Application In Live Cell Imaging Research Articles

A thioether containing reversible fluorescence “turn-on” chemosensor for selective detection of zinc(II): Applications in live cell imaging and inhibit logic gate

A new fluorescence probe (H2L) is designed and synthesized for efficient and selective detection of Zn(II). H2L exhibits a “turn-on” fluorescence response with substantial enhancement of emission intensity with Zn2+ even in presence of other coexisting cations found in various environmental and biological samples. The calculated value of limit of detection (LOD) is 1.73 × 10−9 M. Exploiting the reversibility of the probe in presence of EDTA, an INHIBIT logic gate is constructed with Zn2+ and EDTA as chemical inputs. DFT and TDDFT calculations are used to interpret electronic structures and elucidate the sensing mechanism. Cytotoxicity study by MTT method with human breast cancer cell lines (MCF-7) reveals that H2L has negligible toxicity in low concentration levels and can be effectively used for live cell imaging.

Using unnatural amino acids to selectively label proteins for cellular imaging: a cell biologist viewpoint.

Twenty-five years ago, GFP revolutionized the field of cell biology by enabling scientists to visualize, for the first time, proteins in living cells. However, when it comes to current, state-of-the-art imaging technologies, fluorescent proteins (such as GFP) have several limitations that result from their size and photophysics. Over the past decade, an elegant, alternative approach, which is based on the direct labeling of proteins with fluorescent dyes and is compatible with live-cell and super-resolution imaging applications, has been introduced. In this approach, an unnatural amino acid that can covalently bind a fluorescent dye is incorporated into the coding sequence of a protein. The protein of interest is thereby site-specifically fluorescently labeled inside the cell, eliminating the need for protein- or peptide-labeling tags. Whether this labeling approach will change cell biology research is currently unclear, but it clearly has the potential to do so. In this short review, a general overview of this approach is provided, focusing on the imaging of site-specifically labeled proteins in mammalian tissue culture cells, and highlighting its advantages and limitations for cellular imaging.

Synthesis of a far-red emitting flavonoid-based lysosome marker for live cell imaging applications

A bright far-red emitting flavonoid derivative (FuraET) was synthesized in good yields by inserting a π extension group (i.e., furan) into the flavonoid skeleton, via using the Suzuki-Miyaura cross-coupling reaction. FuaraET exhibited optical absorption at λab ≈ 450 nm and emission λem ≈ 660 nm by recognizing as the first far-red emitting flavonoid derivative reported. FuraET exhibited a large Stokes shift (Δλ > 150 nm) high fluorescent quantum yield (φfl ≈ 0.2–0.4), and good photostability indicating excellent characteristics for an imaging probe. Live cell fluorescent confocal microscopy imaging revealed the exceptional selectivity of the FuraET towards cellular lysosomes (Mander’s overlap coefficients >0.9). The observed non-alkalinizing nature and high biocompatibility (LC50 > 50 µM) suggested that FuraET can a reliable lysosome marker for live cell imaging experiments. Our further study also indicated that FuraET may likely internalized into hydrophobic regions of the cellular lysosomes in contrast to acidic lysosomal lumen.

Fluorescent chemosensor for Al(III) based on chelation-induced fluorescence enhancement and its application in live cells imaging

A tridentate Schiff base receptor BPB was synthesized by condensation of 2-(benzo[b]thiophen-2-yl)benzenamine with salicylaldehyde in ethanolic medium and characterized by various spectral (FT-IR, 1H NMR, 13C NMR and mass) data. The methanolic solution of BPB was applied for the fluorescent sensing of metal ions dissolved in aqueous medium. The selectivity experiment revealed that the receptor BPB showed significant fluorescence enhancement at 430 nm in the presence of A(III) due to the chelation-induced fluorescence enhancement (CHEF) mechanism. Receptor BPB formed complex with Al(III) in 2:1 binding ratio with the estimated binding constant of K = 3.02 × 108 M−2. Without any interference from other tested metal ions, the receptor BPB can detect the concentration of Al(III) down to 256 nM. The receptor BPB showed good cell permeability and was applied for the qualitative and quantitative detection of intracellular Al(III) in A549 cell line (adenocarcinomic human alveolar basal epithelial cells) by using a confocal imaging technique.

Naphthyl hydrazone anchored with nitrosalicyl moiety as fluorogenic and chromogenic receptor for heavy metals (Ag+, Hg2+) and biologically important F− ion and its live cell imaging applications in HeLa cells and Zebrafish embryos

A new naphthyl hydrazone anchored nitrosalicyl unit has been developed as a host for the selective and sensitive detection of Ag+, Hg2+ and F− ions in DMSO/water (9:1) medium. An obvious fluorescence enhancement was observed towards Ag+, Hg2+ and F− ions due to the inhibition of both photoinduced electron transfer (PET) process and CN isomerization phenomenon. Moreover, the binding stoichiometry of probe with Ag+, Hg2+ and F− ions was established based on Job’s plot, 1H NMR, 19F NMR titrations and ESI-MS analysis. Furthermore, the sensing behavior of probe for Ag+, Hg2+ and F− ions was supported by Density Functional Theory (DFT) calculations. Besides, cell viability of the probe was demonstrated using live cell imaging experiment and the probe is proved to be an excellent chemical tool for mapping F− and Ag+ ions distribution in HeLa cells and Zebrafish embryos respectively in biological environments.

Self-Healing Dyes-Keeping the Promise?

Self-healing dyes have emerged as a new promising class of fluorescent labels. They consist of two units, a fluorescent dye and a photostabilizer. The latter heals whenever the fluorescent dye is in danger of taking a reaction pathway toward photobleaching. We describe the underlying concepts and summarize the developmental history and state-of-the-art, including latest applications in high-resolution microscopy, live-cell, and single-molecule imaging. We further discuss remaining limitations, which are (i) lower photostabilization of most self-healing dyes when compared to solution additives, (ii) limited mechanistic understanding on the influence of the biochemical environment and molecular oxygen on self-healing, and (iii) the lack of cheap and facile bioconjugation strategies. Finally, we provide ideas on how to further advance self-healing dyes, show new data on redox blinking caused by double-stranded DNA, and highlight forthcoming work on intramolecular photostabilization of fluorescent proteins.

A highly sensitive and selective chemosensors for detection of Zn2+ and its application in live cell imaging

A novel salen derivative, named as 6,6′-((1E,1′E)-(1,2-phenylenebis(azaneylylidene))bis(methaneylylidene))bis(4-(4-iodobutyl)-2-methylphenol) (PA), was obtained for the detection of Zn2+. PA exhibited highly sensitive and selective via a chelation mechanism, which was promoted by Zn2+, accompanied by enhanced fluorescence. After adding various metal ions in the PA solution, only Zn2+ caused PA fluorescence enhancement. Other cations (such as Ag+, Al3+, Ba2+, Ca2+, Cd2+, Cr3+, Cu2+, Fe3+, Hg2+, K+, La3+, Li+, Mg2+, Na+, Ni2+ and Pb2+) cannot induce similar response. The binding stoichiometry and detection limit was 1:1 and 16.3 nM, respectively, which is superior to the reported Zn2+ fluorescent probes based on salen derivatives. The binding process was reasonably speculated by fluorescence measurements, infrared spectroscopy, 1H NMR titrations, FT-IR, mass spectroscopy (MS) and density functional theory (DFT) calculation. In addition, PA can be used for Zn2+ fluorescence imaging in HeLa cells. This work indicated that this probe would be of great application prospect in medical diagnosis.

Picomolar Biosensing and Conformational Analysis Using Artificial Bidomain Proteins and Terbium-to-Quantum Dot Förster Resonance Energy Transfer.

Although antibodies remain a primary recognition element in all forms of biosensing, functional limitations arising from their size, stability, and structure have motivated the development and production of many different artificial scaffold proteins for biological recognition. However, implementing such artificial binders into functional high-performance biosensors remains a challenging task. Here, we present the design and application of Förster resonance energy transfer (FRET) nanoprobes comprising small artificial proteins (αRep bidomains) labeled with a Tb complex (Tb) donor on the C-terminus and a semiconductor quantum dot (QD) acceptor on the N-terminus. Specific binding of one or two protein targets to the αReps induced a conformational change that could be detected by time-resolved Tb-to-QD FRET. These single-probe FRET switches were used in a separation-free solution-phase assay to quantify different protein targets at sub-nanomolar concentrations and to measure the conformational changes with sub-nanometer resolution. Probing ligand-receptor binding under physiological conditions at very low concentrations in solution is a special feature of FRET that can be efficiently combined with other structural characterization methods to develop, understand, and optimize artificial biosensors. Our results suggest that the αRep FRET nanoprobes have a strong potential for their application in advanced diagnostics and intracellular live-cell imaging of ligand-receptor interactions.

Deep adversarial network for super stimulated emission depletion imaging

Stimulated emission depletion (STED), as one of the emerging super-resolution techniques, defines a state-of-the-art image resolution method. It has been developed into a universal fluorescent imaging tool over the past several years. The currently best available lateral resolution offered by STED is around 20 nm, but in real live cell imaging applications, the regular resolution offered through this mechanism is around 100 nm, limited by phototoxicity. Many critical biological structures are below this resolution level. Hence, it will be invaluable to improve the STED resolution through postprocessing techniques. We propose a deep adversarial network for improving the STED resolution significantly, which takes an STED image as an input, relies on physical modeling to obtain training data, and outputs a “self-refined” counterpart image at a higher resolution level. In other words, we use the prior knowledge on the STED point spread function and the structural information about the cells to generate simulated labeled data pairs for network training. Our results suggest that 30-nm resolution can be achieved from a 60-nm resolution STED image, and in our simulation and experiments, the structural similarity index values between the label and output result reached around 0.98, significantly higher than those obtained using the Lucy–Richardson deconvolution method and a state-of-the-art UNet-based super-resolution network.

Interaction of endothelial cells with plasma-polymer modified surfaces

Endothelialisation of implantable vascular grafts and stents is directed by the adsorbed protein layer. Chemical and mechanical cues sensed at the biomaterial surface by endothelial cells determine their attachment, survival and proliferation. Given the multiplicity of possible interactions we describe the novel application of live cell imaging, factorial analysis and single molecule imaging to investigate higher order interactions between surface chemistry and adsorbed proteins influencing endothelial cell (EC) adhesion dynamics. EC fates were tracked by time-lapse imaging of cell contact area on plasma polymer modified surfaces. The combinatorial effect of plasma polymer chemistry and adsorbed proteins was characterised by factorial experimental design and analysis. Single molecule imaging and counting was used for the first time to quantify binding of fluorescently labelled albumin to plasma polymerized surfaces: Modified glass surfaces with thin plasma polymer coatings rich in primary amine groups had a high affinity for albumin (~2, 650 molecules/μm2) while plasma polymer surfaces functionalised with hydroxyl groups had very low levels of albumin binding (22–30 molecules/μm2). Plasma polymer coatings rich in primary amine groups also promoted endothelial cell adhesion and was superior to tissue culture plastic. An interaction between albumin, heparin and fibronectin promoted adhesion to amine plasma polymer coated glass, while hydroxyl plasma polymer coated glass prevented EC attachment. Tracking cell-specific interactions with the adsorbed protein layer by time-lapse microscopy is more predictive of in vivo cellular responses to biomaterials compared to studies that only measure protein adsorption.

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.