Application In Live Cell Imaging Research Articles

An anthracene–quinoline based dual-mode fluorometric–colorimetric sensor for the detection of Fe3+ and its application in live cell imaging

A highly sensitive anthracene–quinoline based dual-mode sensor has been synthesized and used for the fluorometric and colorimetric detection of Fe3+ and in live cell imaging.

A highly selective pyridoxal-based chemosensor for the detection of Zn(ii) and application in live-cell imaging; X-ray crystallography of pyridoxal-TRIS Schiff-base Zn(ii) and Cu(ii) complexes.

In a simple, one-step reaction, we have synthesized a pyridoxal-based chemosensor by reacting tris(hydroxymethyl)aminomethane (TRIS) together with pyridoxal hydrochloride to yield a Schiff-base ligand that is highly selective for the detection of Zn(ii) ion. Both the ligand and the Zn(ii) complex have been characterized by 1H & 13C NMR, ESI-MS, CHN analyses, and X-ray crystallography. The optical properties of the synthesized ligand were investigated in an aqueous buffer solution and found to be highly selective and sensitive toward Zn(ii) ion through a fluorescence turn-on response. The competition studies reveal the response for zinc ion is unaffected by all alkali and alkaline earth metals; and suppressed by Cu(ii) ion. The ligand itself shows a weak fluorescence intensity (quantum yield, Φ = 0.04), and the addition of zinc ion enhanced the fluorescence intensity 12-fold (quantum yield, Φ = 0.48). The detection limit for zinc ion was 2.77 × 10−8 M, which is significantly lower than the WHO's guideline (76.5 μM). Addition of EDTA to a solution containing the ligand–Zn(ii) complex quenched the fluorescence, indicating the reversibility of Zn(ii) binding. Stoichiometric studies indicated the formation of a 2 : 1 L2Zn complex with a binding constant of 1.2 × 109 M−2 (±25%). The crystal structure of the zinc complex shows the same hydrated L2Zn complex, with Zn(ii) ion binding with an octahedral coordination geometry. We also synthesized the copper(ii) complex of the ligand, and the crystal structure showed the formation of a 1 : 1 adduct, revealing 1-dimensional polymeric networks with octahedral coordinated Cu(ii). The ligand was employed as a sensor to detect zinc ion in HEK293 cell lines derived from human embryonic kidney cells grown in tissue culture which showed strong luminescence in the presence of Zn(ii). We believe that the outstanding turn-on response, sensitivity, selectivity, lower detection limit, and reversibility toward zinc ion will find further application in chemical and biological science.

Strategic engineering of alkyl spacer length for a pH-tolerant lysosome marker and dual organelle localization.

Long-term visualization of lysosomal properties is extremely crucial to evaluate diseases related to their dysfunction. However, many of the reported lysotrackers are less conducive to imaging lysosomes precisely because they suffer from fluorescence quenching and other inherent drawbacks such as pH-sensitivity, polarity insensitivity, water insolubility, slow diffusibility, and poor photostability. To overcome these limitations, we have utilized an alkyl chain length engineering strategy and synthesized a series of lysosome targeting fluorescent derivatives namely NIMCs by attaching a morpholine moiety at the peri position of the 1,8-naphthalimide (NI) ring through varying alkyl spacers between morpholine and 1,8-naphthalimide. The structural and optical properties of the synthesized NIMCs were explored by 1H-NMR, single-crystal X-ray diffraction, UV-Vis, and fluorescence spectroscopy. Afterward, optical spectroscopic measurements were carefully performed to identify a pH-tolerant, polarity sensitive, and highly photostable fluoroprobes for further live-cell imaging applications. NIMC6 displayed excellent pH-tolerant and polarity-sensitive properties. Consequently, all NIMCs were employed in kidney fibroblast cells (BHK-21) to investigate their applicability for lysosome targeting and probing lysosomal micropolarity. Interestingly, a switching of localization from lysosomes to the endoplasmic reticulum (ER) was also achieved by controlling the linker length and this phenomenon was subsequently applied in determining ER micropolarity. Additionally, the selected probe NIMC6 was also employed in BHK-21 cells for 3-D spheroid imaging and in Caenorhabditis elegans (C. elegans) for in vivo imaging, to evaluate its efficacy for imaging animal models.

A novel dehydroabietic acid-based turn-on fluorescent probe for the detection of bisulfite and its application in live-cell and zebrafish imaging

A novel “Turn-on” fluorescent probe, which displayed prominent sensitivity and selectivity for the detection of HSO3−, was synthesized from dehydroabietic acid. The probe also showed high lysosome-targeting properties when sensing HSO3− in MCF-7 cells.

Benzene Linked Dipodal Naphthalene: Chemosensor with Colorimetric Enhancement and Fluorimetric Quenching for Fe3+ Ion and its Application in Live Cell Imaging

In this work, two naphthalene scaffolds dangling around a benzene moiety as a colorimetric and fluorimetric probe for Fe3+ in aqueous media have been reported. Recognition processes of Fe3+ were shown to be scarcely influenced by other coexisting metal ions. The probe exhibits a turn on colorimetric and a turn off fluorimetric detection of Fe3+ ions. The association constant for sensor 1–Fe3+ complex was found as Ka = 1.12 × 105 M–1 with the detection limit of 1.7 nM. The chemosensor has been utilized for bioimaging of E. coli cells.

C-tag TNF: a reporter system to study TNF shedding

TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow the live visualization of shedding events. Here, we generated C-tag TNF as a genetically encoded reporter to study TNF shedding at the single-cell level. The functionality of the C-tag TNF reporter is based on the exposure of a cryptic epitope on the C terminus of the transmembrane portion of pro-TNF on cleavage. In both denatured and nondenatured samples, this epitope can be detected by a nanobody in a highly sensitive and specific manner only upon TNF shedding. As such, C-tag TNF can successfully be used for the detection of TNF cleavage in flow cytometry and live-cell imaging applications. We furthermore demonstrate its applicability in a forward genetic screen geared toward the identification of genetic regulators of TNF maturation. In summary, the C-tag TNF reporter can be employed to gain novel insights into the complex regulation of ADAM-dependent TNF shedding.

A Boron Dipyrromethene-Based Fluorescence 'OFF-ON' Probe for Sensitive and Selective Detection of Palladium(II) Ions and Its Application in Live Cell Imaging.

A novel boron dipyrromethene (BODIPY)-based fluorescent probe BDP-Pd was designed and synthesized. Upon coordination with Pd2+ , the emission of the probe at 508 nm significantly increased, showing an 'OFF-ON' fluorescence response. The complexation of BDP-Pd with Pd2+ in both acetonitrile and aqueous solution were then studied by absorption and fluorescence spectra. The binding stoichiometry between the probe and Pd2+ was found to be 1 : 2, and the binding constant was determined to be 8.5×1010 M-2 and 8.2×1010 M-2 in acetonitrile and aqueous solution, respectively. The probe exhibited a detection limit as low as 0.72 ppb toward Pd2+ with no obvious interference from up to 21 species of common metal ions, suggesting BDP-Pd as a sensitive and selective fluorescent probe for Pd2+ detection. The fast fluorescence 'OFF-ON' phenomenon of the probe upon coordination with Pd2+ ions could be easily observed by a hand-hold UV lamp under naked eye in solution as well as on homemade test trips. Density functional theory (DFT) calculations were carried out to give the optimized structure of complex BDP-Pd : 2Pd2+ and rationalize the detection mechanism through a prohibited intramolecular photoinduced electron transfer (PET) process. The bio-imaging application of the probe was investigated and it showed excellent cell permeability for fluorescent imaging of Pd2+ ions in A549 human non-small cell lung cancer cells.

A novel dehydroabietic acid-based fluorescent probe for detection of Fe3+ and Hg2+ ions and its application in live-cell imaging

A novel dehydroabietic acid-based fluorescent probe (DBH) containing 2,4-diarylbenzimidazole moiety was designed and synthesized, and the compound was characterized by its UV–Vis, FT-IR, 1H NMR, 13C NMR and ESI-MS spectra. DBH could be used for the detection of Fe3+ and Hg2+ through the selective fluorescence quenching in EtOH/H2O solution. The probe DBH showed excellent selectivity and sensitivity towards Fe3+ and Hg2+ over other metal ions. The limits of detection were calculated as 3.12 × 10−8 M and 3.10 × 10−8 M, respectively. Especially, DBH have a wide pH range for ion detection (5 ~ 12), and a short response time (10 s). Job’s plot method in combination with ESI-MS and FT-IR spectra was used to determine the stoichiometry of DBH-Fe3+/Hg2+ complexes as both 2:1. Moreover, DBH exhibited low cytotoxicity to MCF-7 and SMCC-7721 cells (IC50 > 100 μM). In the fluorescent cell imaging experiments, DBH could be used as a prominent intracellular probe for the detection of Fe3+ and Hg2+ in living cells.

Live-cell imaging with Aspergillus fumigatus-specific fluorescent siderophore conjugates

Live-cell imaging allows the in vivo analysis of subcellular localisation dynamics of physiological processes with high spatial–temporal resolution. However, only few fluorescent dyes have been custom-designed to facilitate species-specific live-cell imaging approaches in filamentous fungi to date. Therefore, we developed fluorescent dye conjugates based on the sophisticated iron acquisition system of Aspergillus fumigatus by chemical modification of the siderophore triacetylfusarinine C (TAFC). Various fluorophores (FITC, NBD, Ocean Blue, BODIPY 630/650, SiR, TAMRA and Cy5) were conjugated to diacetylfusarinine C (DAFC). Gallium-68 labelling enabled in vitro and in vivo characterisations. LogD, uptake assays and growth assays were performed and complemented by live-cell imaging in different Aspergillus species. Siderophore conjugates were specifically recognised by the TAFC transporter MirB and utilized as an iron source in growth assays. Fluorescence microscopy revealed uptake dynamics and differential subcellular accumulation patterns of all compounds inside fungal hyphae.[Fe]DAFC-NBD and -Ocean Blue accumulated in vacuoles, whereas [Fe]DAFC-BODIPY, -SiR and -Cy5 localised to mitochondria. [Fe]DAFC -FITC showed a uniform cytoplasmic distribution, whereas [Fe]DAFC-TAMRA was not internalised at all. Co-staining experiments with commercially available fluorescent dyes confirmed these findings. Overall, we developed a new class of fluorescent dyes that vary in intracellular fungal targeting , thereby providing novel tools for live-cell imaging applications for Aspergillus fumigatus.

Design and synthesis of new salicylhydrazone tagged indole derivative for fluorometric sensing of Zn2+ ion and colorimetric sensing of F− ion: Applications in live cell imaging

A novel, bifunctional, colorimetric, highly selective and sensitive “turn-on” fluorescent probe, 2-((E)-(((E)-(5-bromo-1H-indol-3-yl) methylene) hydrazono) methyl) phenol (IHP) has been designed and synthesized for the detection of Zn2+/F− ions in DMSO: H2O (9:1, v/v) medium. The probe IHP exposed an excellent fluorescence enhancement towards Zn2+ ion owing to the inhibition of ESIPT with concurrent enhancement of LMCT operated on IHP. Upon addition of F− ion, the probe IHP displayed an obvious colorimetric change and it can be simply observed by naked eye. Further, 1:1 binding stoichiometric ratio of probe with Zn2+/F− ions was confirmed via Job’s plot and highly supported by 1H NMR titration and 19F NMR analysis for F− ion. The plausible mechanistic pathway and the fluorescence enhancement of IHP with Zn2+ ions were highly gauged by density functional theoretical (DFT) calculations. Moreover, the probe IHP has been successfully employed for monitoring Zn2+ ion in HeLa cells and live cell imaging analysis in Zebrafish embryos.

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.