Application In Live Cell Imaging Research Articles

A new 5-bromoindolehydrazone anchored diiodosalicylaldehyde derivative as efficient fluoro and chromophore for selective and sensitive detection of tryptamine and F− ions: Applications in live cell imaging

A novel indole hydrazone tagged moiety, 2-((5-bromo-1H-indol-2-yl) methylene) hydrazono) methyl)-4, 6-diiodophenol (BHDL) has been developed for the selective and sensitive detection of biogenic tryptamine and F− ions. The binding dexterity of probe BHDL towards F−/tryptamine (TryptA) has been investigated by UV–visible/fluorescence spectroscopy. In the presence of TryptA, probe exhibits strong enhancement in the emission band at 433 nm and the band at 555 nm underwent a blue shift accompanied by a decrease in intensity by the inhibition of Excited State Intramolecular Proton Transfer (ESIPT) on BHDL. Excitingly, complexation with F− ions as well triggers an enhancement in a fluorescence band at 430 nm with the concomitant disappearance of the emission band at 555 nm due to the inhibition of ESIPT and deprotonation process initiated by the hydrogen bonding complex formation. Further, Density Functional Theoretical (DFT) calculations have been performed to support the mechanism functioned on the probe BHDL in the presence of TryptA/F−.

Engineered HaloTag variants for fluorescence lifetime multiplexing

Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications.

Knotting Two Donor−π-Acceptor AIEgens Using a Nonconjugated Linker: Tunable and Switchable Fluorescence and Fingerprinting and Live Cell Imaging Applications

Aggregation-induced emissive fluorophores (AIEgens) have drawn widespread attention over the years because of their structure-controlled tunable and external-stimuli-responsive solid-state fluorescence, which have stimulated rapid development in the optoelectronics and biomedical fields. However, precise control of organic π-conjugated molecular fluorescence remains a great challenge and depends on the molecular structure and organization. Herein, we have interlinked two triphenylamine-based donor−π acceptor (D−π-A) AIEgens using a nonconjugate spacer (xylene) and explored the effect of interlinking, positional isomerism, and acceptor structure on the tunable and switchable solid-state fluorescence. A change of the acceptor group resulted in tuning of fluorescence between 534 and 614 nm. Interestingly, interspacing modulation from o-xylene (1) to the meta (2) and para isomers (3) showed a decrease of fluorescence efficiency in the solid state. A closer spacing between the AIEgens and tighter molecular packing could restrict the intramolecular rotation in ortho isomers that resulted in enhanced fluorescence in comparison to para isomers. In contrast, more spatial freedom in para isomers allowed the molecules to respond to external pressure and produced mechanofluorochromism (MFC) in comparison to ortho isomers. Strong tunable solid-state fluorescence of ortho isomers was used to obtain clear latent fingerprinting images on three different surfaces with tunable fluorescence color. 1a,b,e (orange, green, and red, respectively) were further utilized for live cell imaging applications using E. coli cells that demonstrated clear labeling of the cell membrane and cytoplasm with strong green/red fluorescence in the cell structures. In vitro and in vivo toxicity studies of 1a,b,e confirmed the nontoxic nature of these fluorophores and indicated their potential for live cell imaging applications.

High efficiency branched thermal activated delayed fluorescent probe based on cyanogroup for detecting Fe3+ with low limit of detection

A multipolarization branched thermal activated delayed fluorescence sensor BrACCN was designed and synthesized based on cyano-carbazole. The classical molecule structure 4CzCN was used as luminescent nucleus, which was progressively branched for photophysical optimization. On the basis of the thermal activated delayed fluorescence material 4CzCN and 4Cz-CzCN, the molecular modification was optimized to achieve long fluorescence life and high quantum yield. Through research and analysis, the fluorescence quantum yield of the branched probe was 73%, which was 1.5 times higher than that of unbranched 4CzCN. The fluorescence lifetime of the probe BrACCN was doubled up to 3 μs based on the introduction of peripheral Br atoms. Meanwhile, the probe has high selectivity and sensitivity for Fe3+ ions. In the presence of multiple ions, the probe responded only to Fe3+ with a complexing mode of 1:1 and the fluorescence was turned off. The probe has a low limit of detection (LOD) of 2.9 × 10−8 mol/L for Fe3+ ions. The probe also has a strong AIE characteristic, which is extremely beneficial for the application range of the probe. Moreover, these excellent properties of the probe will offer promising applications in live-cell confocal fluorescence imaging and time-resolved fluorescence imaging.

A hydrazone dual-functional fluorescent probe based on carbazole and coumarin groups for the detection of Cu2+ and ClO−: Application in live cell imaging and actual water samples

A new hydrazone derivative LL3 based on carbazole and coumarin was prepared by the condensation of 7-diethylaminocoumarin-3-carbohydrazide with 3-aldehyde-N-butyl carbazole and its structure was characterized using FT-IR, 1H NMR, 13C NMR, MS and elemental analysis techniques. LL3 could be used as a dual-functional fluorescent probe to identify Cu2+ and ClO−. Probe LL3 could recognize Cu2+ and formed a 2:1 complex. Under a 365 nm UV lamp, the color changed from yellowish to blue-green which could be readily distinguished by the naked eye. Meanwhile, under the catalysis of ClO−, the imine bond of probe LL3 was hydrolyzed to obtain carbazolealdehyde, resulting in specific UV-Vis and fluorescence phenomena, so as to realize the detection of ClO−. The recognition mechanism was proved by spectrometric titrations, Job’s plots, FT-IR, 1H NMR, mass spectroscopy (MS) and density functional theory (DFT) calculation analysis. The results of live cell imaging showed that LL3 could be used as a fluorescent probe to detect Cu2+ and ClO− in Hela cells. In addition, the probe LL3 could also be made into a test strip for the detection of Cu2+ in actual water samples.

Adenosine triphosphate (ATP) and zinc(II) ions responsive pyrene based turn-on fluorescent probe and its application in live cell imaging.

A novel highly selective and sensitive turn-on fluorescent chemosensor PCE to recognize Zn2+ has been developed. The sensor PCE displays a remarkable fluorescent enhancement at 456nm (λex=340nm) with Zn2+ without the interference of other biologically important relevant metal ions in aqueous acetonitrile solution. Job's plot and mass spectral studies divulge such the interaction of PCE by Zn2+ was 1:1 binding stoichiometry. The association constant and detection limit of PCE to recognize Zn2+ was found to be 0.948×104M-1 and 4.82×10-7M respectively. The nature of turn-on fluorescence sensor was supported by TD-DFT calculations. And the synthesized probe PCE was able to image intracellular Zn2+ in living cells using confocal imaging techniques. PCE-Zn ensemble showed the remarkable fluorescence enhancement with ATP selectively among other biologically important phosphates. 31P NMR experiments suggesting that the triphosphates unit of ATP is intact with the PCEZn. PCE-Zn ensemble can be utilized for monitoring ATP in live cells.

A highly selective colorimetric and fluorescent probe for cysteine sensing: Application in live cell imaging and test strips

Here, a coumarin-based probe 4-chloro-3-formyl-2-oxo-2H-chromen-7-yl acrylate (named CFCA), which performed high efficient and specific recognition for cysteine (Cys), was successfully synthesized and characterized by NMR and MS. The CFCA probe showed colorimetric and “turn-on” fluorescent signals to Cys, and its color changes could be recognized by naked eyes. CFCA responded to Cys with the characteristics of rapid response time (less than 40 s), high selectivity, and excellent anti-interference. Among all kinds of samples, only Cys could significantly enhance the fluorescence intensity of the solution, which was not affected by the presence of other interferences. At the same time, within a certain concentration range, there was a good linear relationship between relative intensity of fluorescence and Cys concentration. The detection limit has been calculated to be as low as 0.33 μM. And the sensing mechanism of CFCA with Cys has been well confirmed by 1H NMR and HR-MS spectra. Moreover, the fluorescence probe CFCA was successfully used for imaging Cys in living cells and processed into test strips for on-site analysis and testing.

Graphene Oxide Biosensors Based on Hybridization Chain Reaction Signal Amplification for Detecting Biomarkers of Radiation-Resistant Nasopharyngeal Carcinoma and Imaging in Living Cells.

Since microRNA-205 (miRNA-205) is a predictive biomarker for antiradiation of nasopharyngeal carcinoma (NPC), quantitative detection of miRNA-205 is important for developing personalized strategies for the treatment of NPC. In this investigation, based on the graphene oxide (GO) sensor and hybridization chain reaction (HCR) for fluorescence signal amplification, a highly sensitive and selective detection method for miRNA-205 was designed. A target-recycling mechanism is employed, where a single miRNA-205 target triggers the signal amplification of many DNA signal probes. The biosensor shows the ability to analyze miRNA-205 in solution, and it can detect miRNA-205 at concentrations as low as 311.96 pM. Furthermore, the method is specific in that it distinguishes between a target miRNA and a sequence with single-, double-, and three-base mismatches, as well as other miRNAs. Considering its simplicity and superior sensitivity, it was also verified in 1‰ serum with a detection limit of 111.65 pM. Importantly, the method successfully demonstrated that miRNA-205 could be imaged in living cells, which provided the possibility of localizing target molecules in live cell imaging applications. This method has great clinical application potential in the determination of miRNA-205, a biomarker for radiation-resistant NPC.

Development of novel blue emissive carbon dots for sensitive detection of dual metal ions and their potential applications in bioimaging and chelation therapy

In recent years, there has been an increased interest in the use of fluorescent carbon dots (C-dots) as an alternative to toxic synthetic organic dyes in live cell imaging, medical diagnostics and other diverse biomedical applications. In the present work, highly fluorescent C-dots have been prepared from commercially available peptic digest of animal tissue, without using any additional chemical reagent for surface functionalization. The synthesized C-dots are monodispersed nanomaterials with hydroxyl, carboxyl and amino surface functionalities and remain stable in high ionic strength media and over a wide range of pH. They exhibit a strong blue fluorescence upon excitation at 350 nm with a high quantum yield of 61%. The potential use of these C-dots as fluorescent nanoprobe for specific metal ions (Cu2+ and Hg2+) detection through chelation, radical scavenging and cellular imaging has been investigated. These highly fluorescent labels show rapid cellular internalization without hampering cellular integrity. Cell proliferation assay demonstrates that these C-dots have minimal cell toxicity even at a high concentration of 3 mg/mL. They show significant quenching on incubation with cells pretreated with Cu2+ and Hg2+ ions. They also show rapid exocytosis from cells showcasing their high potential for chelation therapy. Thus, this study offers a scalable synthesis approach for highly fluorescent and bio-compatible C-dots from a cost-effective precursor, which can be used for specific metal ions detection and their removal from cells through chelation via exocytosis. They can also be used as an alternate to synthetic organic dyes for fluorescence-based cell-imaging.

3D Structured Illumination Microscopy via Channel Attention Generative Adversarial Network

Three-dimensional (3D) structured illumination microscopy (SIM) plays an important role in biological volumetric imaging with the capabilities of doubling the lateral and axial resolution and optical sectioning. However, 3D-SIM suffers from more photobleaching and phototoxicity compared to other volumetric imaging modalities, such as light-sheet microscopy, because it requires 15 raw images per axial slice, which hampers its widespread application in live cell imaging. Here we report the design of a channel attention generative adversarial network (caGAN) that improves the quality of 3D-SIM reconstruction under low signal-to-noise-ratio (SNR) condition and enables reconstruction using fewer raw images. Compared to the conventional algorithm, caGAN-SIM achieves comparable or higher reconstruction fidelity while using 15-fold less signal level. We demonstrate the superior performance of caGAN-SIM for various subcellular structures and its ability in long-term multi-color 3D super-resolution imaging using the example of dynamic interactions between microtubules and lysosomes in live cells.

Fluorogenic recognition of Zn2+, Cd2+ by a new Pyrazoline-based Multi-Analyte chemosensor and its application in live cell imaging

A novel multi-response pyrazoline-based fluorescent probe 1 was designed and synthesized with a very simple molecular structure, which exhibited high sensitivity for detecting Zn2+ and Cd2+ in ethanol aqueous solution state based on the dissuasion of intramolecular charge transfer (ICT) and chelation-enhanced fluorescence (CHEF) effect. The binding modes of the complexes were determined to have 1:1 complexation stoichiometry and the binding constants were calculated to be 1.284 × 104 M−1 for Zn2+ and 4.313 × 103 M−1 for Cd2+ using the nonlinear curve-fitting method. Zn2+ and Cd2+ sensor have high anti-interference ability. Under optimal conditions, the detection limits were determined to be 0.9274 μM for Zn2+ and 0.8158 μM for Cd2+. Notably, the probe was found to have good biological compatibility and was employed to detect Zn2+/ Cd2+ in living Hela cells.

A benzimidazole-based new fluorogenic differential/sequential chemosensor for Cu2+, Zn2+, CN-, P2O74-, DNA, its live-cell imaging and pyrosequencing applications

A benzimidazole–based chemosensor ( DFB) for Cu 2+ and Zn 2+ was developed, and its sequential anions (CN − and P 2 O 7 4- ) and DNA sensing capability and its live-cell imaging, as well as its PPi detection in PCR-amplified DNA products, has been demonstrated. • A benzimidazole-based chemosensor ( DFB ) shows “ off-on ” responses upon differential detection of Cu 2+ and Zn 2+ . • The DFB derived Cu 2+ & Zn 2+ complexes act as efficient sensors toward CN − , PPi and DNA in an aqueous medium. • DFB acts as a sequential fluorescent probe to detect Cu 2+ , Zn 2+ and PPi in living cancer cells. • DFB-Zn 2 + can detect the PPi in the PCR amplified DNA products. Differential chemosensors have emerged as next-generation systems due to their simplicity and favourable responsive properties to produce different signals upon selective binding of various analytes simultaneously. Nevertheless, given their inadequate fluorescence response and laborious synthetic procedures, only a few differential chemosensors have been developed so far. In this work, we have employed a single pot synthesis strategy to establish a new benzimidazole-based Schiff base type fluorogenic chemosensor ( DFB ) which differentially detects Cu 2+ (detection limit (LOD) = 24.4 ± 0.5 nM) and Zn 2+ (LOD = 2.18 ± 0.1 nM) through fluorescence “ off-on ” manner over the library of other metal cations in an aqueous medium. The DFB -derived ‘ in situ ’ complexes DFB-Cu 2+ and DFB-Zn 2+ showed fluorescence revival “ on-off ” responses toward cyanide (CN − ) and bio-relevant pyrophosphate (P 2 O 7 4- -PPi) ions with a significantly low LOD of 9.43 ± 0.2 and 2.9 ± 0.1 nM, respectively, in water. We have demonstrated the phosphate group-specific binding capability of DFB-Zn 2+ , by testing it with both ssDNA and dsDNA samples which displayed fluorescence “ turn-off ” response (LOD ∼10 -7 M), similar to the PPi binding in an aqueous medium, indicating that it interacts explicitly with the phosphate backbone of DNA. We have also harnessed the DFB as a sequential fluorescent probe to detect Cu 2+ , Zn 2+ , CN − and P 2 O 7 4- ions in human cervical (HeLa) and breast (MCF-7 and MDA-MB-231 (aggressive and invasive)) cancer cell lines. Moreover, we have explored the PPi recognition capability of DFB-Zn 2+ in the polymerase-chain-reaction (PCR) products where PPi is one of the primary by-products during amplification of DNA.

A dual responsive probe based on bromo substituted salicylhydrazone moiety for the colorimetric detection of Cd2+ ions and fluorometric detection of F‒ ions: Applications in live cell imaging

A dual responsive probe based on bromo substituted salicylhydrazone moiety for the colorimetric detection of Cd2+ ions and fluorometric detection of F‒ ions: Applications in live cell imaging

Dual color triads: synthesis, photophysics and applications in live cell imaging

Fluorescent labels and probes constitute an important class of organic materials used in the development of sensor systems and imaging platforms for various chemical and molecular biology applications.

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.