Probes For Live Cell Imaging Research Articles

Water-soluble BODIPY-conjugated glycopolymers as fluorescent probes for live cell imaging

Fluorescent probes have attracted ongoing attention in early detection of tumor cells. The development of novel fluorescent materials with high cellular internalization efficiency, good photostability, and high specificity for tumor cells is in urgent demand. Herein, a highly water-soluble, multivalent and highly specific BODIPY-conjugated glycopolymer was synthesized by Atom Transfer Radical Polymerization (ATRP) for direct tumor cell imaging, which showed good photostability. The fluorescence quantum yield of a BODIPY-conjugated glycopolymer increased to 0.52 compared with 0.31 of BODIPYMA due to the introduction of galactose. The cell viability of BODIPY-conjugated glycopolymers against HepG2 and NIH3T3 cells was more than 80%, indicating that the glycopolymers have low cytotoxicity to living cells. Moreover, simple incubation of living cells with a BODIPY-conjugated glycopolymer led to efficient internalization into HepG2 and clear visualization in cytoplasm, due to the high brightness of BODIPY and good specificity between HepG2 and galactose as compared to NIH3T3 cells. These results suggest that BODIPY-conjugated glycopolymers have potential use as fluorescent probes in live cell imaging. Further fine-tuning the fluorescent property and targeting ability of BODIPY-conjugated glycopolymers can lead to more complicated imaging applications and subcellular target detection.

Development of live-cell imaging probes for monitoring histone modifications

The combination of histone posttranslational modifications occurring in nucleosomal histones determines the epigenetic code. Histone modifications such as acetylation are dynamically controlled in response to a variety of signals during the cell cycle and differentiation, but they are paradoxically maintained through cell division to impart tissue specific gene expression patterns to progeny. The dynamics of histone modifications in living cells are poorly understood, because of the lack of experimental tools to monitor them in a real-time fashion. Recently, FRET-based imaging probes for histone H4 acetylation have been developed, which enabled monitoring of changes in histone acetylation during the cell cycle and drug treatment. Further development of this type of fluorescent probes for other modifications will make it possible to visualize complicated epigenetic regulation in living cells.

Microarray-guided discovery of two-photon (2P) small molecule probes for live-cell imaging of cysteinyl cathepsin activities

A microarray immobilized with 105 aldehyde-containing small molecules was screened against mammalian cell lysates over-expressing cathepsin L to identify two potent inhibitors, which were subsequently converted into cell-permeable probes capable of live-cell imaging of endogenous cysteinyl cathepsin activities by two-photon fluorescence microscopy.

Regiospecific Synthesis of 1,2-Disubstituted (Hetero)aryl Fused Imidazoles with Tunable Fluorescent Emission

A palladium-catalyzed two or fourfold amination was established that allows regiospecific synthesis of a diversity-oriented library of 1,2-disubstituted (hetero)aryl fused imidazoles, and provides an exceptional tool for the discovery of fluorescent scaffolds with tunable fluorescence emission. These fluorophores have been applied as fluorescent probes for live cell imaging.

Microwave-Assisted Aqueous Synthesis of Highly Luminescent Carboxymethyl Chitosan-Coated CdTe/CdS Quantum Dots as Fluorescent Probe for Live Cell Imaging

This paper describes the development of a simplified and rapid method for the aqueous synthesis of quantum dots (QDs) with CdTe cores and gradient CdS external shells (CdTe/CdS QDs) aided by microwave irradiation. Several synthesis parameters, such as molar ratio of reagents, pH, reaction temperature, and reaction time, were studied in details. Under the optimized conditions, highly effective CdTe/CdS QDs could be synthesized in aqueous phase in only 15min. In order to improve the biocompatibility of the CdTe/CdS QDs, these QDs were then interacted with carboxymethyl chitosan (CMC) so as they could be used as fluorescent probes in the aqueous phase. With the incorporation of CMC, the stability of modified QDs was found to have improved significantly (from 4months to more than 10months at room temperature). The photoluminescence quantum yield (PLQY) of the modified QDs could reach 75%, other parameters include a full width at half maximum of the emission (FWHM) spectrum as 40 ~ 60nm, and an average size, estimated from electron microscopic images, as 3.5nm. As fluorescent probes, these modified QDs were successfully used for imaging live Madin-Darby canine kidney (MDCK) cells, in which the preliminary results indicated that these modified QDs demonstrated good biocompatibility and showed promising applications for bio-labeling and imaging.

Form follows function – The versatile fungal cytoskeleton

The cytoskeleton plays a major role in the regulation of fungal cell morphogenesis. The fungal cytoskeleton is comprised of three polymers: F-actin, microtubules and septins. Due to the successful application of the newly developed Lifeact probe for live-cell imaging of F-actin it is now possible, in combination with existing microtubule markers and fluorescently labelled septins, to monitor real-time dynamics of the entire fungal cytoskeleton, and reassess the many and integrated roles of F-actin, microtubules and septins throughout fungal growth and development. Evidence is accumulating that functional properties of higher-order structures derived from actin and septin filaments interacting with microtubules are employed in different ways in different cell types. This may reflect marked differences in cytoskeletal architecture that are found, for example, in unicellular yeasts, spore germlings and mature fungal hyphae. In this review we address key aspects of the versatile fungal cytoskeleton, highlight recently gained insights into important roles of F-actin in filamentous fungi, and raise some key questions that are likely to be solved in the coming years based on the new experimental tools that have recently become available.

Biotin tethered homotryptamine derivatives: High affinity probes of the human serotonin transporter (hSERT)

Quantum dot conjugates of compounds capable of inhibiting the serotonin transporter (SERT) could form the basis of fluorescent probes for live cell imaging of membrane bound SERT. Additionally, quantum dot-SERT antagonist conjugates may be amenable to fluorescence-based, high-throughput assays for this transporter. This Letter describes the synthesis of SERT-selective ligands amenable to conjugation to quantum dots via a biotin-streptavidin binding interaction. SERT selectivity and affinity were incorporated into the ligand via a tetrahydropyridine or cyclohexylamine derivative and the affinity of these compounds for SERT was measured by their ability to produce SERT-dependent currents in Xenopus laveis oocytes.

Mechanism of cellular uptake of highly fluorescent conjugated polymer nanoparticles.

Conjugated polymer nanoparticles are formed by precipitation of highly fluorescent conjugated polymers to form small nanoparticles with extremely bright fluorescence. We characterized cellular uptake and cytotoxicity of 18 ± 5 nm PFBT conjugated polymer nanoparticles in J774A.1 cells. Significant nanoparticle uptake was observed, indicating efficient nanoparticle entry into cells, even for short (1 h) incubations. The high fluorescence of these nanoparticles allows extremely low loading concentrations; PFBT nanoparticle fluorescence in cells could be detected with loading concentrations of 155 pM (270 ppb). Cellular uptake slows at low temperature, consistent with endocytic entry. Nanoparticles colocalize with Texas Red dextran and are trafficked to lysosomes, as demonstrated by the location of nanoparticle fluorescence in perinuclear organelles that also stain with an anti-LAMP-1 antibody. Inhibition of uptake by phosphoinositide 3-kinase inhibitors implicates macropinocytosis as the operative endocytic mechanism. No significant cytotoxic or inflammatory effects could be observed, making PFBT nanoparticles attractive probes for live cell imaging.

Calcium Transduces Plasma Membrane Receptor Signals to Produce Diacylglycerol at Golgi Membranes

Protein kinase C and protein kinase D are potently activated by agonist-evoked increases in diacylglycerol. Using live cell-imaging probes for kinase activity, we have shown that both kinases are robustly activated at the Golgi following stimulation of G(q)-coupled receptor signaling pathways, displaying activation signatures at the Golgi that are distinct from those at the plasma membrane. Here we report that Ca(2+) is the mediator that allows signals received at the plasma membrane to activate these two protein kinases at the Golgi. Specifically, using fluorescence resonance energy transfer-based reporters to image diacylglycerol production, we show that Ca(2+) is necessary and sufficient to elevate diacylglycerol levels at the Golgi. First, raising intracellular Ca(2+) by treating cells with thapsigargin induces diacylglycerol production at the Golgi. Second, chelation of intracellular Ca(2+) prevents UTP-stimulated increases in diacylglycerol at the Golgi. Thus, agonist-evoked increases in intracellular Ca(2+) cause increases in Golgi diacylglycerol, allowing this intracellular membrane to serve as a platform for signaling by protein kinases C and D.

Two-color Photoactivatable Probe for Selective Tracking of Proteins and Cells

We report the development and application of photoactivatable Green Cherry (G(PA)C), the first genetically encoded "continuously red-photoactivatable green" two-color probe for live cell imaging. G(PA)C is unique in that it enables real-time tracking of selected subpopulations of proteins and organelles in the cell or of cells within tissues and whole organisms, with constant reference to the entire population of the probe. Using G(PA)C-zyxin as proof of utility, we obtained new insights into the dynamic movement of the cytoskeletal protein zyxin. We show that zyxin is continuously and rapidly recruited from the cytosol into established focal adhesions. It can also move rapidly within a given focal adhesion and "hop" between adjacent focal adhesions, emphasizing the dynamic nature of proteins within these structures. The in vivo utility of G(PA)C is exemplified by tracking hemocyte movements using a versatile transgenic Drosophila model engineered to express G(PA)C in tissues and cells of interest under the control of the GAL4-inducible promoter.

Diacylglycerol production at the Golgi: regulation by Ca2+

Protein kinase C and protein kinase D are potently activated by agonist‐evoked increases in diacylglycerol. Using live cell‐imaging probes for kinase activity, we have previously shown that both kinases are robustly active at the Golgi following stimulation of Gq‐coupled receptor signaling pathways, displaying activation signatures at the Golgi that are distinct from those at the plasma membrane. Here we address the mechanism by which signals received at the plasma membrane cause these two protein kinases to be activated at the Golgi. Using FRET‐based reporters to image diacylglycerol production, we show that Ca2+ is necessary and sufficient to transduce signals from the plasma membrane to the Golgi. First, chelation of intracellular Ca2+ prevents UTP‐dependent increases in diacylglycerol at the Golgi, but not plasma membrane. Second, raising intracellular Ca2+ by treating cells with thapsigargin induces diacylglycerol production at the Golgi. Thus, agonist‐evoked increases in intracellular Ca2+ cause increases in Golgi diacylglycerol, allowing this intracellular membrane to serve as a platform for signaling by protein kinases C and D.This work was supported by NIH GM43154 and NIH DK54441.

Tetrac-Conjugated Quantum Dots for Diagnosis and Treatment of Pancreatic Cancer: An In Vitro Evaluation

We report a preliminary investigation of the use of pegylated quantum dots (PEG-QDs) conjugated to tetraiodothyroacetic acid (tetrac), a thyroid hormone antagonist and anti-proliferative agent, as a luminescent probe for live cell imaging and treatment of human pancreatic cancer. PEG-QDs were conjugated to epoxyactivated tetrac through primary amino groups present on the PEG-QDs. Cellular entry of tetrac-conjugated PEG-QDs (tetrac-PEG-QDs) in vitro was examined by confocal microscopy and effects of PEG-QDs on cell proliferation were examined by CyQUANT® Cell Proliferation Assay. Tetrac-PEG-QDs exhibited a higher level of entry into pancreatic cancer (PANC-1) cells than unconjugated PEG-QDs. In addition, pre-incubation of cells with the thyroid hormone analogue L-thyroxine (T4) increased fluorescent intensity, indicating enhanced uptake of tetrac-PEG-QDs. Proliferation of PANC-1 cells was significantly inhibited by treatment with tetrac-PEG-QDs. Keywords: Quantum dots, tetrac, pancreatic cancer, confocal microscopy, cell proliferation, PANC-1 cells

Folate receptor–targeted quantum dot liposomes as fluorescence probes

In this study, the preparation of the novel imaging agents, folate receptor (FR)–targeted liposomes encapsulating hydrophilic CdTe quantum dots (QDs), and their use as luminescence probes for live cell imaging are reported. Hydrophilic CdTe QDs were directly synthesized in the water phase, and FR-targeted QD liposomes were prepared by hydrating the lipid thin film with CdTe suspension. Formulations were characterized by UV-visible and fluorescent measurements, liposomal particle size, and zeta potential. The targeting and imaging ability of FR-targeted liposomes were investigated against the human uterine cervix cancer cell line (HeLa). Furthermore, the cytotoxicity of QD liposomes was evaluated by HeLa cells incubated with FR-targeted QD liposomes, nontargeted QD liposomes, and free QDs. The results showed that FR-targeted QD liposomes were spherically shaped with high fluorescence yield, excellent photochemical stability, good cancer targeting, and minimal cytotoxicity. The average size of FR-targeted fluorescence liposomes was ~105 nm, and their size distribution was rather narrow. After storage at 4°C for 11 months, QD liposomes maintained similar size and did not show any leakage of QDs. FR-targeted CdTe QD liposomes, which can target tumor cells via FR-mediated endocytosis, would become an attractive probe for tumor cell or tissue imaging for a long-time monitoring.

Development of 2,6-carboxy-substituted boron dipyrromethene (BODIPY) as a novel scaffold of ratiometric fluorescent probes for live cell imaging

Ratiometric fluorescent probes based on boron dipyrromethene (BODIPY) were developed based on a novel design strategy, in which a change of the electron-withdrawing character of the 2,6-substituents resulting from reaction with a target molecule generates a fluorescence wavelength change.

Thioflavin T Displays Enhanced Fluorescence Selectively Inside Anionic Micelles and Mammalian Cells

Thioflavin T (ThT) has been widely employed to detect amyloid fibrils in tissues and recently in presence of SDS micelles. However, the contribution of membranes or micelles to ThT fluorescence has never been investigated. In this paper, we show for the first time that the anionic micellar microenvironment of SDS has a profound impact on the absorption and fluorescence spectra of ThT in sharp contrast to cationic (CTAB) and neutral micelles (Triton X-100 & Tween 20). Unlike CTAB or Triton X-100 or Tween 20 micelles, formation of SDS micelles shifts the lambdamax for ThT absorption from 412 nm in buffer to 428 nm inside the micelle, with a 28% increase in the peak molar absorptivity and an approximately 13 fold increase in ThT fluorescence (lambdamax=489 nm). Extending these observations to cell plasma membranes, we show that ThT can quickly enter and appear selectively fluorescent inside mammalian cells like BHK21 and HT29, against a dark background owing to negligible fluorescence from free ThT in aqueous medium. The above results suggest that ThT can be a useful probe for live cell imaging and for selectively labeling micelles on the basis of the charge in the polar headgroup.

Simple synthesis of carbon-11 labeled styryl dyes as new potential PET RNA-specific, living cell imaging probes

A new type of styryl dyes have been developed as RNA-specific, live cell imaging probes for fluorescent microscopy technology to study nuclear structure and function. This study was designed to develop carbon-11 labeled styryl dyes as new probes for biomedical imaging technique positron emission tomography (PET) imaging of RNA in living cells. Precursors ( E)-2-(2-(1-(triisopropylsilyl)-1 H-indol-3-yl)vinyl)quinoline ( 2), ( E)-2-(2,4,6-trimethoxystyryl)quinoline ( 3) and ( E)-4-(2-(6-methoxyquinolin-2-yl)vinyl)- N, N-diemthylaniline ( 4), and standards styryl dyes E36 ( 6), E144 ( 7) and F22 ( 9) were synthesized in multiple steps with moderate to high chemical yields. Precursor 2 was labeled by [ 11C]CH 3OTf, trapped on a cation-exchange CM Sep-Pak cartridge following a quick deprotecting reaction by addition of ( n-Bu) 4NF in THF, and isolated by solid-phase extraction (SPE) purification to provide target tracer [ 11C]E36 ([ 11C] 6) in 40–50% radiochemical yields, decay corrected to end of bombardment (EOB), based on [ 11C]CO 2. The target tracers [ 11C]E144 ([ 11C] 7) and [ 11C]F22 ([ 11C] 9) were prepared by N-[ 11C]methylation of the precursors 3 and 4, respectively, using [ 11C]CH 3OTf and isolated by SPE method in 50–70% radiochemical yields at EOB. The specific activity of the target tracers [ 11C] 6, [ 11C] 7 and [ 11C] 9 was in a range of 74–111 GBq/μmol at the end of synthesis (EOS).

Cyanine dye conjugates as probes for live cell imaging

A series of fluorescent compounds suitable for live cell imaging is described. Functionalized forms of four different asymmetric cyanine dyes are reported that are amenable to peptide conjugation. The photophysical properties of the modified dyes and conjugates and the use of the compounds as cellular imaging agents are described. The results obtained indicate that these spectrally versatile compounds, which have absorption and emission profiles spanning the visible spectrum, are useful probes for cellular imaging.

A protocol for preparing, characterizing and using three RNA-specific, live cell imaging probes: E36, E144 and F22

This protocol outlines a methodology for the preparation and characterization of three RNA-specific fluorescent probes (E36, E144 and F22) and their use in live cell imaging. It describes a detailed procedure for their chemical synthesis and purification; serial product characterization and quality control tests, including measurements of their fluorescence properties in solution, measurement of RNA specificity and analysis of cellular toxicity; and live cell staining and counterstaining with Hoechst or DAPI. Preparation and application of these RNA imaging probes takes 1 week.

RNA-Selective, Live Cell Imaging Probes for Studying Nuclear Structure and Function

The higher-order structural organization of the cell nucleus reflects the underlying genome-wide transcriptional activity and macromolecular transport processes. To study the microscopic organization of RNA distribution within the nucleus, a combinatorial library of fluorescent styryl molecules was synthesized and screened for an in vitro RNA response and live cell nuclear imaging. Four different cell lines (HeLa, A549, 3T3, and 3T3-L1) were analyzed in terms of higher-order nuclear organization. We identified RNA-selective dyes with better imaging properties relative to commercially available SYTORNASelect dye; the selected dyes were also cell permeant, photostable, and well tolerated by the cells. Our dyes also had very good counterstain compatibility with Hoechst and DAPI, which could help to image the DNA distribution in relation to RNA distribution in live cells and therefore reveal different patterns of RNA-DNA colocalization.

Folate-Receptor-Mediated Delivery of InP Quantum Dots for Bioimaging Using Confocal and Two-Photon Microscopy

A novel method for the synthesis of highly monodispersed hydrophillic InP-ZnS nanocrystals and their use as luminescence probes for live cell imaging is reported. Hydrophobic InP-ZnS nanocrystals are prepared by a new method that yields high-quality, luminescent core-shell nanocrystals within 6-8 h of total reaction time. Then by carefully manipulating the surface of these passivated nanocrystals, aqueous dispersions of folate-conjugated nanocrystals (folate-QDs) with high photostability are prepared. By use of confocal microscopy, we demonstrate the receptor-mediated delivery of folic acid conjugated quantum dots into folate-receptor-positive cell lines such as KB cells. These folate-QDs tend to accumulate in multi-vescicular bodies of KB cells after 6 h of incubation. Receptor-mediated delivery was confirmed by comparison with the uptake of these particles in folate-receptor-negative cell lines such as A549. Efficient two-photon excitation of these particles and two-photon imaging using these particles are also demonstrated. The use of these InP-ZnS nanoparticles and their efficient two-photon excitation can be potentially useful for deep tissue imaging for future in vivo studies.