEF) in water-acetonitrile medium. The probe L can be used to detect Al3+ in food and environmental samples. What’s more interesting is that the L could detect intracellular Al3+ in live cells by bioimaging.

Pyrene Based Fluorescent Turn-on Chemosensor for Sequential Detection of Fe3+ and Fe2+ Ions and its Application in Live Cell Imaging.

A simple pyrene-based "turn-on" chemosensor bearing isonizide namely 3-(pyren-1-yl methylene) pentane-2,4-diyldene di(isonicotinohydrazide) (PMPD) was designed and synthesized for detecting Fe3+and Fe2+ ions in HEPES buffer solution at pH7.4 (DMSO: H2O (1:9v/v)) medium. The probe shows conducive selectivity for Fe2+ and Fe3+ ions over the other competitive metal cations. The detection limit was found to be 1.67μM for Fe3+ and 2.02μM for Fe2+. The recognition mechanism of PMPD towards Fe3+ and Fe2+ has been examined in detail by absorption, emission, and ESI-MS studies. Moreover, "turn-on" fluorescence behavior of the probe was used to track iron ions in living cells.

A rationally designed flavone-based ESIPT fluorescent chemodosimeter for highly selective recognition towards fluoride and its application in live-cell imaging

Fluoride recognition and sensing via artificial receptors have attracted a great deal of attention since it plays an important and indispensable role in biological and environmental processes. Herein, a novel colorimetric and fluorescent chemodosimeter (3-triisopropylsilylflavone 1) based on 3-hydroxyflavone framework has been rationally designed and synthesized for selective recognition towards fluoride. As confirmed by UV–vis and fluorescence techniques, the fluorescent probe 1 exhibited a highly sensitive and selective as well as rapidly responsive (20 s) recognition towards fluoride with a large Stokes shift of 180 nm. The detection limit was deduced to be 0.68 μM, which is much lower than the limit concentration level in drinking water specified by USEPA. The distinctive sensing mechanism is attributed to fluoride-triggered desilylation reaction on the cleavage of SiO bonds, leading to the strong excited-state intramolecular proton transfer emission of 3-hydroxyflavone. In addition, test strips experiment based on the sensor demonstrated the excellently practical feasibility of 1 to sense fluoride even in ppm level. Moreover, the probe is cell permeable and can be capable of reliably detecting fluoride in living cells.

An ESIPT-based fluorescent probe for Hg2+ in aqueous solution and its application in live-cell imaging

A new Excited-State Intramolecular Proton Transfer (ESIPT) based fluorescent probe for the detection of Hg2+ has been rationally designed and developed. Based on the specific reactivity of mercury-promoted hydrolysis, the probe exhibits high selectivity and sensitivity for mercury ions in almost pure aqueous solution (containing only 1% DMSO) with a low detection limit of 1.9 ppb. Furthermore, the probe was also successfully used for fluorescence imaging of Hg2+ in live cells.