Study and selection of in vivo protein interactions by coupling bimolecular fluorescence complementation and flow cytometry.

PubMed

Morell, Montse; Espargaro, Alba; Aviles, Francesc Xavier; Ventura, Salvador

2008-01-01

We present a high-throughput approach to study weak protein-protein interactions by coupling bimolecular fluorescent complementation (BiFC) to flow cytometry (FC). In BiFC, the interaction partners (bait and prey) are fused to two rationally designed fragments of a fluorescent protein, which recovers its function upon the binding of the interacting proteins. For weak protein-protein interactions, the detected fluorescence is proportional to the interaction strength, thereby allowing in vivo discrimination between closely related binders with different affinity for the bait protein. FC provides a method for high-speed multiparametric data acquisition and analysis; the assay is simple, thousands of cells can be analyzed in seconds and, if required, selected using fluorescence-activated cell sorting (FACS). The combination of both methods (BiFC-FC) provides a technically straightforward, fast and highly sensitive method to validate weak protein interactions and to screen and identify optimal ligands in biologically synthesized libraries. Once plasmids encoding the protein fusions have been obtained, the evaluation of a specific interaction, the generation of a library and selection of active partners using BiFC-FC can be accomplished in 5 weeks.

Bimolecular fluorescence complementation and targeted biotinylation provide insight into the topology of the skeletal muscle Ca2+ channel β1a subunit

PubMed Central

Sheridan, David C.; Moua, Ong; Lorenzon, Nancy M.; Beam, Kurt G.

2012-01-01

In skeletal muscle, L-type calcium channels (DHPRs), localized to plasma membrane sarcoplasmic reticulum junctions, are tightly packed into groups of four termed tetrads. Here, we have used bimolecular fluorescence complementation (BiFC) and targeted biotinylation to probe the structure and organization of β1a subunits associated with native CaV1.1 in DHPRs of myotubes. The construct YN-β1a-YC, in which the non-fluorescent fragments of YFP (“YN” corresponding to YFP residues 1–158, and “YC” corresponding to YFP residues 159–238) were fused, respectively, to the N- and C-termini of β1a, was fully functional and displayed yellow fluorescence within DHPR tetrads after expression in β1-knockout (β1KO) myotubes; this yellow fluorescence demonstrated the occurrence of BiFC of YN and YC on the β1a N- and C-termini. In these experiments, we avoided overexpression because control experiments in non-muscle cells indicated that this could result in non-specific BiFC. BiFC of YN-β1a-YC in DHPR tetrads appeared to be intramolecular between N- and C-termini of individual β1a subunits rather than between adjacent DHPRs because BiFC (1) was observed for YN-β1a-YC co-expressed with CaV1.2 (which does not form tetrads) and (2) was not observed after co-expression of YN-β1a-YN plus YC-β1a-YC in β1KO myotubes. Thus, β1a function is compatible with N- and C-termini being close enough together to allow BiFC. However, both termini appeared to have positional freedom and not to be closely opposed by other junctional proteins since both were accessible to gold-streptavidin conjugates. Based on these results, a model is proposed for the arrangement of β1a subunits in DHPR tetrads. PMID:22522946

Synthesis of di-functional ligand and fluorescently labeling SiO2 microspheres

NASA Astrophysics Data System (ADS)

Chen, Kexu; Kang, Ming; Liu, Min; Shen, Simin; Sun, Rong

2018-05-01

In order to complete the fluorescent labeling of SiO2 microspheres, a kind of di-functional ligand was synthesized and purified, which could not only coordinate rare earth ions but also react with the active groups to bond host materials with an alkoxysilane groups. Fourier transform infrared spectroscopy (FT-IR), 1H NMR spectra, MS spectra, field emission scanning electron microscope (FESEM), transmission electron microscope (TEM), X-ray photoelectron spectroscopy (XPS) and luminescence spectrophotometer were used to study the structure of di-functional ligand and properties of fluorescent coupling agent and fluorescent labeled SiO2 microspheres. The optimal experiment conditions were acquired as follows: molar ratio as 1: 4 (MDBM: MICPTES), reaction time at 6 h and reaction temperature as 65 °C (yield up to 40%) through the orthogonal experiment and purification process. The results indicated that fluorescent coupling agent presented red photoluminesence of Eu3+ ions at 610 nm, and the absolute quantum yield was 11%. On the other hand, the hydrolysis of the coupling agent reacted on the surface of SiO2 microspheres and presented fluorescent labeling homogeneously.

Bi-directional exchange of membrane components occurs during co-culture of mesenchymal stem cells and nucleus pulposus cells.

PubMed

Strassburg, Sandra; Hodson, Nigel W; Hill, Patrick I; Richardson, Stephen M; Hoyland, Judith A

2012-01-01

Mesenchymal stem cell (MSC)-based therapies have been proposed as novel treatments for intervertebral disc (IVD) degeneration. We have previously demonstrated that when MSCs are co-cultured with nucleus pulposus (NP) cells with direct cell-cell contact, they differentiate along the NP lineage and simultaneously stimulate the degenerate NP cell population to regain a normal (non-degenerate) phenotype, an effect which requires cell-cell communication. However, the mechanisms by which NP cells and MSCs interact in this system are currently unclear. Thus, in this study we investigated a range of potential mechanisms for exchange of cellular components or information that may direct these changes, including cell fusion, gap-junctional communication and exchange of membrane components by direct transfer or via microvesicle formation. Flow cytometry of fluorescently labeled MSCs and NP cells revealed evidence of some cell fusion and formation of gapjunctions, although at the three timepoints studied these phenomena were detectable only in a small proportion of cells. While these mechanisms may play a role in cell-cell communication, the data suggests they are not the predominant mechanism of interaction. However, flow cytometry of fluorescently dual-labeled cells showed that extensive bi-directional transfer of membrane components is operational during direct co-culture of MSCs and NP cells. Furthermore, there was also evidence for secretion and internalization of membrane-bound microvesicles by both cell types. Thus, this study highlights bi-directional intercellular transfer of membrane components as a possible mechanism of cellular communication between MSC and NP cells.

Carbon "Quantum" Dots for Fluorescence Labeling of Cells.

PubMed

Liu, Jia-Hui; Cao, Li; LeCroy, Gregory E; Wang, Ping; Meziani, Mohammed J; Dong, Yiyang; Liu, Yuanfang; Luo, Pengju G; Sun, Ya-Ping

2015-09-02

The specifically synthesized and selected carbon dots of relatively high fluorescence quantum yields were evaluated in their fluorescence labeling of cells. For the cancer cell lines, the cellular uptake of the carbon dots was generally efficient, resulting in the labeling of the cells with bright fluorescence emissions for both one- and two-photon excitations from predominantly the cell membrane and cytoplasm. In the exploration on labeling the live stem cells, the cellular uptake of the carbon dots was relatively less efficient, though fluorescence emissions could still be adequately detected in the labeled cells, with the emissions again predominantly from the cell membrane and cytoplasm. This combined with the observed more efficient internalization of the same carbon dots by the fixed stem cells might suggest some significant selectivity of the stem cells toward surface functionalities of the carbon dots. The needs and possible strategies for more systematic and comparative studies on the fluorescence labeling of different cells, including especially live stem cells, by carbon dots as a new class of brightly fluorescent probes are discussed.

In vitro labeling strategies for in cellulo fluorescence microscopy of single ribonucleoprotein machines.

PubMed

Custer, Thomas C; Walter, Nils G

2017-07-01

RNA plays a fundamental, ubiquitous role as either substrate or functional component of many large cellular complexes-"molecular machines"-used to maintain and control the readout of genetic information, a functional landscape that we are only beginning to understand. The cellular mechanisms for the spatiotemporal organization of the plethora of RNAs involved in gene expression are particularly poorly understood. Intracellular single-molecule fluorescence microscopy provides a powerful emerging tool for probing the pertinent mechanistic parameters that govern cellular RNA functions, including those of protein coding messenger RNAs (mRNAs). Progress has been hampered, however, by the scarcity of efficient high-yield methods to fluorescently label RNA molecules without the need to drastically increase their molecular weight through artificial appendages that may result in altered behavior. Herein, we employ T7 RNA polymerase to body label an RNA with a cyanine dye, as well as yeast poly(A) polymerase to strategically place multiple 2'-azido-modifications for subsequent fluorophore labeling either between the body and tail or randomly throughout the tail. Using a combination of biochemical and single-molecule fluorescence microscopy approaches, we demonstrate that both yeast poly(A) polymerase labeling strategies result in fully functional mRNA, whereas protein coding is severely diminished in the case of body labeling. © 2016 The Protein Society.

Development and validation of a high-content bimolecular fluorescence complementation assay for small-molecule inhibitors of HIV-1 Nef dimerization.

PubMed

Poe, Jerrod A; Vollmer, Laura; Vogt, Andreas; Smithgall, Thomas E

2014-04-01

Nef is a human immunodeficiency virus 1 (HIV-1) accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to nonfluorescent, complementary fragments of yellow fluorescent protein (YFP) and coexpressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus a monomeric red fluorescent protein (mRFP) reporter were expressed from a single vector, separated by picornavirus "2A" linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type versus dimerization-defective Nef were very clearly separated, with Z factors consistently in the 0.6 to 0.7 range. A fully automated pilot screen of the National Cancer Institute Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function.

Development of a general methodology for labelling peptide-morpholino oligonucleotide conjugates using alkyne-azide click chemistry.

PubMed

Shabanpoor, Fazel; Gait, Michael J

2013-11-11

We describe a general methodology for fluorescent labelling of peptide conjugates of phosphorodiamidate morpholino oligonucleotides (PMOs) by alkyne functionalization of peptides, subsequent conjugation to PMOs and labelling with a fluorescent compound (Cy5-azide). Two peptide-PMO (PPMO) examples are shown. No detrimental effect of such labelled PMOs was seen in a biological assay.

Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

PubMed

Peng, Tao; Hang, Howard C

2016-11-02

Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

Interactions of the EcoRV restriction endonuclease with fluorescent oligodeoxynucleotides.

PubMed

Erskine, S G; Halford, S E

1995-05-19

A self-complementary dodecadeoxyribonucleotide that contains the recognition sequence for the R.EcoRV ENase was synthesised with a primary amino group at its 5' terminus. The 5' amino function was labeled with the fluorescent dye 5-[dimethylamino] napthalene-1-sulfonyl chloride. The labeled oligodeoxyribonucleotide in its duplex form was shown to be a suitable substrate for kinetic studies on the ENase and that no significant dye-DNA or dye-protein interactions occurred. Finally, the binding of R.EcoRV to the labeled DNA was followed by detecting the fluorescence resonance energy transfer between the tryptophans of the protein and the fluorescent labels of the DNA.

Enzyme-enhanced fluorescence detection of DNA on etched optical fibers.

PubMed

Niu, Shu-yan; Li, Quan-yi; Ren, Rui; Zhang, Shu-sheng

2009-05-15

A novel DNA biosensor based on enzyme-enhanced fluorescence detection on etched optical fibers was developed. The hybridization complex of DNA probe and biotinylated target was formed on the etched optical fiber, and was then bound with streptavidin labeled horseradish peroxidase (streptavidin-HRP). The target DNA was quantified through the fluorescent detection of bi-p,p'-4-hydroxyphenylacetic acid (DBDA) generated from the substrate 4-hydroxyphenylacetic acid (p-HPA) under the catalysis of HRP, with a detection limit of 1 pM and a linear range from 1.69 pM to 169 pM. It is facile to regenerate this sensor through surface treatment with concentrated urea solution. It was discovered that the sensor can retain 70% of its original activity after three detection-regeneration cycles.

Volume labeling with Alexa Fluor dyes and surface functionalization of highly sensitive fluorescent silica (SiO2) nanoparticles

NASA Astrophysics Data System (ADS)

Wang, Wei; Nallathamby, Prakash D.; Foster, Carmen M.; Morrell-Falvey, Jennifer L.; Mortensen, Ninell P.; Doktycz, Mitchel J.; Gu, Baohua; Retterer, Scott T.

2013-10-01

A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and a polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or ``free'' surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface-modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems.A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and a polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or ``free'' surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface-modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems. Electronic supplementary information (ESI) available: Cell culture preparation for dose/response imaging experiments. See DOI: 10.1039/c3nr02639f

Intense upconversion luminescence and effect of local environment for Tm3+/Yb3+ co-doped novel TeO2-BiCl3 glass system.

PubMed

Wang, Guonian; Dai, Shixun; Zhang, Junjie; Wen, Lei; Yang, Jianhu; Jiang, Zhonghong

2006-05-15

We present the results of a study that uses theoretical and experimental methods to investigate the characteristics of the upconversion luminescence of Tm3+/Yb3+ codoped TeO2-BiCl3 glass system as a function of the BiCl3 fraction. These glasses are potentially important in the design of upconversion fiber lasers. Effect of local environment around Tm3+ on upconversion fluorescence intensity was analyzed by theoretical calculations. The structure and spectroscopic properties were investigated in the experiments by measuring the Raman spectra, IR transmission spectra, and absorption and fluorescence intensities at room temperature. The results indicate that blue luminescence quantum efficiency increases with increasing BiCl3 content from 10 to 60 mol%, which were interpreted by the increase of asymmetry of glass structure, decrease of phonon energy and removing of OH- groups.

Protein-binding aptamer assisted signal amplification for the detection of influenza A (H1N1) DNA sequences based on quantum dot fluorescence polarization analysis.

PubMed

Zhang, Juanni; Tian, Jianniao; He, Yanlong; Chen, Sheng; Jiang, Yixuan; Zhao, Yanchun; Zhao, Shulin

2013-09-07

We report a fluorescence polarization platform for H1N1 detection based on the construction of a DNA functional QD fluorescence polarization probe and a bi-functional protein binding aptamer (Apt-DNA). The assay has a linear range from 10 nM to 100 nM with a detection limit of 3.45 nM and is selective over the mismatched bases.

Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

PubMed

Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

2014-01-15

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

DNA-length-dependent quenching of fluorescently labeled iron oxide nanoparticles with gold, graphene oxide and MoS2 nanostructures.

PubMed

Balcioglu, Mustafa; Rana, Muhit; Robertson, Neil; Yigit, Mehmet V

2014-08-13

We controlled the fluorescence emission of a fluorescently labeled iron oxide nanoparticle using three different nanomaterials with ultraefficient quenching capabilities. The control over the fluorescence emission was investigated via spacing introduced by the surface-functionalized single-stranded DNA molecules. DNA molecules were conjugated on different templates, either on the surface of the fluorescently labeled iron oxide nanoparticles or gold and nanographene oxide. The efficiency of the quenching was determined and compared with various fluorescently labeled iron oxide nanoparticle and nanoquencher combinations using DNA molecules with three different lengths. We have found that the template for DNA conjugation plays significant role on quenching the fluorescence emission of the fluorescently labeled iron oxide nanoparticles. We have observed that the size of the DNA controls the quenching efficiency when conjugated only on the fluorescently labeled iron oxide nanoparticles by setting a spacer between the surfaces and resulting change in the hydrodynamic size. The quenching efficiency with 12mer, 23mer and 36mer oligonucleotides decreased to 56%, 54% and 53% with gold nanoparticles, 58%, 38% and 32% with nanographene oxide, 46%, 38% and 35% with MoS2, respectively. On the other hand, the presence, not the size, of the DNA molecules on the other surfaces quenched the fluorescence significantly with different degrees. To understand the effect of the mobility of the DNA molecules on the nanoparticle surface, DNA molecules were attached to the surface with two different approaches. Covalently immobilized oligonucleotides decreased the quenching efficiency of nanographene oxide and gold nanoparticles to ∼22% and ∼21%, respectively, whereas noncovalently adsorbed oligonucleotides decreased it to ∼25% and ∼55%, respectively. As a result, we have found that each nanoquencher has a powerful quenching capability against a fluorescent nanoparticle, which can be tuned with surface functionalized DNA molecules.

Volume Labeling with Alexa-Fluor Dyes and Surface Functionalization of Highly Sensitive Fluorescent SiO2 Nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Foster, Carmen M; Morrell-Falvey, Jennifer L

2013-01-01

A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and amore » polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or free surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems.« less

Live Cell Visualization of Multiple Protein-Protein Interactions with BiFC Rainbow.

PubMed

Wang, Sheng; Ding, Miao; Xue, Boxin; Hou, Yingping; Sun, Yujie

2018-05-18

As one of the most powerful tools to visualize PPIs in living cells, bimolecular fluorescence complementation (BiFC) has gained great advancement during recent years, including deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins. However, little progress has been made toward simultaneous detection and visualization of multiple PPIs in the same cell, mainly due to the spectral crosstalk. In this report, we developed novel BiFC assays based on large-Stokes-shift fluorescent proteins (LSS-FPs) to detect and visualize multiple PPIs in living cells. With the large excitation/emission spectral separation, LSS-FPs can be imaged together with normal Stokes shift fluorescent proteins to realize multicolor BiFC imaging using a simple illumination scheme. We also further demonstrated BiFC rainbow combining newly developed BiFC assays with previously established mCerulean/mVenus-based BiFC assays to achieve detection and visualization of four PPI pairs in the same cell. Additionally, we prove that with the complete spectral separation of mT-Sapphire and CyOFP1, LSS-FP-based BiFC assays can be readily combined with intensity-based FRET measurement to detect ternary protein complex formation with minimal spectral crosstalk. Thus, our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in living cells.

Optical Properties of CdSe/ZnS Nanocrystals

PubMed Central

Gaigalas, Adolfas K; DeRose, Paul; Wang, Lili; Zhang, Yu-Zhong

2014-01-01

Measurements are presented of the absorbance, fluorescence emission, fluorescence quantum yield, and fluorescence lifetime of CdSe/ZnS nanocrystals, also known as quantum dots (QDs). The study included three groups of nanocrystals whose surfaces were either passivated with organic molecules, modified further with carboxyl groups, or conjugated with CD14 mouse anti-human antibodies. The surface modifications had observable effects on the optical properties of the nanocrystals. The oscillator strength (OS) of the band edge transition was about 1.0 for the nanocrystals emitting at 565 nm, 605 nm, and 655 nm. The OS could not be determined for QDs with emission at 700 nm and 800 nm. The fluorescence lifetimes varied from 26 ns for nanocrystals emitting near 600 nm to 150 ns for nanocrystals emitting near 800 nm. The quantum yield ranged between 0.4 and 0.9 for the nanocrystals in this study. A brightness index (BI) was used to evaluate the suitability of the nanocrystal labels for flow cytometer measurements. Most QD labels are at least as bright as fluorescein for applications in flow cytometer assays with 488 nm excitation. For optimal brightness the QDs should be excited with 405 nm light. We observed a strong dependence of the QD absorbance at 250 nm on the surface modification of the QD. PMID:26601047

HaloTag technology for specific and covalent labeling of fusion proteins.

PubMed

Benink, Hélène A; Urh, Marjeta

2015-01-01

Appending proteins of interest to fluorescent protein tags such as GFP has revolutionized how proteins are studied in the cellular environment. Over the last few decades many varieties of fluorescent proteins have been generated, each bringing new capability to research. However, taking full advantage of standard fluorescent proteins with advanced and differential features requires significant effort on the part of the researcher. This approach necessitates that many genetic fusions be generated and confirmed to function properly in cells with the same protein of interest. To lessen this burden, a newer category of protein fusion tags termed "self-labeling protein tags" has been developed. This approach utilizes a single protein tag, the function of which can be altered by attaching various chemical moieties (fluorescent labels, affinity handles, etc.). In this way a single genetically encoded protein fusion can easily be given functional diversity and adaptability as supplied by synthetic chemistry. Here we present protein labeling methods using HaloTag technology; comprised of HaloTag protein and the collection of small molecules designed to bind it specifically and provide it with varied functionalities. For imaging purposes these small molecules, termed HaloTag ligands, contain distinct fluorophores. Due to covalent and rapid binding between HaloTag protein and its ligands, labeling is permanent and efficient. Many of these ligands have been optimized for permeability across cellular membranes allowing for live cell labeling and imaging analysis. Nonpermeable ligands have also been developed for specific labeling of surface proteins. Overall, HaloTag is a versatile technology that empowers the end user to label a protein of interest with the choice of different fluorophores while alleviating the need for generation of multiple genetic fusions.

Instant live-cell super-resolution imaging of cellular structures by nanoinjection of fluorescent probes.

PubMed

Hennig, Simon; van de Linde, Sebastian; Lummer, Martina; Simonis, Matthias; Huser, Thomas; Sauer, Markus

2015-02-11

Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.

Time-Domain Fluorescence Lifetime Imaging Techniques Suitable for Solid-State Imaging Sensor Arrays

PubMed Central

Li, David Day-Uei; Ameer-Beg, Simon; Arlt, Jochen; Tyndall, David; Walker, Richard; Matthews, Daniel R.; Visitkul, Viput; Richardson, Justin; Henderson, Robert K.

2012-01-01

We have successfully demonstrated video-rate CMOS single-photon avalanche diode (SPAD)-based cameras for fluorescence lifetime imaging microscopy (FLIM) by applying innovative FLIM algorithms. We also review and compare several time-domain techniques and solid-state FLIM systems, and adapt the proposed algorithms for massive CMOS SPAD-based arrays and hardware implementations. The theoretical error equations are derived and their performances are demonstrated on the data obtained from 0.13 μm CMOS SPAD arrays and the multiple-decay data obtained from scanning PMT systems. In vivo two photon fluorescence lifetime imaging data of FITC-albumin labeled vasculature of a P22 rat carcinosarcoma (BD9 rat window chamber) are used to test how different algorithms perform on bi-decay data. The proposed techniques are capable of producing lifetime images with enough contrast. PMID:22778606

A novel multiplexed fluorescence polarisation immunoassay based on a recombinant bi-specific single-chain diabody for simultaneous detection of fluoroquinolones and sulfonamides in milk.

PubMed

Chen, Min; Wen, Kai; Tao, Xiaoqi; Ding, Shuangyang; Xie, Jie; Yu, Xuezhi; Li, Jiancheng; Xia, Xi; Wang, Yang; Xie, Sanlei; Jiang, Haiyang

2014-01-01

Major research efforts are focusing on the development of simultaneous multiplexed immunoassays. In this study, a novel dual-binding fluorescence polarisation immunoassay (DB-FPIA) using a broad-specificity bi-specific single-chain diabody (scDb) and two fluorescent-labelled tracers (sulfamethoxypyridazine-fluorescein isothiocyanate (SMP-FITC) and sarafloxacin-Texas Red (SAR-TR)) with different excitation and emission wavelengths was developed for simultaneous and high-throughput detection of 19 fluoroquinolones (FQs) and 13 sulfonamides (SAs) at the maximum residue limits in milk samples. Recoveries for spiked milk samples were from 76.4% to 128.4%, with a relative standard deviation lower than 13.9%. The developed DB-FPIA was then applied to field samples, followed by confirmation by LC-MS/MS. All three instances in which FQs and SAs were present at concentrations near or above the assay limit of detection were identified as positive by the developed DB-FPIA, demonstrating that the method is suitable for rapid screening of FQs and SAs contamination. The novel methodology combines the advantage of the FPIA and the broad sensitivity of scDb and shows great promise for fast multi-analyte screening of low-molecular weight chemical residues in food samples.

Rapid Covalent Fluorescence Labeling of Membrane Proteins on Live Cells via Coiled-Coil Templated Acyl Transfer.

PubMed

Reinhardt, Ulrike; Lotze, Jonathan; Mörl, Karin; Beck-Sickinger, Annette G; Seitz, Oliver

2015-10-21

Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. In labeling reactions targeted against specific tag sequences, the size of the fluorophore-tag is of major concern. The tag should be small to prevent interference with protein function. Furthermore, rapid and covalent labeling methods are desired to enable the analysis of fast biological processes. Herein, we describe the development of a method in which the formation of a parallel coiled coil triggers the transfer of a fluorescence dye from a thioester-linked coil peptide conjugate onto a cysteine-modified coil peptide. This labeling method requires only small tag sequences (max 23 aa) and occurs with high tag specificity. We show that size matching of the coil peptides and a suitable thioester reactivity allow the acyl transfer reaction to proceed within minutes (rather than hours). We demonstrate the versatility of this method by applying it to the labeling of different G-protein coupled membrane receptors including the human neuropeptide Y receptors 1, 2, 4, 5, the neuropeptide FF receptors 1 and 2, and the dopamine receptor 1. The labeled receptors are fully functional and able to bind the respective ligand with high affinity. Activity is not impaired as demonstrated by activation, internalization, and recycling experiments.

Photophysical properties of fluorescently-labeled peptoids.

PubMed

Rudat, Birgit; Birtalan, Esther; Vollrath, Sidonie B L; Fritz, Daniel; Kölmel, Dominik K; Nieger, Martin; Schepers, Ute; Müllen, Klaus; Eisler, Hans-Jürgen; Lemmer, Uli; Bräse, Stefan

2011-09-01

Fluorescently-labeled biomolecules are often utilized in biochemical or cellular experiments without further detailed spectroscopical characterization. This report is intended to narrow this gap and therefore presents the photophysical investigation of a library of 17 fluorescently-labeled molecules, namely peptoid transporters. First, one peptoid structure is labeled with seven different fluorophores and the spectroscopical properties are examined. Absorption and fluorescence maxima are almost identical for free dyes and conjugated dyes, suggesting free choice of a spectrally suitable fluorophore for different applications. Otherwise, extinction coefficients and quantum yields, and therefore the brightness of all seven dyes are strongly influenced. For the fluorophores, e.g. rhodamine B, the extent of this influence depends on the peptoid itself. This is shown by comparing different structures in the second part of this report. Especially the side chain functionalities influence the brightness. And finally, peptoids having two identical fluorescent labels are presented, which show decreased quantum yields. Possible reasons for the observed photophysical properties are discussed. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Adapter reagents for protein site specific dye labeling.

PubMed

Thompson, Darren A; Evans, Eric G B; Kasza, Tomas; Millhauser, Glenn L; Dawson, Philip E

2014-05-01

Chemoselective protein labeling remains a significant challenge in chemical biology. Although many selective labeling chemistries have been reported, the practicalities of matching the reaction with appropriately functionalized proteins and labeling reagents is often a challenge. For example, we encountered the challenge of site specifically labeling the cellular form of the murine Prion protein with a fluorescent dye. To facilitate this labeling, a protein was expressed with site specific p-acetylphenylalanine. However, the utility of this acetophenone reactive group is hampered by the severe lack of commercially available aminooxy fluorophores. Here we outline a general strategy for the efficient solid phase synthesis of adapter reagents capable of converting maleimido-labels into aminooxy or azide functional groups that can be further tuned for desired length or solubility properties. The utility of the adapter strategy is demonstrated in the context of fluorescent labeling of the murine Prion protein through an adapted aminooxy-Alexa dye. © 2014 Wiley Periodicals, Inc.

Adapter Reagents for Protein Site Specific Dye Labeling

PubMed Central

Thompson, Darren A.; Evans, Eric G. B.; Kasza, Tomas; Millhauser, Glenn L.; Dawson, Philip E.

2016-01-01

Chemoselective protein labeling remains a significant challenge in chemical biology. Although many selective labeling chemistries have been reported, the practicalities of matching the reaction with appropriately functionalized proteins and labeling reagents is often a challenge. For example, we encountered the challenge of site specifically labeling the cellular form of the murine Prion protein with a fluorescent dye. To facilitate this labeling, a protein was expressed with site specific p-acetylphenylalanine. However, the utility of this aceto-phenone reactive group is hampered by the severe lack of commercially available aminooxy fluorophores. Here we outline a general strategy for the efficient solid phase synthesis of adapter reagents capable of converting maleimido-labels into aminooxy or azide functional groups that can be further tuned for desired length or solubility properties. The utility of the adapter strategy is demonstrated in the context of fluorescent labeling of the murine Prion protein through an adapted aminooxy-Alexa dye. PMID:24599728

Development and Validation of a High-Content Bimolecular Fluorescence Complementation Assay for Small Molecule Inhibitors of HIV-1 Nef Dimerization

PubMed Central

Poe, Jerrod A.; Vollmer, Laura; Vogt, Andreas; Smithgall, Thomas E.

2014-01-01

Nef is an HIV-1 accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to non-fluorescent, complementary fragments of YFP and co-expressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus an mRFP reporter were expressed from a single vector, separated by picornavirus ‘2A’ linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type vs. dimerization-defective Nef were very clearly separated, with Z-factors consistently in the 0.6–0.7 range. A fully automated pilot screen of the NIH Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function. PMID:24282155

Comparison of fluorescence-based methods to determine nanoparticle uptake by phagocytes and non-phagocytic cells in vitro.

PubMed

Claudia, Meindl; Kristin, Öhlinger; Jennifer, Ober; Eva, Roblegg; Eleonore, Fröhlich

2017-03-01

At many portals of entry the relative uptake by phagocytes and non-phagocytic cells has a prominent effect on availability and biological action of nanoparticles (NPs). Cellular uptake can be determined for fluorescence-labeled NPs. The present study compares three methods (plate reader, flow cytometry and image analysis) in order to investigate the influence of particle size and functionalization and medium content on cellular uptake of fluorescence-labeled polystyrene particles and to study the respective method́s suitability for uptake studies. For comparison between the techniques, ratios of macrophage to alveolar epithelial cell uptakes were used. Presence of serum protein in the exposure solution decreased uptake of carboxyl-functionalized and non-functionalized particles; there was no clear effect for the amine-functionalized particles. The 200nm non- or carboxyl-functionalized NPs were taken up preferentially by phagocytes while for amine-functionalized particles preference was lowest. The presence of the serum slightly increased the preference for these particles. In conclusion, due to the possibility of calibration, plate reader measurements might present a better option than the other techniques to (semi)quantify differences between phagocytes and non-phagocytic cells for particles with different fluorescence. In order to obtain unbiased data the fluorescent labeling has to fulfill certain requirements. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Comparison of fluorescence-based methods to determine nanoparticle uptake by phagocytes and non-phagocytic cells in vitro

PubMed Central

Claudia, Meindl; Kristin, Öhlinger; Jennifer, Ober; Eva, Roblegg; Eleonore, Fröhlich

2017-01-01

At many portals of entry the relative uptake by phagocytes and non-phagocytic cells has a prominent effect on availability and biological action of nanoparticles (NPs). Cellular uptake can be determined for fluorescence-labeled NPs. The present study compares three methods (plate reader, flow cytometry and image analysis) in order to investigate the influence of particle size and functionalization and medium content on cellular uptake of fluorescence–labeled polystyrene particles and to study the respective method’s suitability for uptake studies. For comparison between the techniques, ratios of macrophage to alveolar epithelial cell uptakes were used. Presence of serum protein in the exposure solution decreased uptake of carboxyl-functionalized and non-functionalized particles; there was no clear effect for the amine-functionalized particles. The 200 nm non- or carboxyl-functionalized NPs were taken up preferentially by phagocytes while for amine-functionalized particles preference was lowest. The presence of the serum slightly increased the preference for these particles. In conclusion, due to the possibility of calibration, plate reader measurements might present a better option than the other techniques to (semi)quantify differences between phagocytes and non-phagocytic cells for particles with different fluorescence. In order to obtain unbiased data the fluorescent labeling has to fulfill certain requirements. PMID:28065592

Small-molecule-based protein-labeling technology in live cell studies: probe-design concepts and applications.

PubMed

Mizukami, Shin; Hori, Yuichiro; Kikuchi, Kazuya

2014-01-21

The use of genetic engineering techniques allows researchers to combine functional proteins with fluorescent proteins (FPs) to produce fusion proteins that can be visualized in living cells, tissues, and animals. However, several limitations of FPs, such as slow maturation kinetics or issues with photostability under laser illumination, have led researchers to examine new technologies beyond FP-based imaging. Recently, new protein-labeling technologies using protein/peptide tags and tag-specific probes have attracted increasing attention. Although several protein-labeling systems are com mercially available, researchers continue to work on addressing some of the limitations of this technology. To reduce the level of background fluorescence from unlabeled probes, researchers have pursued fluorogenic labeling, in which the labeling probes do not fluoresce until the target proteins are labeled. In this Account, we review two different fluorogenic protein-labeling systems that we have recently developed. First we give a brief history of protein labeling technologies and describe the challenges involved in protein labeling. In the second section, we discuss a fluorogenic labeling system based on a noncatalytic mutant of β-lactamase, which forms specific covalent bonds with β-lactam antibiotics such as ampicillin or cephalosporin. Based on fluorescence (or Förster) resonance energy transfer and other physicochemical principles, we have developed several types of fluorogenic labeling probes. To extend the utility of this labeling system, we took advantage of a hydrophobic β-lactam prodrug structure to achieve intracellular protein labeling. We also describe a small protein tag, photoactive yellow protein (PYP)-tag, and its probes. By utilizing a quenching mechanism based on close intramolecular contact, we incorporated a turn-on switch into the probes for fluorogenic protein labeling. One of these probes allowed us to rapidly image a protein while avoiding washout. In the future, we expect that protein-labeling systems with finely designed probes will lead to novel methodologies that allow researchers to image biomolecules and to perturb protein functions.

Noncharged and Charged Monodendronised Perylene Bisimides as Highly Fluorescent Labels and their Bioconjugates.

PubMed

Huth, Katharina; Heek, Timm; Achazi, Katharina; Kühne, Christian; Urner, Leonhard H; Pagel, Kevin; Dernedde, Jens; Haag, Rainer

2017-04-06

A series of water-soluble, hydroxylated and sulphated, polyglycerol (PG) dendronised, monofunctional perylene bisimides (PBIs) were synthesised in three generations. Their photophysical properties were determined by absorption and emission spectroscopy and their suitability as potential biolabels examined by biological in vitro studies after bioconjugation. It could be shown that the photophysical properties of the PBI labels can be improved by increasing the sterical demand and ionic charge of the attached dendron. Thereby, charged labels show superior suppression of aggregation over charge neutral labels owing to electrostatic repulsion forces on the PG-dendron. The ionic charges also enabled a reduction in dendron generation while retaining the labels' outstanding fluorescence quantum yields (FQYs) up to 100 %. These core-unsubstituted perylene derivatives were successfully applied as fluorescent labels upon bioconjugation to the therapeutic antibody cetuximab. The dye-antibody conjugates showed a strongly enhanced aggregation tendency compared to the corresponding free dyes. Biological evaluation by receptor-binding, cellular uptake, and cytotoxicity studies revealed that labelling did not affect the antibody's function, which renders the noncharged and charged dendronised PBIs suitable candidates as fluorescent labels in biological imaging. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single-neuron labeling with inducible cre-mediated knockout in transgenic mice

PubMed Central

Young, Paul; Qiu, Li; Wang, Dongqing; Zhao, Shengli; Gross, James; Feng, Guoping

2011-01-01

To facilitate functional analysis of neuronal connectivity in a mammalian nervous system tightly packed with billions of cells, we developed a new technique that allows inducible genetic manipulations within fluorescently labeled single neurons in mice. We term this technique SLICK for Single-neuron Labeling with Inducible Cre-mediated Knockout. SLICK is achieved by co-expressing a drug-inducible form of cre recombinase and a fluorescent protein within the same small subsets of neurons. Thus, SLICK combines the powerful cre recombinase system for conditional genetic manipulation and the fluorescent labeling of single neurons for imaging. We demonstrate efficient inducible genetic manipulation in several types of neurons using SLICK. Furthermore, we apply SLICK to eliminate synaptic transmission in a small subset of neuromuscular junctions. Our results provide evidence for the long-term stability of inactive neuromuscular synapses in adult animals. More broadly, these studies demonstrate a cre-LoxP compatible system for dissecting gene functions in single identifiable neurons. PMID:18454144

Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells.

PubMed

Kerppola, Tom K

2008-01-01

Protein interactions are a fundamental mechanism for the generation of biological regulatory specificity. The study of protein interactions in living cells is of particular significance because the interactions that occur in a particular cell depend on the full complement of proteins present in the cell and the external stimuli that influence the cell. Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the association between two nonfluorescent fragments of a fluorescent protein when they are brought in proximity to each other by an interaction between proteins fused to the fragments. Numerous protein interactions have been visualized using the BiFC assay in many different cell types and organisms. The BiFC assay is technically straightforward and can be performed using standard molecular biology and cell culture reagents and a regular fluorescence microscope or flow cytometer.

Fluorescent Labeling and Biodistribution of Latex Nanoparticles Formed by Surfactant-Free RAFT Emulsion Polymerization.

PubMed

Poon, Cheuk Ka; Tang, Owen; Chen, Xin-Ming; Kim, Byung; Hartlieb, Matthias; Pollock, Carol A; Hawkett, Brian S; Perrier, Sébastien

2017-10-01

The authors report the preparation of a novel range of functional polyacrylamide stabilized polystyrene nanoparticles, obtained by surfactant-free reversible addition-fragmentation chain transfer (RAFT) emulsion polymerization, their fluorescent tagging, cellular uptake, and biodistribution. The authors show the versatility of the RAFT emulsion process for the design of functional nanoparticles of well-defined size that can be used as drug delivery vectors. Functionalization with a fluorescent tag offers a useful visualization tool for tracing, localization, and clearance studies of these carriers in biological models. The studies are carried out by labeling the sterically stabilized latex particles chemically with rhodamine B. The fluorescent particles are incubated in a healthy human renal proximal tubular cell line model, and intravenously injected into a mouse model. Cellular localization and biodistribution of these particles on the biological models are explored. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Experimental assessment and analysis of super-resolution in fluorescence microscopy based on multiple-point spread function fitting of spectrally demultiplexed images

NASA Astrophysics Data System (ADS)

Nishimura, Takahiro; Kimura, Hitoshi; Ogura, Yusuke; Tanida, Jun

2018-06-01

This paper presents an experimental assessment and analysis of super-resolution microscopy based on multiple-point spread function fitting of spectrally demultiplexed images using a designed DNA structure as a test target. For the purpose, a DNA structure was designed to have binding sites at a certain interval that is smaller than the diffraction limit. The structure was labeled with several types of quantum dots (QDs) to acquire their spatial information as spectrally encoded images. The obtained images are analyzed with a point spread function multifitting algorithm to determine the QD locations that indicate the binding site positions. The experimental results show that the labeled locations can be observed beyond the diffraction-limited resolution using three-colored fluorescence images that were obtained with a confocal fluorescence microscope. Numerical simulations show that labeling with eight types of QDs enables the positions aligned at 27.2-nm pitches on the DNA structure to be resolved with high accuracy.

Dynamic labelling of neural connections in multiple colours by trans-synaptic fluorescence complementation

PubMed Central

Macpherson, Lindsey J.; Zaharieva, Emanuela E.; Kearney, Patrick J.; Alpert, Michael H.; Lin, Tzu-Yang; Turan, Zeynep; Lee, Chi-Hon; Gallio, Marco

2015-01-01

Determining the pattern of activity of individual connections within a neural circuit could provide insights into the computational processes that underlie brain function. Here, we develop new strategies to label active synapses by trans-synaptic fluorescence complementation in Drosophila. First, we demonstrate that a synaptobrevin-GRASP chimera functions as a powerful activity-dependent marker for synapses in vivo. Next, we create cyan and yellow variants, achieving activity-dependent, multi-colour fluorescence reconstitution across synapses (X-RASP). Our system allows for the first time retrospective labelling of synapses (rather than whole neurons) based on their activity, in multiple colours, in the same animal. As individual synapses often act as computational units in the brain, our method will promote the design of experiments that are not possible using existing techniques. Moreover, our strategies are easily adaptable to circuit mapping in any genetic system. PMID:26635273

Labeling RNAs in Live Cells Using Malachite Green Aptamer Scaffolds as Fluorescent Probes.

PubMed

Yerramilli, V Siddartha; Kim, Kyung Hyuk

2018-03-16

RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamer-fluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells. However, these studies also exhibited low fluorescence signals and high background noise. Here we constructed and tested RNA scaffolds containing multiple tandem repeats of MGA as a strategy to increase the brightness of the MGA aptamer-fluorogen system as well as to make the system fluoresce when tagging various RNA molecules, in live cells. We demonstrate that our MGA scaffolds can induce fluorescence signals by up to ∼20-fold compared to the basal level as a genetic tag for other RNA molecules. We also show that our scaffolds function reliably as genetically encoded fluorescent tags for mRNAs of fluorescent proteins and other RNA aptamers.

Cytidine-stabilized gold nanocluster as a fluorescence turn-on and turn-off probe for dual functional detection of Ag(+) and Hg(2+).

PubMed

Zhang, Yuanyuan; Jiang, Hui; Wang, Xuemei

2015-04-22

In this study, we have developed a label-free, dual functional detection strategy for highly selective and sensitive determination of aqueous Ag(+) and Hg(2+) by using cytidine stabilized Au NCs and AuAg NCs as fluorescent turn-on and turn off probes, respectively. The Au NCs and AuAg NCs showed a remarkably rapid response and high selectivity for Ag(+) and Hg(2+) over other metal ions, and relevant detection limit of Ag(+) and Hg(2+) is ca. 10 nM and 30 nM, respectively. Importantly, the fluorescence enhanced Au NCs by doping Ag(+) can be conveniently reusable for the detection of Hg(2+) based on the corresponding fluorescence quenching. The sensing mechanism was based on the high-affinity metallophilic Hg(2+)-Ag(+) interaction, which effectively quenched the fluorescence of AuAg NCs. Furthermore, these fluorescent nanoprobes could be readily applied to Ag(+) and Hg(2+) detection in environmental water samples, indicating their possibility to be utilized as a convenient, dual functional, rapid response, and label-free fluorescence sensor for related environmental and health monitoring. Copyright © 2015 Elsevier B.V. All rights reserved.

Functionalized magnetic-fluorescent hybrid nanoparticles for cell labelling.

PubMed

Lou, Lei; Yu, Ke; Zhang, Zhengli; Li, Bo; Zhu, Jianzhong; Wang, Yiting; Huang, Rong; Zhu, Ziqiang

2011-05-01

A facile method of synthesizing 60 nm magnetic-fluorescent core-shell bifunctional nanocomposites with the ability to label cells is presented. Hydrophobic trioctylphosphine oxide (TOPO)-capped CdSe@ZnS quantum dots (QDs) were assembled on polyethyleneimine (PEI)-coated Fe(3)O(4) nanoparticles (MNP). Polyethyleneimine was utilized for the realization of multifunction, including attaching 4 nm TOPO capped CdSe@ZnS quantum dots onto magnetite particles, altering the surface properties of quantum dots from hydrophobic to hydrophilic as well as preventing the formation of large aggregates. Results show that these water-soluble hybrid nanocomposites exhibit good colloidal stability and retain good magnetic and fluorescent properties. Because TOPO-capped QDs are assembled instead of their water-soluble equivalents, the nanocomposites are still highly luminescent with no shift in the PL peak position and present long-term fluorescence stability. Moreover, TAT peptide (GRKKRRQRRRPQ) functionalized hybrid nanoparticles were also studied due to their combined magnetic enrichment and optical detection for cell separation and rapid cell labelling. A cell viability assay revealed good biocompatibility of these hybrid nanoparticles. The potential application of the new magnetic-fluorescent nanocomposites in biological and medicine is demonstrated. © The Royal Society of Chemistry 2011

Saturation Fluorescence Labeling of Proteins for Proteomic Analyses

PubMed Central

Pretzer, Elizabeth; Wiktorowicz, John E.

2008-01-01

We present here an optimized and cost-effective approach to saturation fluorescence labeling of protein thiols for proteomic analysis. We investigated a number of conditions and reagent concentrations including a disulfide reducing agent (TCEP), pH, incubation time, linearity of labeling, and saturating dye: protein thiol ratio with protein standards to gauge specific and non-specific labeling. Efficacy of labeling under these conditions was quantified using specific fluorescence estimation, defined as the ratio of fluorescence pixel intensities and Coomassie-stained pixel intensities of bands after digital imaging. Factors leading to specific vs. non-specific labeling in the presence of thiourea are also discussed. We have found that reproducible saturation of available Cys residues of the proteins used as labeling standards (human carbonic anhydrase I, enolase, α-lactalbumin) is achieved at 50-100-fold excess of the uncharged maleimide-functionalized BODIPY™ dyes over Cys. We confirm our previous findings and those of others that the maleimide dyes are not impacted by the presence of 2M thiourea. Moreover, we establish that 2 mM TCEP used as reductant is optimal. We also establish further that labeling is optimal at pH 7.5 and complete after 30 min. Low non-specific labeling was gauged by the inclusion of non-Cys containing proteins (horse myoglobin, bovine carbonic anhydrase) to the labeling mixture. We also show that the dye exhibits little to no effect on the two dimensional mobilities of labeled proteins derived from cells. PMID:18191033

Application of fluorescently labeled tracer technique for detection of natural active macromolecules in Chinese medicine.

PubMed

Zeng, Qiao-Hui; Zhang, Xue-Wu; Xu, Kai-Peng; Jiang, Jian-Guo

2014-02-01

Active substances in traditional Chinese Medicine (TCM) contain not only a variety of small molecules, but also many other macromolecules (TCMMs), such as proteins, peptides and polysaccharides. Active TCMM can achieve good therapeutic effects by regulating the body's overall function with lower side effects. This review summarized the literatures published in recent years on the application of fluorescently labeled tracer technique for detection of natural active macromolecules in TCM. Classified by fluorescent markers, applications of fluorescein, rhodamine, and quantum dots (QDs) in TCMM active tracer are reviewed, and the methods and principles of TCMM fluorescent marker are illustrated. Studies on active TCMMs and their action mechanism are quite difficult due to a multitarget, multicomponent, and multipath system of TCM. However, the development of fluorescently labeled active tracer technique (FLATT) provides this research with new tools. Traditional fluorescent markers have many deficiencies, such as easily quenched, short luminous cycle, and intrinsic toxicity. Relatively, FLATT has many obvious advantages, and its application in TCMM is still at the early stage. In order to improve the overall level of fluorescence labeling in TCMM active tracer, the improvement on FLATT's detection sensitivity and biological affinity is urgent and critical to allow study of these interesting molecules.

Hydrogen sulfide deactivates common nitrobenzofurazan-based fluorescent thiol labeling reagents.

PubMed

Montoya, Leticia A; Pluth, Michael D

2014-06-17

Sulfhydryl-containing compounds, including thiols and hydrogen sulfide (H2S), play important but differential roles in biological structure and function. One major challenge in separating the biological roles of thiols and H2S is developing tools to effectively separate the reactivity of these sulfhydryl-containing compounds. To address this challenge, we report the differential responses of common electrophilic fluorescent thiol labeling reagents, including nitrobenzofurazan-based scaffolds, maleimides, alkylating agents, and electrophilic aldehydes, toward cysteine and H2S. Although H2S reacted with all of the investigated scaffolds, the photophysical response to each scaffold was significantly different. Maleimide-based, alkylating, and aldehydic thiol labeling reagents provided a diminished fluorescence response when treated with H2S. By contrast, nitrobenzofurazan-based labeling reagents were deactivated by H2S addition. Furthermore, the addition of H2S to thiol-activated nitrobenzofurazan-based reagents reduced the fluorescence signal, thus establishing the incompatibility of nitrobenzofurazan-based thiol labeling reagents in the presence of H2S. Taken together, these studies highlight the differential reactivity of thiols and H2S toward common thiol-labeling reagents and suggest that sufficient care must be taken when labeling or measuring thiols in cellular environments that produce H2S due to the potential for both false-positive and eroded responses.

Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

PubMed Central

Ren, Xiaomei; El-Sagheer, Afaf H.; Brown, Tom

2016-01-01

A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX. PMID:26819406

Synthesis of a Fluorescently Labeled 68Ga-DOTA-TOC Analog for Somatostatin Receptor Targeting.

PubMed

Ghosh, Sukhen C; Hernandez Vargas, Servando; Rodriguez, Melissa; Kossatz, Susanne; Voss, Julie; Carmon, Kendra S; Reiner, Thomas; Schonbrunn, Agnes; Azhdarinia, Ali

2017-07-13

Fluorescently labeled imaging agents can identify surgical margins in real-time to help achieve complete resections and minimize the likelihood of local recurrence. However, photon attenuation limits fluorescence-based imaging to superficial lesions or lesions that are a few millimeters beneath the tissue surface. Contrast agents that are dual-labeled with a radionuclide and fluorescent dye can overcome this limitation and combine quantitative, whole-body nuclear imaging with intraoperative fluorescence imaging. Using a multimodality chelation (MMC) scaffold, IRDye 800CW was conjugated to the clinically used somatostatin analog, 68 Ga-DOTA-TOC, to produce the dual-labeled analog, 68 Ga-MMC(IRDye 800CW)-TOC, with high yield and specific activity. In vitro pharmacological assays demonstrated retention of receptor-targeting properties for the dual-labeled compound with robust internalization that was somatostatin receptor (SSTR) 2-mediated. Biodistribution studies in mice identified the kidneys as the primary excretion route for 68 Ga-MMC(IRDye 800CW)-TOC, along with clearance via the reticuloendothelial system. Higher uptake was observed in most tissues compared to 68 Ga-DOTA-TOC but decreased as a function of time. The combination of excellent specificity for SSTR2-expressing cells and suitable biodistribution indicate potential application of 68 Ga-MMC(IRDye 800CW)-TOC for intraoperative detection of SSTR2-expressing tumors.

Label-free NIR reflectance imaging as a complimentary tool for two-photon fluorescence microscopy: multimodal investigation of stroke (Conference Presentation)

NASA Astrophysics Data System (ADS)

Allegra Mascaro, Anna Letizia; Costantini, Irene; Margoni, Emilia; Iannello, Giulio; Bria, Alessandro; Sacconi, Leonardo; Pavone, Francesco S.

2016-03-01

Two-photon imaging combined with targeted fluorescent indicators is extensively used for visualizing critical features of brain functionality and structural plasticity. Back-scattered photons from the NIR laser provide complimentary information without introducing any exogenous labelling. Here, we describe a versatile approach that, by collecting the reflected NIR light, provides structural details on the myelinated axons and blood vessels in the brain, both in fixed samples and in live animals. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from Thy1-GFPm mice, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Label-free detection of axonal elongations over the layer 2/3 of mouse cortex under a cranial window was also possible in live brain. Finally, blood flow could be measured in vivo, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated simultaneously.

Novel 1:1 labeling and purification process for C-terminal thioester and single cysteine recombinant proteins using generic peptidic toolbox reagents.

PubMed

Portal, Christophe F; Seifert, Jan-Marcus; Buehler, Christof; Meisner-Kober, Nicole-Claudia; Auer, Manfred

2014-07-16

We developed a versatile set of chemical labeling reagents which allow dye ligation to the C-terminus of a protein or a single internal cysteine and target purification in a simple two-step process. This simple process results in a fully 1:1 labeled conjugate suitable for all quantitative fluorescence spectroscopy and imaging experiments. We refer to a "generic labeling toolbox" because of the flexibility to choose one of many available dyes, spacers of different lengths and compositions which increase the target solubility, a variety of affinity purification tags, and different cleavage chemistries to release the 1:1 labeled proteins. Studying protein function in vitro or in the context of live cells and organisms is of vital importance in biological research. Although label free detection technologies gain increasing interest in molecular recognition science, fluorescence spectroscopy is still the most often used detection technique for assays and screens both in academic as well as in industrial groups. For generations, fluorescence spectroscopists have labeled their proteins of interest with small fluorescent dyes by random chemical linking on the proteins' exposed lysines and cysteines. Chemical reactions with a certain excess of activated esters or maleimides of longer wavelength dyes hardly ever result in quantitative labeling of the target protein. Most of the time, more than one exposed amino acid side chain reacts. This results in a mixture of dye-protein complexes of different labeling stoichiometries and labeling sites. Only mass spectrometry allows resolving the precise chemical composition of the conjugates. In "classical" ensemble averaging fluorescent experiments, these labeled proteins are still useful, and quantification of, e.g., ligand binding experiments, is achieved via knowledge of the overall protein concentration and a fluorescent signal change which is proportional to the amount of complex formed. With the development of fluorescence fluctuation analysis techniques working at single molecule resolution, like fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), fluorescence intensity diffusion analysis (FIDA), etc., it became important to work with homogeneously labeled target proteins. Each molecule participating in a binding equilibrium should be detectable when it freely fluctuates through the confocal focus of a microscope. The measured photon burst for each transition contains information about the size and the stoichiometry of a protein complex. Therefore, it is important to work with reagents that contain an exact number of tracers per protein at identical positions. The ideal fluorescent tracer-protein complex stoichiometry is 1:1. While genetic tags such as fluorescent proteins (FPs) are widely used to detect proteins, FPs have several limitations compared to chemical tags. For example, FPs cannot easily compete with organic dyes in the flexibility of modification and spectral range; moreover, FPs have disadvantages in brightness and photostability and are therefore not ideal for most biochemical single molecule studies. We present the synthesis of a series of exemplaric toolbox reagents and labeling results on three target proteins which were needed for high throughput screening experiments using fluorescence fluctuation analysis at single molecule resolution. On one target, Hu-antigen R (HuR), we demonstrated the activity of the 1:1 labeled protein in ribonucleic acid (RNA) binding, and the ease of resolving the stoichiometry of an RNA-HuR complex using the same dye on protein and RNA by Fluorescence Intensity Multiple Distribution Analysis (FIMDA) detection.

Breaking the color barrier - a multi-selective antibody reporter offers innovative strategies of fluorescence detection.

PubMed

Gallo, Eugenio; Jarvik, Jonathan W

2017-08-01

A novel bi-partite fluorescence platform exploits the high affinity and selectivity of antibody scaffolds to capture and activate small-molecule fluorogens. In this report, we investigated the property of multi-selectivity activation by a single antibody against diverse cyanine family fluorogens. Our fluorescence screen identified three cell-impermeant fluorogens, each with unique emission spectra (blue, green and red) and nanomolar affinities. Most importantly, as a protein fusion tag to G-protein-coupled receptors, the antibody biosensor retained full activity - displaying bright fluorogen signals with minimal background on live cells. Because fluorogen-activating antibodies interact with their target ligands via non-covalent interactions, we were able to perform advanced multi-color detection strategies on live cells, previously difficult or impossible with conventional reporters. We found that by fine-tuning the concentrations of the different color fluorogen molecules in solution, a user may interchange the fluorescence signal (onset versus offset), execute real-time signal exchange via fluorogen competition, measure multi-channel fluorescence via co-labeling, and assess real-time cell surface receptor traffic via pulse-chase experiments. Thus, here we inform of an innovative reporter technology based on tri-color signal that allows user-defined fluorescence tuning in live-cell applications. © 2017. Published by The Company of Biologists Ltd.

Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.

PubMed

Kerppola, Tom K

2006-01-01

Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the discoveries that two non-fluorescent fragments of a fluorescent protein can form a fluorescent complex and that the association of the fragments can be facilitated when they are fused to two proteins that interact with each other. BiFC must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. It is not necessary for the interaction partners to juxtapose the fragments within a specific distance of each other because they can associate when they are tethered to a complex with flexible linkers. It is also not necessary for the interaction partners to form a complex with a long half-life or a high occupancy since the fragments can associate in a transient complex and un-associated fusion proteins do not interfere with detection of the complex. Many interactions can be visualized when the fusion proteins are expressed at levels comparable to their endogenous counterparts. The BiFC assay has been used for the visualization of interactions between many types of proteins in different subcellular locations and in different cell types and organisms. It is technically straightforward and can be performed using a regular fluorescence microscope and standard molecular biology and cell culture reagents.

C-Terminal Fluorescent Labeling Impairs Functionality of DNA Mismatch Repair Proteins

PubMed Central

Brieger, Angela; Plotz, Guido; Hinrichsen, Inga; Passmann, Sandra; Adam, Ronja; Zeuzem, Stefan

2012-01-01

The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency. PMID:22348133

Development of cadmium-free quantum dot for intracellular labelling through electroporation or lipid-calcium-phosphate

NASA Astrophysics Data System (ADS)

Liu, Ying-Feng; Hung, Wei-Ling; Hou, Tzh-Yin; Huang, Hsiu-Ying; Lin, Cheng-An J.

2016-04-01

Traditional fluorescent labelling techniques has severe photo-bleaching problem such as organic dyes and fluorescent protein. Quantum dots made up of traditional semiconductor (CdSe/ZnS) material has sort of biological toxicity. This research has developed novel Cd-free quantum dots divided into semiconductor (Indium phosphide, InP) and noble metal (Gold). Former has lower toxicity compared to traditional quantum dots. Latter consisting of gold (III) chloride (AuCl3) and toluene utilizes sonochemical preparation and different stimulus to regulate fluorescent wavelength. Amphoteric macromolecule surface technology and ligand Exchange in self-Assembled are involved to develop hydrophilic nanomaterials which can regulate the number of grafts per molecule of surface functional groups. Calcium phosphate (CaP) nanoparticle (NP) with an asymmetric lipid bilayer coating technology developed for intracellular delivery and labelling has synthesized Cd-free quantum dots possessing high brightness and multi-fluorescence successfully. Then, polymer coating and ligand exchange transfer to water-soluble materials to produce liposome nanomaterials as fluorescent probes and enhancing medical applications of nanotechnology.

Separation-oriented derivatization of native fluorescent compounds through fluorous labeling followed by liquid chromatography with fluorous-phase.

PubMed

Sakaguchi, Yohei; Yoshida, Hideyuki; Todoroki, Kenichiro; Nohta, Hitoshi; Yamaguchi, Masatoshi

2009-06-15

We have developed a new and simple method based on "fluorous derivatization" for LC of native fluorescent compounds. This method involves the use of a column with a fluorous stationary phase. Native fluorescent analytes with target functional groups are precolumn derivatized with a nonfluorescent fluorous tag, and the fluorous-labeled analytes are retained in the column, whereas underivatized substances are not. Only the retained fluorescent analytes are detected fluorometrically at appropriate retention times, and retained substrates without fluorophores are not detected. In this study, biologically important carboxylic acids (homovanillic acid, vanillylmandelic acid, and 5-hydroxyindoleacetic acid) and drugs (naproxen, felbinac, flurbiprofen, and etodolac) were used as model native fluorescent compounds. Experimental results indicate that the fluorous-phase column can selectively retain fluorous compounds including fluorous-labeled analytes on the basis of fluorous separation. We believe that separation-oriented derivatization presented here is the first step toward the introduction of fluorous derivatization in quantitative LC analysis.

A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure

PubMed Central

Hauser, Christina T.; Tsien, Roger Y.

2007-01-01

Site-specific fluorescent labeling of proteins in vivo remains one of the most powerful techniques for imaging complex processes in live cells. Although fluorescent proteins in many colors are useful tools for tracking expression and localization of fusion proteins in cells, these relatively large tags (>220 aa) can perturb protein folding, trafficking and function. Much smaller genetically encodable domains (<15 aa) offer complementary advantages. We introduce a small fluorescent chelator whose membrane-impermeant complex with nontoxic Zn2+ ions binds tightly but reversibly to hexahistidine (His6) motifs on surface-exposed proteins. This live-cell label helps to resolve a current controversy concerning externalization of the stromal interaction molecule STIM1 upon depletion of Ca2+ from the endoplasmic reticulum. Whereas N-terminal fluorescent protein fusions interfere with surface exposure of STIM1, short His6 tags are accessible to the dye or antibodies, demonstrating externalization. PMID:17360414

Fluorescent Labeling of Proteins.

PubMed

Modesti, Mauro

2018-01-01

Many single-molecule experimental techniques exploit fluorescence as a tool to investigate conformational dynamics, molecular interactions, or track the movement of proteins in order to gain insight into their biological functions. A prerequisite to these experimental approaches is to graft one or more fluorophores on the protein of interest with the desired photophysical properties. Here, we describe procedures for efficient methods used to covalently attach fluorophores to proteins. Alternative direct and indirect labeling strategies are also described.

Fluorescein-labeled stable neurotensin derivatives.

PubMed

Maes, Veronique; Hultsch, Christina; Kohl, Suzann; Bergmann, Ralf; Hanke, Thomas; Tourwé, Dirk

2006-08-01

Neurotensin(8-13) analogs containing a glycine or 5-aminovaleroyl spacer were labeled with fluorescein through formation of an N-terminal thiourea function. The receptor binding was measured in HT-29 cell cultures and showed a substantial decrease in affinity, especially for the metabolically stabilized [MeArg(9), Tle(11)] analog. Using fluorescence microscopy, the internalization of the fluorescent neurotensin analogs into HT-29 cells was observed. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

Multimodal optoacoustic and multiphoton fluorescence microscopy

NASA Astrophysics Data System (ADS)

Sela, Gali; Razansky, Daniel; Shoham, Shy

2013-03-01

Multiphoton microscopy is a powerful imaging modality that enables structural and functional imaging with cellular and sub-cellular resolution, deep within biological tissues. Yet, its main contrast mechanism relies on extrinsically administered fluorescent indicators. Here we developed a system for simultaneous multimodal optoacoustic and multiphoton fluorescence 3D imaging, which attains both absorption and fluorescence-based contrast by integrating an ultrasonic transducer into a two-photon laser scanning microscope. The system is readily shown to enable acquisition of multimodal microscopic images of fluorescently labeled targets and cell cultures as well as intrinsic absorption-based images of pigmented biological tissue. During initial experiments, it was further observed that that detected optoacoustically-induced response contains low frequency signal variations, presumably due to cavitation-mediated signal generation by the high repetition rate (80MHz) near IR femtosecond laser. The multimodal system may provide complementary structural and functional information to the fluorescently labeled tissue, by superimposing optoacoustic images of intrinsic tissue chromophores, such as melanin deposits, pigmentation, and hemoglobin or other extrinsic particle or dye-based markers highly absorptive in the NIR spectrum.

[Sensitive determination of Bi3+ by spectrofluorimetry based on graphene oxide-methylene blue system].

PubMed

Zhai, Qiu-ge; Guo, Peng; Zhou, Lin; Liu, Yan-ming

2014-08-01

Graphene oxide was prepared by the modified Hummers method and characterized by field emission scanning electron microscopy. The interaction of graphene with methylene blue was studied by UV absorption, the intensity of two main absorption peaks of methylene blue decreased significantly after the fluorescence was quenched, and the energy transfer didn't occur because the overlap of the absorption spectrum of GO and the emission spectrum of MB is too small. Therefore, the fluorescence quenching of MB and GO was static. When adding a certain amount of Bi3+ in the graphene-methylene blue system, Bi3+ replaces the methylene blue from the graphene-methylene blue complexes because Bi3+ has the smaller volume and is more positively charged. The methylene blue therefore dissociates from the GO-MB complexes, resulting in the recovery of fluorescence of the system. Furthermore, the fluorescence of the system increases with the increase in the amount of Bi3+ due to the enhanced amount of MB in the system. A novel spectrofluorimetric method was therefore developed for the sensitive determination of Bi3+. Some parameters including the concentration of methylene blue, the amount of graphene oxide, the amount of nitric acid and the sequence of reagent adding were optimized to obtain higher sensitivity. The fluorescence of the system was detected at an emission wavelength of 667 nm with excitation at 690 nm. Under the optimized conditions, the concentration of Bi3+ showed good linear relationships with the fluorescence intensity in the range of 0.5-100 micromol x L(-1), with correlation coefficients of r = 0.9955. The limits of detection for Bi3+ was 1.0 x 10(-8) mol x L(-1) (S/N=3). The selectivity of the proposed method was evaluated and the results showed that 1000-fold K+, Ca+, Na+, Mg2+, Cu2+; 100-fold Fe3+, Be2+, SiO2- Al3+, Ni2+, Sb3+, NO3-, Cl-, F-, and 20-fold Pb2+, Hg2+, Cd2+ had negligible interference with the determination of Bi3+. The method has advantages of sensitivity, rapidness and low cost. It was used for the determination of Bi3+ in environmental water samples inclu- ding pond water, rain water and sewage water with recoveries of 93.4%-105.2%.

Forced Unfolding of Proteins Within Cells

PubMed Central

Johnson, Colin P.; Tang, Hsin-Yao; Carag, Christine; Speicher, David W.; Discher, Dennis E.

2009-01-01

To identify cytoskeletal proteins that change conformation or assembly within stressed cells, in situ labeling of sterically shielded cysteines with fluorophores was analyzed by fluorescence imaging, quantitative mass spectrometry, and sequential two-dye labeling. Within red blood cells, shotgun labeling showed that shielded cysteines in the two isoforms of the cytoskeletal protein spectrin were increasingly labeled as a function of shear stress and time, indicative of forced unfolding of specific domains. Within mesenchymal stem cells—as a prototypical adherent cell—nonmuscle myosin IIA and vimentin are just two of the cytoskeletal proteins identified that show differential labeling in tensed versus drug-relaxed cells. Cysteine labeling of proteins within live cells can thus be used to fluorescently map out sites of molecular-scale deformation, and the results also suggest means to colocalize signaling events such as phosphorylation with forced unfolding. PMID:17673662

Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling.

PubMed

Neville, David C A; Coquard, Virginie; Priestman, David A; te Vruchte, Danielle J M; Sillence, Daniel J; Dwek, Raymond A; Platt, Frances M; Butters, Terry D

2004-08-15

Interest in cellular glycosphingolipid (GSL) function has necessitated the development of a rapid and sensitive method to both analyze and characterize the full complement of structures present in various cells and tissues. An optimized method to characterize oligosaccharides released from glycosphingolipids following ceramide glycanase digestion has been developed. The procedure uses the fluorescent compound anthranilic acid (2-aminobenzoic acid; 2-AA) to label oligosaccharides prior to analysis using normal-phase high-performance liquid chromatography. The labeling procedure is rapid, selective, and easy to perform and is based on the published method of Anumula and Dhume [Glycobiology 8 (1998) 685], originally used to analyze N-linked oligosaccharides. It is less time consuming than a previously published 2-aminobenzamide labeling method [Anal. Biochem. 298 (2001) 207] for analyzing GSL-derived oligosaccharides, as the fluorescent labeling is performed on the enzyme reaction mixture. The purification of 2-AA-labeled products has been improved to ensure recovery of oligosaccharides containing one to four monosaccharide units, which was not previously possible using the Anumula and Dhume post-derivatization purification procedure. This new approach may also be used to analyze both N- and O-linked oligosaccharides.

Synthesis and biological evaluation of radio and dye labeled amino functionalized dendritic polyglycerol sulfates as multivalent anti-inflammatory compounds.

PubMed

Gröger, Dominic; Paulus, Florian; Licha, Kai; Welker, Pia; Weinhart, Marie; Holzhausen, Cornelia; Mundhenk, Lars; Gruber, Achim D; Abram, Ulrich; Haag, Rainer

2013-09-18

Herein we describe a platform technology for the synthesis and characterization of partially aminated, (35)S-labeled, dendritic polyglycerol sulfate (dPG(35)S amine) and fluorescent dPGS indocarbocyanine (ICC) dye conjugates. These polymer conjugates, based on a biocompatible dendritic polyglycerol scaffold, exhibit a high affinity to inflamed tissue in vivo and represent promising candidates for therapeutic and diagnostic applications. By utilizing a one-step sequential copolymerization approach, dendritic polyglycerol (Mn ≈ 4.5 kDa) containing 9.4% N-phthalimide protected amine functionalities was prepared on a large scale. Sulfation and simultaneous radio labeling with (35)SO3 pyridine complex, followed by cleavage of the N-phthalimide protecting groups, yielded dPG(35)S amine as a beta emitting, inflammation specific probe with free amino functionalities for conjugation. Furthermore, efficient labeling procedures with ICC via iminothiolane modification and subsequent "Michael" addition of the maleimide functionalized ICC dye, as well as by amide formation via NHS derivatized ICC on a dPGS amine scaffold, are described. The dPGS-ICC conjugates were investigated with respect to their photophysical properties, and both the radiolabeled and fluorescent compounds were comparatively visualized in histological tissue sections (radio detection and fluorescence microscopy) of animals treated with dPGS. Furthermore, cellular uptake of dPGS-ICC was found in endothelial cord blood (HUVEC) and the epithelial lung cells (A549). The presented synthetic routes allow a reproducible, controlled synthesis of dPGS amine on kilogram scale applying a one-pot batch reaction process. dPGS amine can be used for analysis via radioactivity or fluorescence, thereby creating a new platform for inflammation specific, multimodal imaging purposes using other attachable probes or contrast agents.

Facile one-pot synthesis of visible light-responsive BiPO4/nitrogen doped graphene hydrogel for fabricating label-free photoelectrochemical tetracycline aptasensor.

PubMed

Ge, Lan; Li, Henan; Du, Xiaojiao; Zhu, Mingyue; Chen, Wei; Shi, Tingyan; Hao, Nan; Liu, Qian; Wang, Kun

2018-07-15

It is fundamental to develop highly efficient visible light-responsive photoelectrochemical (PEC) performance material for fabricating PEC biosensor. Herein, BiPO 4 /three-dimensional nitrogen doped graphene hydrogel (3DNGH) nanocomposites were prepared for the first time via a facile one-pot hydrothermal route. In this nanoarchitecture, the BiPO 4 nanorods were anchored onto the porous structure of 3DNGH. Compared with pristine BiPO 4 , the absorption of BiPO 4 /3DNGH has been extend to visible-light region, and the energy band gap of BiPO 4 /3DNGH was calculated to be 2.10 eV, which was greatly narrower than that of pristine BiPO 4 with a band gap of 3.85 eV. Under visible light irradiation, the photocurrent signal of the as-prepared BiPO 4 /3DNGH was 847.2-fold, 4.1-fold and 2.3-fold enhanced comparing to pristine BiPO 4 , BiPO 4 functionalized reduced graphene oxide and BiPO 4 /nitrogen doped graphene. The enhancement of such photocurrent signal was attributed to the introduction of 3DNGH, which was capable to improve the charge transfer rate and also the efficiency of visible-light utilization of BiPO 4 . Based on the excellent PEC properties of BiPO 4 /3DNGH, a label-free PEC aptasensor for selectivity and sensitivity detection of tetracycline (Tc) was successfully established by using Tc aptamer as a biorecognition element. Under optimized conditions, the proposed PEC aptasensor exhibited a wide linear in the range from 0.1 nmol L -1 to 1 μmol L -1 as well as a low detection limit of 0.033 nmol L -1 (S/N = 3). The prepared BiPO 4 /3DNGH nanocomposites would serve as a promising visible light-responsive photoactive material for fabrication of PEC biosensors with high performance. Copyright © 2018 Elsevier B.V. All rights reserved.

Noncovalent labeling of biomolecules with red and near- infrared dyes.

PubMed

Patonay, Gabor; Salon, Jozef; Sowell, John; Strekowski, Lucjan

2004-02-28

Biopolymers such as proteins and nucleic acids can be labeled with a fluorescent marker to allow for their detection. Covalent labeling is achieved by the reaction of an appropriately functionalized dye marker with a reactive group on a biomolecule. The recent trend, however, is the use of noncovalent labeling that results from strong hydrophobic and/or ionic interactions between the marker and biomolecule of interest. The main advantage of noncovalent labeling is that it affects the functional activity of the biomolecule to a lesser extent. The applications of luminescent cyanine and squarylium dyes are reviewed.

Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines

PubMed Central

Vuong, Helen E.; de Sevilla Müller, Luis Pérez; Hardi, Claudia N.; McMahon, Douglas G.; Brecha, Nicholas C.

2015-01-01

Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) line with three catecholamine-related Cre recombinase lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ~6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium somal diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines were generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing. PMID:26335381

Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines.

PubMed

Vuong, H E; Pérez de Sevilla Müller, L; Hardi, C N; McMahon, D G; Brecha, N C

2015-10-29

Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) mouse line with three catecholamine-related Cre recombinase mouse lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ∼ 6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines was generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing. Published by Elsevier Ltd.

Study and development of label-free optical biosensors for biomedical applications

NASA Astrophysics Data System (ADS)

Choi, Charles J.

For the majority of assays currently performed, fluorescent or colorimetric chemical labels are commonly attached to the molecules under study so that they may be readily visualized. The methods of using labels to track biomolecular binding events are very sensitive and effective, and are employed as standardized assay protocol across research labs worldwide. However, using labels induces experimental uncertainties due to the effect of the label on molecular conformation, active binding sites, or inability to find an appropriate label that functions equivalently for all molecules in an experiment. Therefore, the ability to perform highly sensitive biochemical detection without the use of fluorescent labels would further simplify assay protocols and would provide quantitative kinetic data, while removing experimental artifacts from fluorescent quenching, shelf-life, and background fluorescence phenomena. In view of the advantages mentioned above, the study and development of optical label-free sensor technologies have been undertaken here. In general, label-free photonic crystal (PC) biosensors and metal nanodome array surface-enhanced Raman scattering (SERS) substrates, both of which are fabricated by nanoreplica molding process, have been used as the method to attack the problem. Chapter 1 shows the work on PC label-free biosensor incorporated microfluidic network for bioassay performance enhancement and kinetic reaction rate constant determination. Chapter 2 describes the work on theoretical and experimental comparison of label-free biosensing in microplate, microfluidic, and spot-based affinity capture assays. Chapter 3 shows the work on integration of PC biosensor with actuate-to-open valve microfluidic chip for pL-volume combinatorial mixing and screening application. In Chapter 4, the development and characterization of SERS nanodome array is shown. Lastly, Chapter 5 describes SERS nanodome sensor incorporated tubing for point-of-care monitoring of intravenous drugs and metabolites.

Near-infrared emitting fluorescent nanocrystals-labeled natural killer cells as a platform technology for the optical imaging of immunotherapeutic cells-based cancer therapy

NASA Astrophysics Data System (ADS)

Taik Lim, Yong; Cho, Mi Young; Noh, Young-Woock; Chung, Jin Woong; Chung, Bong Hyun

2009-11-01

This study describes the development of near-infrared optical imaging technology for the monitoring of immunotherapeutic cell-based cancer therapy using natural killer (NK) cells labeled with fluorescent nanocrystals. Although NK cell-based immunotherapeutic strategies have drawn interest as potent preclinical or clinical methods of cancer therapy, there are few reports documenting the molecular imaging of NK cell-based cancer therapy, primarily due to the difficulty of labeling of NK cells with imaging probes. Human natural killer cells (NK92MI) were labeled with anti-human CD56 antibody-coated quantum dots (QD705) for fluorescence imaging. FACS analysis showed that the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 have no effect on the cell viability. The effect of anti-human CD56 antibody-coated QD705 labeling on the NK92MI cell function was investigated by measuring interferon gamma (IFN- γ) production and cytolytic activity. Finally, the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 showed a therapeutic effect similar to that of unlabeled NK92MI cells. Images of intratumorally injected NK92MI cells labeled with anti-human CD56 antibody-coated could be acquired using near-infrared optical imaging both in vivo and in vitro. This result demonstrates that the immunotherapeutic cells labeled with fluorescent nanocrystals can be a versatile platform for the effective tracking of injected therapeutic cells using optical imaging technology, which is very important in cell-based cancer therapies.

Next-Generation Theranostic Agents Based on Polyelectrolyte Microcapsules Encoded with Semiconductor Nanocrystals: Development and Functional Characterization

NASA Astrophysics Data System (ADS)

Nifontova, Galina; Zvaigzne, Maria; Baryshnikova, Maria; Korostylev, Evgeny; Ramos-Gomes, Fernanda; Alves, Frauke; Nabiev, Igor; Sukhanova, Alyona

2018-01-01

Fabrication of polyelectrolyte microcapsules and their use as carriers of drugs, fluorescent labels, and metal nanoparticles is a promising approach to designing theranostic agents. Semiconductor quantum dots (QDs) are characterized by extremely high brightness and photostability that make them attractive fluorescent labels for visualization of intracellular penetration and delivery of such microcapsules. Here, we describe an approach to design, fabricate, and characterize physico-chemical and functional properties of polyelectrolyte microcapsules encoded with water-solubilized and stabilized with three-functional polyethylene glycol derivatives core/shell QDs. Developed microcapsules were characterized by dynamic light scattering, electrophoretic mobility, scanning electronic microscopy, and fluorescence and confocal microscopy approaches, providing exact data on their size distribution, surface charge, morphological, and optical characteristics. The fluorescence lifetimes of the QD-encoded microcapsules were also measured, and their dependence on time after preparation of the microcapsules was evaluated. The optimal content of QDs used for encoding procedure providing the optimal fluorescence properties of the encoded microcapsules was determined. Finally, the intracellular microcapsule uptake by murine macrophages was demonstrated, thus confirming the possibility of efficient use of developed system for live cell imaging and visualization of microcapsule transportation and delivery within the living cells.

HDL particles incorporate into lipid bilayers - a combined AFM and single molecule fluorescence microscopy study.

PubMed

Plochberger, Birgit; Röhrl, Clemens; Preiner, Johannes; Rankl, Christian; Brameshuber, Mario; Madl, Josef; Bittman, Robert; Ros, Robert; Sezgin, Erdinc; Eggeling, Christian; Hinterdorfer, Peter; Stangl, Herbert; Schütz, Gerhard J

2017-11-21

The process, how lipids are removed from the circulation and transferred from high density lipoprotein (HDL) - a main carrier of cholesterol in the blood stream - to cells, is highly complex. HDL particles are captured from the blood stream by the scavenger receptor, class B, type I (SR-BI), the so-called HDL receptor. The details in subsequent lipid-transfer process, however, have not yet been completely understood. The transfer has been proposed to occur directly at the cell surface across an unstirred water layer, via a hydrophobic channel in the receptor, or after HDL endocytosis. The role of the target lipid membrane for the transfer process, however, has largely been overlooked. Here, we studied at the single molecule level how HDL particles interact with synthetic lipid membranes. Using (high-speed) atomic force microscopy and fluorescence correlation spectroscopy (FCS) we found out that, upon contact with the membrane, HDL becomes integrated into the lipid bilayer. Combined force and single molecule fluorescence microscopy allowed us to directly monitor the transfer process of fluorescently labelled amphiphilic lipid probe from HDL particles to the lipid bilayer upon contact.

Sub-micron scale patterning of fluorescent silver nanoclusters using low-power laser.

PubMed

Kunwar, Puskal; Hassinen, Jukka; Bautista, Godofredo; Ras, Robin H A; Toivonen, Juha

2016-04-05

Noble metal nanoclusters are ultrasmall nanomaterials with tunable properties and huge application potential; however, retaining their enhanced functionality is difficult as they readily lose their properties without stabilization. Here, we demonstrate a facile synthesis of highly photostable silver nanoclusters in a polymer thin film using visible light photoreduction. Furthermore, the different stages of the nanocluster formation are investigated in detail using absorption and fluorescence spectroscopy, fluorescence microscopy, and atomic force microscopy. A cost-effective fabrication of photostable micron-sized fluorescent silver nanocluster barcode is demonstrated in silver-impregnated polymer films using a low-power continuous-wave laser diode. It is shown that a laser power of as low as 0.75 mW is enough to write fluorescent structures, corresponding to the specifications of a commercially available laser pointer. The as-formed nanocluster-containing microstructures can be useful in direct labeling applications such as authenticity marking and fluorescent labeling.

Bismuth@US-tubes as a Potential Contrast Agent for X-ray Imaging Applications

PubMed Central

Rivera, Eladio J.; Tran, Lesa A.; Hernández-Rivera, Mayra; Yoon, Diana; Mikos, Antonios G.; Rusakova, Irene A.; Cheong, Benjamin Y.; Cabreira-Hansen, Maria da Graça; Willerson, James T.; Perin, Emerson C.; Wilson, Lon J.

2013-01-01

The encapsulation of bismuth as BiOCl/Bi2O3 within ultra-short (ca. 50 nm) single-walled carbon nanocapsules (US-tubes) has been achieved. The Bi@US-tubes have been characterized by high-resolution transmission electron microscopy (HR-TEM), energy-dispersive X-ray spectroscopy (EDS), thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. Bi@US-tubes have been used for intracellular labeling of pig bone marrow-derived mesenchymal stem cells (MSCs) to show high X-ray contrast in computed tomography (CT) cellular imaging for the first time. The relatively high contrast is achieved with low bismuth loading (2.66% by weight) within the US-tubes and without compromising cell viability. X-ray CT imaging of Bi@US-tubes-labeled MSCs showed a nearly two-fold increase in contrast enhancement when compared to unlabeled MSCs in a 100 kV CT clinical scanner. The CT signal enhancement from the Bi@US-tubes is 500 times greater than polymer-coated Bi2S3 nanoparticles and several-fold that of any clinical iodinated contrast agent (CA) at the same concentration. Our findings suggest that the Bi@US-tubes can be used as a potential new class of X-ray CT agent for stem cell labeling and possibly in vivo tracking. PMID:24288589

Protein oligomerization monitored by fluorescence fluctuation spectroscopy: Self-assembly of Rubisco activase

USDA-ARS?s Scientific Manuscript database

A methodology is presented to characterize complex protein assembly pathways by fluorescence correlation spectroscopy. We have derived the total autocorrelation function describing the behavior of mixtures of labeled and unlabeled protein under equilibrium conditions. Our modeling approach allows us...

Comparison of cardioprotective effects of labeled and unlabeled oleanoic acids with new BOPIM dye on primary neonatal rat cardiomyocytes following hypoxia/reoxygenation injury.

PubMed

Wang, Sa; He, Hai-bo; Xiao, Shu-zhang; Wang, Jun-zhi; Bai, Cai-hong; Wei, Na; Zou, Kun

2014-08-01

It is well known that fluorescent labeling has recently become a major research tool in molecular and cellular biology for demonstrating therapeutic mechanisms and metabolic pathways. However, few studies have reported the use of fluorescent labeling of natural products. We recently explored the boron 2-(2'-pyridyl) imidazole (BOPIM) derivative analogs, which are highly fluorescent, non-aggregated, and nontoxic. In the present study, the natural product oleanolic acid (OA) was functionalized and labeled with BOPIM, thus yielding a highly fluorescent probe, the comparison of cardioprotective effects of labeled and unlabeled OAs with BOPIM on primary neonatal rat cardiomyocytes with hypoxia/reoxygenation (H/R) injury were investigated. Pretreatment with OA and BOPIM-OA significantly prevented the H/R induced cell death in primary neonatal rat cardiomyocytes. However, BOPIM exhibited no improvements on the H/R injury cardiomyocytes, and which were similar to those of the H/R group. The results of comparison of cardioprotective effects between labeled and unlabeled OAs with BOPIM showed that introducing the BOPIM chromophore did not make a difference with H/R injury cardiomyocytes. BOPIM chromophore is a suitable probe for investigating the pharmacological mechanisms of natural products. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Ratiometric Fluorescence Azide-Alkyne Cycloaddition for Live Mammalian Cell Imaging.

PubMed

Fu, Hongxia; Li, Yanru; Sun, Lingbo; He, Pan; Duan, Xinrui

2015-11-17

Click chemistry with metabolic labeling has been widely used for selectively imaging biomacromolecules in cells. The first example of azide-alkyne cycloaddition for ratiometric fluorescent imaging of live cells is reported. The precursor of the azido fluorophore (cresyl violet) has a fluorescence emission peak at 620 nm. The electron-rich nitrogen of the azido group blue-shifts the emission peak to 566 nm. When the click reaction occurs, an emission peak appears at 620 nm due to the lower electronic density of the newly formed triazole ring, which allows us to ratiometrically record fluorescence signals. This emission shift was applied to ratiometric imaging of propargylcholine- and dibenzocyclooctyne-labeled human breast cancer cells MCF-7 under laser confocal microscopy. Two typical triazole compounds were isolated for photophysical parameter measurements. The emission spectra presented a fluorescence emission peak around 620 nm for both click products. The results further confirmed the emission wavelength change was the result of azide-alkyne cycloaddition reaction. Since nearly all biomolecules can be metabolically labeled by reported alkyne-functionalized derivatives of native metabolites, our method can be readily applied to image these biomacromolecules.

Biodistributions, Myelosuppression, and Toxicities in Mice Treated with an Anti-CD45 Antibody Labeled with the alpha-Emitting Radionuclides Bismuth-213 or Astatine-211

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakamae, Hirohisa; Wilbur, D. Scott; Hamlin, Donald K.

2009-03-15

We previously investigated 213Bi-labeled anti-CD45 antibody to replace total body irradiation as conditioning for hematopoietic cell transplantation in a canine model. While this allowed sustained engraftment of marrow, limited availability and high cost of 213Bi led to a preliminary investigation in mice of 211At-labeled antibody for the same application. To gain an understanding of the differences between the two radionuclides, biodistribution and myelosuppression/toxicity studies were conducted with 213Bi- and 211At-labeled rat anti-murine CD45 antibody, 30F11, conjugates. After injecting mice with 2-50 μCi on 10 μg 30F11 conjugate or 20 μCi on 2 or 40 μg conjugate, biodistributions, myelosuppression and non-hematologicalmore » toxicities were evaluated. Biodistribution studies showed that the spleen had the highest concentration of radioactivity, ranging from167-417 % injected dose/gram (%ID/g) at 24 h after injection in the 211At studies and 45-166 %ID/g at 3 h after injection in the 213Bi studies. The higher concentrations observed for 211At-labeled 30F11 was likely due to its longer half-life which, permitted more localization of antibody to the spleen before decay. 211At was more effective at myelosuppression for the same (mCi) quantity of injected radioactivity. Injection of only 20 or 50 μCi 211At resulted in lethal myeloablation. There was severe reversible acute hepatic toxicity with 50 μCi 213Bi, but not with lower doses or any dose of 211At. No significant renal toxicity occurred with either radionuclide. The data suggested that considerably lower quantities of 211At-labeled anti-CD45 antibody than 213Bi-labeled antibody might be effective for myelosuppression.« less

Determination of protein secondary structure and solvent accessibility using site-directed fluorescence labeling. Studies of T4 lysozyme using the fluorescent probe monobromobimane.

PubMed

Mansoor, S E; McHaourab, H S; Farrens, D L

1999-12-07

We report an investigation of how much protein structural information could be obtained using a site-directed fluorescence labeling (SDFL) strategy. In our experiments, we used 21 consecutive single-cysteine substitution mutants in T4 lysozyme (residues T115-K135), located in a helix-turn-helix motif. The mutants were labeled with the fluorescent probe monobromobimane and subjected to an array of fluorescence measurements. Thermal stability measurements show that introduction of the label is substantially perturbing only when it is located at buried residue sites. At buried sites (solvent surface accessibility of <40 A(2)), the destabilizations are between 3 and 5.5 kcal/mol, whereas at more exposed sites, DeltaDeltaG values of < or = 1.5 kcal/mol are obtained. Of all the fluorescence parameters that were explored (excitation lambda(max), emission lambda(max), fluorescence lifetime, quantum yield, and steady-state anisotropy), the emission lambda(max) and the steady-state anisotropy values most accurately reflect the solvent surface accessibility at each site as calculated from the crystal structure of cysteine-less T4 lysozyme. The parameters we identify allow the classification of each site as buried, partially buried, or exposed. We find that the variations in these parameters as a function of residue number reflect the sequence-specific secondary structure, the determination of which is a key step for modeling a protein of unknown structure.

Proteomic detection of oxidized and reduced thiol proteins in cultured cells.

PubMed

Cuddihy, Sarah L; Baty, James W; Brown, Kristin K; Winterbourn, Christine C; Hampton, Mark B

2009-01-01

The oxidation and reduction of cysteine residues is emerging as an important post-translational control of protein function. We describe a method for fluorescent labelling of either reduced or oxidized thiols in combination with two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2DE) to detect changes in the redox proteome of cultured cells. Reduced thiols are labelled with the fluorescent compound 5-iodoacetamidofluorescein. To monitor oxidized thiols, the reduced thiols are first blocked with N-ethyl-maleimide, then the oxidized thiols reduced with dithiothreitol and labelled with 5-iodoacetamidofluorescein. The method is illustrated by treating Jurkat T-lymphoma cells with hydrogen peroxide and monitoring increased labelling of oxidized thiol proteins. A decrease in labelling can also be detected, and this is attributed to the formation of higher oxidation states of cysteine that are not reduced by dithiothreitol.

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

PubMed Central

Valades Cruz, Cesar Augusto; Shaban, Haitham Ahmed; Kress, Alla; Bertaux, Nicolas; Monneret, Serge; Mavrakis, Manos; Savatier, Julien; Brasselet, Sophie

2016-01-01

Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells. PMID:26831082

Laser induced fluorescence technique for detecting organic matter in East China Sea

NASA Astrophysics Data System (ADS)

Chen, Peng; Wang, Tianyu; Pan, Delu; Huang, Haiqing

2017-10-01

A laser induced fluorescence (LIF) technique for fast diagnosing chromophoric dissolved organic matter (CDOM) in water is discussed. We have developed a new field-portable laser fluorometer for rapid fluorescence measurements. In addtion, the fluorescence spectral characteristics of fluorescent constituents (e.g., CDOM, chlorophyll-a) were analyzed with a spectral deconvolution method of bi-Gaussian peak function. In situ measurements by the LIF technique compared well with values measured by conventional spectrophotometer method in laboratory. A significant correlation (R2 = 0.93) was observed between fluorescence by the technique and absorption by laboratory spectrophotometer. Influence of temperature variation on LIF measurement was investigated in lab and a temperature coefficient was deduced for fluorescence correction. Distributions of CDOM fluorescence measured using this technique in the East China Sea coast were presented. The in situ result demonstrated the utility of the LIF technique for rapid detecting dissolved organic matter.

Dual-labeling method for electron microscopy to characterize synaptic connectivity using genetically encoded fluorescent reporters in Drosophila

PubMed Central

Tanaka, Nobuaki K.; Dye, Louis; Stopfer, Mark

2010-01-01

Light and electron microscopy (LM and EM) both offer important advantages for characterizing neuronal circuitry in intact brains: LM can reveal the general patterns neurons trace between brain areas, and EM can confirm synaptic connections between identified neurons within a small area. In a few species, genetic labeling with fluorescent proteins has been used with LM to visualize many kinds of neurons and to analyze their morphologies and projection patterns. However, combining these large-scale patterns with the fine detail available in EM analysis has been a technical challenge. To analyze the synaptic connectivity of neurons expressing fluorescent markers with EM, we developed a dual-labeling method for use with pre-embedded brains. In Drosophila expressing genetic labels and also injected with markers we visualized synaptic connections among two populations of neurons in the AL, one of which has been shown to mediate a specific function, odor evoked neural oscillation. PMID:21074556

Buprenorphine Implants for Treatment of Opioid Dependence: Randomized Comparison to Placebo and Sublingual Buprenorphine/Naloxone

PubMed Central

Rosenthal, Richard N.; Ling, Walter; Casadonte, Paul; Vocci, Frank; Bailey, Genie L.; Kampman, Kyle; Patkar, Ashwin; Chavoustie, Steven; Blasey, Christine; Sigmon, Stacey; Beebe, Katherine L.

2015-01-01

Aims To evaluate safety and efficacy of buprenorphine implants (BI) versus placebo implants (PI) for the treatment of opioid dependence. A secondary aim compared BI to open-label sublingual buprenorphine/naloxone tablets (BNX). Design Randomized, double-blind, placebo-controlled trial. Subjects received either 4 buprenorphine implants (80 mg/implant) (n=114), 4 placebo implants (n=54), or open-label BNX (12–16 mg/d) (n=119). Setting 20 addiction treatment centers. Participants Adult outpatients (ages 18 to 65) with DSM-IV-TR opioid dependence. Measurements The primary efficacy endpoint was the percent of urine samples negative for opioids collected from weeks 1 to 24, examined as a cumulative distribution function (CDF). Findings The BI CDF was significantly different from placebo (P<.0001). Mean (95% CI) proportions of urines negative for opioids were: BI: 31.2% (25.3, 37.1) and PI: 13.4% (8.3, 18.6). BI subjects had a higher study completion rate relative to placebo (64% vs. 26%, P<.0001), lower clinician-rated (P<.0001) and patient-rated (P<.0001) withdrawal, lower patient-ratings of craving (P<.0001), and better subjects’ (P=.031) and clinicians’ (P=.022) global ratings of improvement. BI also resulted in significantly lower cocaine use (P=.0016). Minor implant-site reactions were comparable in the buprenorphine (27.2% [31/114]) and placebo groups (25.9% [14/54]). BI were non-inferior to BNX on percent urines negative for opioids [mean (95% CI): 33.5 (27.3, 39.6); CI for the difference of proportions, (−10.7, 6.2)]. Conclusions Compared with placebo, buprenorphine implants result in significantly less frequent opioid use, and are non-inferior to sublingual buprenorphine/naloxone tablets. PMID:23919595

Buprenorphine implants for treatment of opioid dependence: randomized comparison to placebo and sublingual buprenorphine/naloxone.

PubMed

Rosenthal, Richard N; Ling, Walter; Casadonte, Paul; Vocci, Frank; Bailey, Genie L; Kampman, Kyle; Patkar, Ashwin; Chavoustie, Steven; Blasey, Christine; Sigmon, Stacey; Beebe, Katherine L

2013-12-01

To evaluate the safety and efficacy of buprenorphine implants (BI) versus placebo implants (PI) for the treatment of opioid dependence. A secondary aim compared BI to open-label sublingual buprenorphine/naloxone tablets (BNX). Randomized, double-blind, placebo-controlled trial. Subjects received either four buprenorphine implants (80 mg/implant) (n = 114), four placebo implants (n = 54) or open-label BNX (12-16 mg/day) (n = 119). Twenty addiction treatment centers. Adult out-patients (ages 18-65) with DSM-IV-TR opioid dependence. The primary efficacy end-point was the percentage of urine samples negative for opioids collected from weeks 1 to 24, examined as a cumulative distribution function (CDF). The BI CDF was significantly different from placebo (P < 0.0001). Mean [95% confidence interval (CI)] proportions of urines negative for opioids were: BI = 31.2% (25.3, 37.1) and PI = 13.4% (8.3, 18.6). BI subjects had a higher study completion rate relative to placebo (64 versus 26%, P < 0.0001), lower clinician-rated (P < 0.0001) and patient-rated (P < 0.0001) withdrawal, lower patient-ratings of craving (P < 0.0001) and better subjects' (P = 0.031) and clinicians' (P = 0.022) global ratings of improvement. BI also resulted in significantly lower cocaine use (P = 0.0016). Minor implant-site reactions were comparable in the buprenorphine [27.2% (31 of 114)] and placebo groups [25.9% (14 of 54)]. BI were non-inferior to BNX on percentage of urines negative for opioids [mean (95% CI) = 33.5 (27.3, 39.6); 95% CI for the difference of proportions = (-10.7, 6.2)]. Compared with placebo, buprenorphine implants result in significantly less frequent opioid use and are non-inferior to sublingual buprenorphine/naloxone tablets. © 2013 Society for the Study of Addiction.

Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background.

PubMed

Stöhr, Katharina; Siegberg, Daniel; Ehrhard, Tanja; Lymperopoulos, Konstantinos; Öz, Simin; Schulmeister, Sonja; Pfeifer, Andrea C; Bachmann, Julie; Klingmüller, Ursula; Sourjik, Victor; Herten, Dirk-Peter

2010-10-01

Recent developments in fluorescence microscopy raise the demands for bright and photostable fluorescent tags for specific and background free labeling in living cells. Aside from fluorescent proteins and other tagging methods, labeling of SNAP-tagged proteins has become available thereby increasing the pool of potentially applicable fluorescent dyes for specific labeling of proteins. Here, we report on novel conjugates of benzylguanine (BG) which are quenched in their fluorescence and become highly fluorescent upon labeling of the SNAP-tag, the commercial variant of the human O(6)-alkylguanosyltransferase (hAGT). We identified four conjugates showing a strong increase, i.e., >10-fold, in fluorescence intensity upon labeling of SNAP-tag in vitro. Moreover, we screened a subset of nine BG-dye conjugates in living Escherichia coli and found them all suited for labeling of the SNAP-tag. Here, quenched BG-dye conjugates yield a higher specificity due to reduced contribution from excess conjugate to the fluorescence signal. We further extended the application of these conjugates by labeling a SNAP-tag fusion of the Tar chemoreceptor in live E. coli cells and the eukaryotic transcription factor STAT5b in NIH 3T3 mouse fibroblast cells. Aside from the labeling efficiency and specificity in living cells, we discuss possible mechanisms that might be responsible for the changes in fluorescence emission upon labeling of the SNAP-tag, as well as problems we encountered with nonspecific labeling with certain conjugates in eukaryotic cells.

Fluorescein isothiocyanate-labeled human plasma fibronectin in extracellular matrix remodeling.

PubMed

Hoffmann, Celine; Leroy-Dudal, Johanne; Patel, Salima; Gallet, Olivier; Pauthe, Emmanuel

2008-01-01

Fluorescein isothiocyanate (FITC) is a well-known probe for labeling biologically relevant proteins. However, the impact of the labeling procedure on protein structure and biological activities remains unclear. In this work, FITC-labeled human plasma fibronectin (Fn) was developed to gain insight into the dynamic relationship between cells and Fn. The similarities and differences concerning the structure and function between Fn-FITC and standard Fn were evaluated using biochemical as well as cellular approaches. By varying the FITC/Fn ratio, we demonstrated that overlabeling (>10 FITC molecules/Fn molecule) induces probe fluorescence quenching, protein aggregation, and cell growth modifications. A correct balance between reliable fluorescence for detection and no significant modifications to structure and biological function compared with standard Fn was obtained with a final ratio of 3 FITC molecules per Fn molecule (Fn-FITC3). Fn-FITC3, similar to standard Fn, is correctly recruited into the cell matrix network. Also, Fn-FITC3 is proposed to be a powerful molecular tool to investigate Fn organization and cellular behavior concomitantly.

Labelling Polymers and Micellar Nanoparticles via Initiation, Propagation and Termination with ROMP

PubMed Central

Thompson, Matthew P.; Randolph, Lyndsay M.; James, Carrie R.; Davalos, Ashley N.; Hahn, Michael E.

2014-01-01

In this paper we compare and contrast three approaches for labelling polymers with functional groups via ring-opening metathesis polymerization (ROMP). We explored the incorporation of functionality via initiation, termination and propagation employing an array of novel initiators, termination agents and monomers. The goal was to allow the generation of selectively labelled and well-defined polymers that would in turn lead to the formation of labelled nanomaterials. Norbornene analogues, prepared as functionalized monomers for ROMP, included fluorescent dyes (rhodamine, fluorescein, EDANS, and coumarin), quenchers (DABCYL), conjugatable moieties (NHS esters, pentafluorophenyl esters), and protected amines. In addition, a set of symmetrical olefins for terminally labelling polymers, and for the generation of initiators in situ is described. PMID:24855496

Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques

PubMed Central

Ghisaidoobe, Amar B. T.; Chung, Sang J.

2014-01-01

Förster resonance energy transfer (FRET) occurs when the distance between a donor fluorophore and an acceptor is within 10 nm, and its application often necessitates fluorescent labeling of biological targets. However, covalent modification of biomolecules can inadvertently give rise to conformational and/or functional changes. This review describes the application of intrinsic protein fluorescence, predominantly derived from tryptophan (λEX ∼ 280 nm, λEM ∼ 350 nm), in protein-related research and mainly focuses on label-free FRET techniques. In terms of wavelength and intensity, tryptophan fluorescence is strongly influenced by its (or the protein’s) local environment, which, in addition to fluorescence quenching, has been applied to study protein conformational changes. Intrinsic Förster resonance energy transfer (iFRET), a recently developed technique, utilizes the intrinsic fluorescence of tryptophan in conjunction with target-specific fluorescent probes as FRET donors and acceptors, respectively, for real time detection of native proteins. PMID:25490136

Optimizing labelling conditions of 213Bi-DOTATATE for preclinical applications of peptide receptor targeted alpha therapy.

PubMed

Chan, Ho Sze; de Blois, Erik; Konijnenberg, Mark W; Morgenstern, Alfred; Bruchertseifer, Frank; Norenberg, Jeffrey P; Verzijlbergen, Fred J; de Jong, Marion; Breeman, Wouter A P

2017-01-01

213 Bismuth ( 213 Bi, T 1/2 = 45.6 min) is one of the most frequently used α-emitters in cancer research. High specific activity radioligands are required for peptide receptor radionuclide therapy. The use of generators containing less than 222 MBq 225 Ac (actinium), due to limited availability and the high cost to produce large-scale 225 Ac/ 213 Bi generators, might complicate in vitro and in vivo applications though.Here we present optimized labelling conditions of a DOTA-peptide with an 225 Ac/ 213 Bi generator (< 222 MBq) for preclinical applications using DOTA-Tyr 3 -octreotate (DOTATATE), a somatostatin analogue. The following labelling conditions of DOTATATE with 213 Bi were investigated; peptide mass was varied from 1.7 to 7.0 nmol, concentration of TRIS buffer from 0.15 mol.L -1 to 0.34 mol.L -1 , and ascorbic acid from 0 to 71 mmol.L -1 in 800 μL. All reactions were performed at 95 °C for 5 min. After incubation, DTPA (50 nmol) was added to stop the labelling reaction. Besides optimizing the labelling conditions, incorporation yield was determined by ITLC-SG and radiochemical purity (RCP) was monitored by RP-HPLC up to 120 min after labelling. Dosimetry studies in the reaction vial were performed using Monte Carlo and in vitro clonogenic assay was performed with a rat pancreatic tumour cell line, CA20948. At least 3.5 nmol DOTATATE was required to obtain incorporation ≥ 99 % with 100 MBq 213 Bi (at optimized pH conditions, pH 8.3 with 0.15 mol.L -1 TRIS) in a reaction volume of 800 μL. The cumulative absorbed dose in the reaction vial was 230 Gy/100 MBq in 30 min. A minimal final concentration of 0.9 mmol.L -1 ascorbic acid was required for ~100 MBq (t = 0) to minimize radiation damage of DOTATATE. The osmolarity was decreased to 0.45 Osmol/L.Under optimized labelling conditions, 213 Bi-DOTATATE remained stable up to 2 h after labelling, RCP was ≥ 85 %. In vitro showed a negative correlation between ascorbic acid concentration and cell survival. 213 Bismuth-DOTA-peptide labelling conditions including peptide amount, quencher and pH were optimized to meet the requirements needed for preclinical applications in peptide receptor radionuclide therapy.

CdTe/CdS-MPA quantum dots as fluorescent probes to label yeast cells: synthesis, characterization and conjugation with Concanavalin A

NASA Astrophysics Data System (ADS)

Kato, Ilka T.; Santos, Camila C.; Benetti, Endi; Tenório, Denise P. L. A.; Cabral Filho, Paulo E.; Sabino, Caetano P.; Fontes, Adriana; Santos, Beate S.; Prates, Renato A.; Ribeiro, Martha S.

2012-03-01

Candida albicans is the most frequent human opportunistic pathogenic fungus and one of the most important causes of nosocomial infections. In fact, diagnosis of invasive candidiasis presents unique problems. The aim of this work was to evaluate, by fluorescence image analysis, cellular labeling of C. albicans with CdTe/CdS quantum dots conjugated or not to concanavalin A (ConA). Yeast cells were incubated with CdTe/CdS quantum dots (QD) stabilized with mercaptopropionic acid (MPA) (emission peak at 530 nm) for 1 hour. In the overall study we observed no morphological alterations. The fluorescence microscopic analysis of the yeast cells showed that the non-functionalized QDs do not label C. albicans cells, while for the QD conjugated to ConA the cells showed a fluorescence profile indicating that the membrane was preferentially marked. This profile was expected since Concanavalin A is a protein that binds specifically to terminal carbohydrate residues at the membrane cell surface. The results suggest that the QD-labeled Candida cells represent a promising tool to open new possibilities for a precise evaluation of fungal infections in pathological conditions.

Sequential strand displacement beacon for detection of DNA coverage on functionalized gold nanoparticles.

PubMed

Paliwoda, Rebecca E; Li, Feng; Reid, Michael S; Lin, Yanwen; Le, X Chris

2014-06-17

Functionalizing nanomaterials for diverse analytical, biomedical, and therapeutic applications requires determination of surface coverage (or density) of DNA on nanomaterials. We describe a sequential strand displacement beacon assay that is able to quantify specific DNA sequences conjugated or coconjugated onto gold nanoparticles (AuNPs). Unlike the conventional fluorescence assay that requires the target DNA to be fluorescently labeled, the sequential strand displacement beacon method is able to quantify multiple unlabeled DNA oligonucleotides using a single (universal) strand displacement beacon. This unique feature is achieved by introducing two short unlabeled DNA probes for each specific DNA sequence and by performing sequential DNA strand displacement reactions. Varying the relative amounts of the specific DNA sequences and spacing DNA sequences during their coconjugation onto AuNPs results in different densities of the specific DNA on AuNP, ranging from 90 to 230 DNA molecules per AuNP. Results obtained from our sequential strand displacement beacon assay are consistent with those obtained from the conventional fluorescence assays. However, labeling of DNA with some fluorescent dyes, e.g., tetramethylrhodamine, alters DNA density on AuNP. The strand displacement strategy overcomes this problem by obviating direct labeling of the target DNA. This method has broad potential to facilitate more efficient design and characterization of novel multifunctional materials for diverse applications.

Analysis of the 22-NBD-cholesterol transfer between liposome membranes and its relation to the intermembrane exchange of 25-hydroxycholesterol.

PubMed

Ishii, Haruyuki; Shimanouchi, Toshinori; Umakoshi, Hiroshi; Walde, Peter; Kuboi, Ryoichi

2010-05-01

The transfer of 22-NBD-cholesterol (22-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol) between two liposome membranes was quantitatively analyzed by using the fluorescence resonance energy transfer (FRET) method. Liposomes labeled with both 22-NBD-cholesterol and a rhodamine-labeled phosphatidylethanolamine (Rh-DHPE) were used as donor liposomes, and the 22-NBD-cholesterol transfer from these donor liposomes to acceptor liposomes prepared from same type of phosphatidylcholine was monitored. The transfer kinetics was found to be composed of a fast and a slow phase, and all kinetic measurements could be fitted with a bi-exponential model. The results obtained indicate that the 22-NBD-cholesterol transfer kinetics between liposome membranes depends on the fluidity of the liposome used and that the curvature may affect the kinetics. Furthermore, the behavior of 22-NBD-cholesterol in lipid membrane is similar to that of the oxysterol 25-hydroxycholesterol rather than cholesterol. It is proposed that 22-NBD-cholesterol can be a useful fluorescent probe to mimic the intermembrane transfer of oxidized cholesterols like 25-hydroxycholesterol, rather than that of cholesterol itself. 2010 Elsevier B.V. All rights reserved.

Streptavidin in Antibody Pretargeting. 5. Chemical Modification of Recombinant Streptavidin for Labeling with the α-Particle Emitting Radionuclides 213Bi and 211At

PubMed Central

Wilbur, D. Scott; Hamlin, Donald K.; Chyan, Ming-Kuan; Brechbiel, Martin W.

2008-01-01

We are investigating the use of recombinant streptavidin (rSAv) as a carrier molecule for the short-lived α-particle emitting radionuclides 213Bi (t1/2 = 45.6 min) and 211At (t1/2 = 7.21 h) in cancer therapy. To utilize rSAv as a carrier, it must be modified in a manner that permits rapid chelation or bonding with these short-lived radionuclides, and also modified in a manner that diminishes its natural propensity for localization in kidney. Modification for labeling with 213Bi was accomplished by conjugation of rSAv with the DTPA derivative p-isothiocyanato-benzyl-CHX-A″ (CHX-A″), 3a. Modification for direct labeling with 211At was accomplished by conjugation of rSAv with an isothiocyanatophenyl derivative of a nido-carborane (nCB), 3b, or an isothiocyanatophenyl-dPEG™/decaborate(2-) derivative, 3c. After conjugation of the chelating or bonding moiety, rSAv was further modified by reaction with an excess (50–100 equivalents) of succinic anhydride. Succinylation of the lysine amines has previously been shown to greatly diminish kidney localization. rSAv modified by conjugation with 3a and succinylated radiolabeled rapidly with 213Bi (< 5 min), providing a 72% isolated yield. 211At labeling of modified rSAv was accomplished in aqueous solution using chloramine-T as the oxidant. Astatination of rSAv conjugated with 3b and succinylated occurred very rapidly (<1 min), providing a 50% isolated radiochemical yield. Astatination of rSAv conjugated with 3c and succinylated was also very rapid (<1 min) providing 66–71% isolated radiochemical yields. Astatination of succinylated rSAv, 2a, which did not have conjugated borane cage moieties, resulted in much lower radiolabeling yield (18%). The 213Bi- or 211At-labeled modified rSAv preparations were mixed with the corresponding 125I-labeled rSAv, and dual-label in vivo distributions were obtained in athymic mice. The in vivo data show that 213Bi-labeled succinylated rSAv [213Bi]6a has tissue concentrations similar to 125I-labeled modified rSAv [125I]6b, suggesting that 213Bi is quite stable towards release from the chelate in vivo. In vivo data also indicate that the 211At-labeled rSAv conjugated with 3b or 3c and succinylated are stable to in vivo deastatination, whereas succinylated rSAv lacking a boron cage moiety is subject to some deastatination. The modified rSAv conjugated with nido-carborane derivative 3b has a higher retention in many tissues than rSAv without the carborane conjugated. Interestingly, the rSAv conjugated with 3c, which also contains a m-dPEG12™ moiety, has significantly decreased concentrations in blood and other tissues when compared with direct labeled rSAv, suggesting that it may be a good candidate for further study. In conclusion, rSAv that has been modified with CHX-A″ and succinylated (i.e. 5a) may be useful as a carrier of 213Bi. The encouraging results obtained with the PEGylated decaborate(2-) derivative 3c and succinylated (i.e. 5c) suggests that its further study as a carrier of 211At in pretargeting protocols is warranted. PMID:18072725

Morpholine Derivative-Functionalized Carbon Dots-Based Fluorescent Probe for Highly Selective Lysosomal Imaging in Living Cells.

PubMed

Wu, Luling; Li, Xiaolin; Ling, Yifei; Huang, Chusen; Jia, Nengqin

2017-08-30

The development of a suitable fluorescent probe for the specific labeling and imaging of lysosomes through the direct visual fluorescent signal is extremely important for understanding the dysfunction of lysosomes, which might induce various pathologies, including neurodegenerative diseases, cancer, and Alzheimer's disease. Herein, a new carbon dot-based fluorescent probe (CDs-PEI-ML) was designed and synthesized for highly selective imaging of lysosomes in live cells. In this probe, PEI (polyethylenimine) is introduced to improve water solubility and provide abundant amine groups for the as-prepared CDs-PEI, and the morpholine group (ML) serves as a targeting unit for lysosomes. More importantly, passivation with PEI could dramatically increase the fluorescence quantum yield of CDs-PEI-ML as well as their stability in fluorescence emission under different excitation wavelength. Consequently, experimental data demonstrated that the target probe CDs-PEI-ML has low cytotoxicity and excellent photostability. Additionally, further live cell imaging experiment indicated that CDs-PEI-ML is a highly selective fluorescent probe for lysosomes. We speculate the mechanism for selective staining of lysosomes that CDs-PEI-ML was initially taken up by lysosomes through the endocytic pathway and then accumulated in acidic lysosomes. It is notable that there was less diffusion of CDs-PEI-ML into cytoplasm, which could be ascribed to the presence of lysosome target group morpholine on surface of CDs-PEI-ML. The blue emission wavelength combined with the high photo stability and ability of long-lasting cell imaging makes CDs-PEI-ML become an alternative fluorescent probe for multicolor labeling and long-term tracking of lysosomes in live cells and the potential application in super-resolution imaging. To best of our knowledge, there are still limited carbon dots-based fluorescent probes that have been studied for specific lysosomal imaging in live cells. The concept of surface functionality of carbon dots will also pave a new avenue for developing carbon dots-based fluorescent probes for subcellular labeling.

Nanoscale Label-free Bioprobes to Detect Intracellular Proteins in Single Living Cells

PubMed Central

Hong, Wooyoung; Liang, Feng; Schaak, Diane; Loncar, Marko; Quan, Qimin

2014-01-01

Fluorescent labeling techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for use in non-transfectable cells, and labels can interfere with protein functions. While label-free biosensors have been realized by nanofabrication, a method to track intracellular protein dynamics in real-time, in situ and in living cells has not been found. Here we present the first demonstration of label-free detection of intracellular p53 protein dynamics through a nanoscale surface plasmon-polariton fiber-tip-probe (FTP). PMID:25154394

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels.

PubMed

Virtanen, Otto L J; Purohit, Ashvini; Brugnoni, Monia; Wöll, Dominik; Richtering, Walter

2016-09-08

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this work, we use single particle tracking of fluorescently labeled PNIPAM microgels as a showcase for tuning microgel size by a rapid non-stirred precipitation polymerization procedure. This approach is well suited for prototyping new reaction compositions and conditions or for applications that do not require large amounts of product. Microgel synthesis, particle size and structure determination by dynamic and static light scattering are detailed in the protocol. It is shown that the addition of functional comonomers can have a large influence on the particle nucleation and structure. Single particle tracking by wide-field fluorescence microscopy allows for an investigation of the diffusion of labeled tracer microgels in a concentrated matrix of non-labeled microgels, a system not easily investigated by other methods such as dynamic light scattering.

Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.

PubMed

Autour, Alexis; C Y Jeng, Sunny; D Cawte, Adam; Abdolahzadeh, Amir; Galli, Angela; Panchapakesan, Shanker S S; Rueda, David; Ryckelynck, Michael; Unrau, Peter J

2018-02-13

Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.

Efficient switching of mCherry fluorescence using chemical caging.

PubMed

Cloin, Bas M C; De Zitter, Elke; Salas, Desiree; Gielen, Vincent; Folkers, Gert E; Mikhaylova, Marina; Bergeler, Maike; Krajnik, Bartosz; Harvey, Jeremy; Hoogenraad, Casper C; Van Meervelt, Luc; Dedecker, Peter; Kapitein, Lukas C

2017-07-03

Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantum-mechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores.

Efficient switching of mCherry fluorescence using chemical caging

PubMed Central

Cloin, Bas M. C.; Salas, Desiree; Gielen, Vincent; Folkers, Gert E.; Mikhaylova, Marina; Bergeler, Maike; Krajnik, Bartosz; Harvey, Jeremy; Hoogenraad, Casper C.; Van Meervelt, Luc; Dedecker, Peter; Kapitein, Lukas C.

2017-01-01

Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantum-mechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores. PMID:28630286

The small molecule probe PT-Yellow labels the renal proximal tubules in zebrafish.

PubMed

Sander, Veronika; Patke, Shantanu; Sahu, Srikanta; Teoh, Chai Lean; Peng, Zhenzhen; Chang, Young-Tae; Davidson, Alan J

2015-01-01

We report the development of a small fluorescent molecule, BDNCA3-D2, herein referred to as PT-Yellow. Soaking zebrafish embryos in PT-Yellow or intraperitoneal injection into adults results in non-toxic in vivo fluorescent labeling of the renal proximal tubules, the major site of blood filtrate reabsorption and a common target of injury in acute kidney injury. We demonstrate the applicability of this new compound as a rapid and simple readout for zebrafish kidney filtration and proximal tubule reabsorption function.

SYNTHESIS AND APPLICATIONS OF Fe3O4/SiO2 CORE-SHELL MATERIALS.

PubMed

Sonmez, Maria; Georgescu, Mihai; Alexandrescu, Laurentia; Gurau, Dana; Ficai, Anton; Ficai, Denisa; Andronescu, Ecaterina

2015-01-01

Multifunctional nanoparticles based on magnetite/silica core-shell, consisting of iron oxides coated with silica matrix doped with fluorescent components such as organic dyes (fluorescein isothiocyanate - FITC, Rhodamine 6G) or quantum dots, have drawn remarkable attention in the last years. Due to the bi-functionality of these types of nanoparticles (simultaneously having magnetic and fluorescent properties), they are successfully used in highly efficient human stem cell labeling, magnetic carrier for photodynamic therapy, drug delivery, hyperthermia and other biomedical applications. Another application of core-shell-based nanoparticles, in which the silica is functionalized with aminosilanes, is for immobilization and separation of various biological entities such as proteins, antibodies, enzymes etc. as well as in environmental applications, as adsorbents for heavy metal ions. In vitro tests on human cancerous cells, such as A549 (human lung carcinoma), breast, human cervical cancer, THP-1 (human acute monocytic leukaemia) etc. , were conducted to assess the potential cytotoxic effects that may occur upon contact of nanoparticles with cancerous tissue. Results show that core-shell nanoparticles doped with cytostatics (cisplatin, doxorubicin, etc.), are easily adsorbed by affected tissue and in some cases lead to an inhibition of cell proliferation and induce cell death by apoptosis. The goal of this review is to summarize the advances in the field of core-shell materials, particularly those based on magnetite/silica with applicability in medicine and environmental protection. This paper briefly describes synthesis methods of silica-coated magnetite nanoparticles (Stöber method and microemulsion), the method of encapsulating functional groups based on aminosilanes in silica shell, as well as applications in medicine of these types of simple or modified nanoparticles for cancer therapy, MRI, biomarker immobilization, drug delivery, biocatalysis etc., and in environmental applications (removal of heavy metal ions and catalysis).

Novel biosensor system model based on fluorescence quenching by a fluorescent streptavidin and carbazole-labeled biotin.

PubMed

Zhu, Xianwei; Shinohara, Hiroaki; Miyatake, Ryuta; Hohsaka, Takahiro

2016-10-01

In the present study, a novel molecular biosensor system model was designed by using a couple of the fluorescent unnatural mutant streptavidin and the carbazole-labeled biotin. BODIPY-FL-aminophenylalanine (BFLAF), a fluorescent unnatural amino acid was position-specifically incorporated into Trp120 position of streptavidin by four-base codon method. On the other hand, carbazole-labeled biotin was synthesized as a quencher for the fluorescent Trp120BFLAF mutant streptavidin. The fluorescence of fluorescent Trp120BFLAF mutant streptavidin was decreased as we expected when carbazole-labeled biotin was added into the mutant streptavidin solution. Furthermore, the fluorescence decrease of Trp120BFLAF mutant streptavidin with carbazole-labeled biotin (100 nM) was recovered by the competitive addition of natural biotin. This result demonstrated that by measuring the fluorescence quenching and recovery, a couple of the fluorescent Trp120BFLAF mutant streptavidin and the carbazole-labeled biotin were successfully applicable for quantification of free biotin as a molecular biosensor system. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

A rapid and fluorogenic TMP-AcBOPDIPY probe for covalent labeling of proteins in live cells.

PubMed

Liu, Wei; Li, Fu; Chen, Xi; Hou, Jian; Yi, Long; Wu, Yao-Wen

2014-03-26

Protein labeling is enormously useful for characterizing protein function in cells and organisms. Chemical tagging methods have emerged as a new generation protein labeling strategy in live cells. Here we have developed a novel and versatile TMP-AcBOPDIPY probe for selective and turn-on labeling of proteins in live cells. A small monomeric tag, E. coli dihydrofolate reductase (eDHFR), was rationally designed to introduce a cysteine in the vicinity of the ligand binding site. Trimethoprim (TMP) that specifically binds to eDHFR was linked to the BOPDIPY fluorophore containing a mildly thiol-reactive acrylamide group. TMP-AcBOPDIPY rapidly labeled engineered eDHFR tags via a reaction termed affinity conjugation (a half-life of ca. 2 min), which is one of the top fast chemical probes for protein labeling. The probe displays 2-fold fluorescence enhancement upon labeling of proteins. We showed that the probe specifically labeled intracellular proteins in live cells without and with washing out the dye. We demonstrated its utility in visualizing intracellular processes by fluorescence-lifetime imaging microscopy (FLIM) measurements.

Incorporation of isotopic, fluorescent, and heavy-atom-modified nucleotides into RNAs by position-selective labeling of RNA.

PubMed

Liu, Yu; Holmstrom, Erik; Yu, Ping; Tan, Kemin; Zuo, Xiaobing; Nesbitt, David J; Sousa, Rui; Stagno, Jason R; Wang, Yun-Xing

2018-05-01

Site-specific incorporation of labeled nucleotides is an extremely useful synthetic tool for many structural studies (e.g., NMR, electron paramagnetic resonance (EPR), fluorescence resonance energy transfer (FRET), and X-ray crystallography) of RNA. However, specific-position-labeled RNAs >60 nt are not commercially available on a milligram scale. Position-selective labeling of RNA (PLOR) has been applied to prepare large RNAs labeled at desired positions, and all the required reagents are commercially available. Here, we present a step-by-step protocol for the solid-liquid hybrid phase method PLOR to synthesize 71-nt RNA samples with three different modification applications, containing (i) a 13 C 15 N-labeled segment; (ii) discrete residues modified with Cy3, Cy5, or biotin; or (iii) two iodo-U residues. The flexible procedure enables a wide range of downstream biophysical analyses using precisely localized functionalized nucleotides. All three RNAs were obtained in <2 d, excluding time for preparing reagents and optimizing experimental conditions. With optimization, the protocol can be applied to other RNAs with various labeling schemes, such as ligation of segmentally labeled fragments.

Labeling the oily core of nanocapsules and lipid-core nanocapsules with a triglyceride conjugated to a fluorescent dye as a strategy to particle tracking in biological studies

NASA Astrophysics Data System (ADS)

Fiel, Luana Almeida; Contri, Renata Vidor; Bica, Juliane Freitas; Figueiró, Fabrício; Battastini, Ana Maria Oliveira; Guterres, Sílvia Stanisçuaski; Pohlmann, Adriana Raffin

2014-05-01

The synthesis of novel fluorescent materials represents a very important step to obtain labeled nanoformulations in order to evaluate their biological behavior. The strategy of conjugating a fluorescent dye with triacylglycerol allows that either particles differing regarding supramolecular structure, i.e., nanoemulsions, nanocapsules, lipid-core nanocapsules, or surface charge, i.e., cationic nanocapsules and anionic nanocapsules, can be tracked using the same labeled material. In this way, a rhodamine B-conjugated triglyceride was obtained to prepare fluorescent polymeric nanocapsules. Different formulations were obtained, nanocapsules (NC) or lipid-core nanocapsules (LNC), using the labeled oil and Eudragit RS100, Eudragit S100, or poly(caprolactone) (PCL), respectively. The rhodamine B was coupled with the ricinolein by activating the carboxylic function using a carbodiimide derivative. Thin layer chromatography, proton nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy (FTIR), UV-vis, and fluorescence spectroscopy were used to identify the new product. Fluorescent nanocapsule aqueous suspensions were prepared by the solvent displacement method. Their pH values were 4.6 (NC-RS100), 3.5 (NC-S100), and 5.0 (LNC-PCL). The volume-weighted mean diameter ( D 4.3) and polydispersity values were 150 nm and 1.05 (NC-RS100), 350 nm and 2.28 (NC-S100), and 270 nm and 1.67 (LNC-PCL). The mean diameters determined by photon correlation spectroscopy (PCS) ( z-average) were around 200 nm. The zeta potential values were +5.85 mV (NC-RS100), -21.12 mV (NC-S100), and -19.25 mV (LNC-PCL). The wavelengths of maximum fluorescence emission were 567 nm (NC-RS100 and LNC-PCL) and 574 nm (NC-S100). Fluorescence microscopy was used to evaluate the cell uptake (human macrophage cell line) of the fluorescent nanocapsules in order to show the applicability of the approach. When the cells were treated with the fluorescent nanocapsules, red emission was detected around the cell nucleus. We demonstrated that the rhodamine B-conjugated triglyceride is a promising new material to obtain versatile dye-labeled nanocarriers presenting different chemical nature in their surfaces.

A novel ion-exchange strategy for the fabrication of high strong BiOI/BiOBr heterostructure film coated metal wire mesh with tunable visible-light-driven photocatalytic reactivity.

PubMed

Wang, Yi; Long, Yang; Yang, Zhiqing; Zhang, Dun

2018-06-05

Visible-light-driven (VLD) BiOI/BiOBr heterostructure films with hierarchical microstructure have been firstly fabricated on 304 stainless steel wire mesh (304SSWM) substrates through a novel ion-exchange method using the BiOI film as precursor. The concentration of tetrabutylammonium bromide (TBAB) is the key factor to control the composition and microstructure of BiOI/BiOBr films. Physical, chemical, and optical properties of BiOI/BiOBr heterostructure films were characterized by X-ray diffraction, scanning electron microscope, energy-dispersive X-ray spectroscopy, high resolution transmittance electron microscopy, X-ray photoelectron spectroscopy, UV-vis diffuse reflectance absorption, and fluorescence spectrophotometer, respectively. The VLD photocatalytic ability of the BiOI/BiOBr heterostructure film coated 304SSWM was studied by degrading rhodamine B and pIRES2-EGFP plasmid as target water organic pollutants and pathogenic bacteria genetic materials. The BiOI/BiOBr heterostructure film coated 304SSWM fabricated with 50 mM TBAB has excellent photocatalytic activity, stability, and reusability in the cycled experiments. The reasons for these unique features can be ascribed to the formation of heterojuction structure and the open framework structure of the 304SSWM. The current work can provide new strategies to construct novel VLD photoactive functional films for water purification and disinfection. Copyright © 2018 Elsevier B.V. All rights reserved.

Activity of cardiorespiratory networks revealed by transsynaptic virus expressing GFP.

PubMed

Irnaten, M; Neff, R A; Wang, J; Loewy, A D; Mettenleiter, T C; Mendelowitz, D

2001-01-01

A fluorescent transneuronal marker capable of labeling individual neurons in a central network while maintaining their normal physiology would permit functional studies of neurons within entire networks responsible for complex behaviors such as cardiorespiratory reflexes. The Bartha strain of pseudorabies virus (PRV), an attenuated swine alpha herpesvirus, can be used as a transsynaptic marker of neural circuits. Bartha PRV invades neuronal networks in the CNS through peripherally projecting axons, replicates in these parent neurons, and then travels transsynaptically to continue labeling the second- and higher-order neurons in a time-dependent manner. A Bartha PRV mutant that expresses green fluorescent protein (GFP) was used to visualize and record from neurons that determine the vagal motor outflow to the heart. Here we show that Bartha PRV-GFP-labeled neurons retain their normal electrophysiological properties and that the labeled baroreflex pathways that control heart rate are unaltered by the virus. This novel transynaptic virus permits in vitro studies of identified neurons within functionally defined neuronal systems including networks that mediate cardiovascular and respiratory function and interactions. We also demonstrate superior laryngeal motorneurons fire spontaneously and synapse on cardiac vagal neurons in the nucleus ambiguus. This cardiorespiratory pathway provides a neural basis of respiratory sinus arrhythmias.

Developmentally Regulated Sphingolipid Synthesis in African Trypanosomes

PubMed Central

Sutterwala, Shaheen S.; Hsu, Fong Fu; Sevova, Elitza S.; Schwartz, Kevin J.; Zhang, Kai; Key, Phillip; Turk, John; Beverley, Stephen M.; Bangs, James D.

2008-01-01

Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase. Procyclic trypanosomes contain IPC, but also sphingomyelin, while surprisingly bloodstream stage parasites contain sphingomyelin and ethanolamine phosphorylceramide (EPC), but no detectable IPC. In vivo fluorescent ceramide labeling confirmed stage specific biosynthesis of both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of TbSLS1-4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels were increased >3-fold by silencing suggesting a toxic downstream effect mediated by this potent intracellular messenger. Topology predictions support a revised six transmembrane domain model for the kinetoplastid sphingolipid synthases consistent with the proposed mammalian SM synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites. PMID:18699867

Detecting RNA/DNA hybridization using double-labeled donor probes with enhanced fluorescence resonance energy transfer signals.

PubMed

Okamura, Yukio; Watanabe, Yuichiro

2006-01-01

Fluorescence resonance energy transfer (FRET) occurs when two fluorophores are in close proximity, and the emission energy of a donor fluorophore is transferred to excite an acceptor fluorophore. Using such fluorescently labeled oligonucleotides as FRET probes, makes possible specific detection of RNA molecules even if similar sequences are present in the environment. A higher ratio of signal to background fluorescence is required for more sensitive probe detection. We found that double-labeled donor probes labeled with BODIPY dye resulted in a remarkable increase in fluorescence intensity compared to single-labeled donor probes used in conventional FRET. Application of this double-labeled donor system can improve a variety of FRET techniques.

A Hierarchical multi-input and output Bi-GRU Model for Sentiment Analysis on Customer Reviews

NASA Astrophysics Data System (ADS)

Zhang, Liujie; Zhou, Yanquan; Duan, Xiuyu; Chen, Ruiqi

2018-03-01

Multi-label sentiment classification on customer reviews is a practical challenging task in Natural Language Processing. In this paper, we propose a hierarchical multi-input and output model based bi-directional recurrent neural network, which both considers the semantic and lexical information of emotional expression. Our model applies two independent Bi-GRU layer to generate part of speech and sentence representation. Then the lexical information is considered via attention over output of softmax activation on part of speech representation. In addition, we combine probability of auxiliary labels as feature with hidden layer to capturing crucial correlation between output labels. The experimental result shows that our model is computationally efficient and achieves breakthrough improvements on customer reviews dataset.

Correlation Functions Quantify Super-Resolution Images and Estimate Apparent Clustering Due to Over-Counting

PubMed Central

Veatch, Sarah L.; Machta, Benjamin B.; Shelby, Sarah A.; Chiang, Ethan N.; Holowka, David A.; Baird, Barbara A.

2012-01-01

We present an analytical method using correlation functions to quantify clustering in super-resolution fluorescence localization images and electron microscopy images of static surfaces in two dimensions. We use this method to quantify how over-counting of labeled molecules contributes to apparent self-clustering and to calculate the effective lateral resolution of an image. This treatment applies to distributions of proteins and lipids in cell membranes, where there is significant interest in using electron microscopy and super-resolution fluorescence localization techniques to probe membrane heterogeneity. When images are quantified using pair auto-correlation functions, the magnitude of apparent clustering arising from over-counting varies inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. In contrast, we demonstrate that over-counting does not give rise to apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (FcεRI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM/dSTORM) and scanning electron microscopy (SEM). We find that apparent clustering of FcεRI-bound IgE is dominated by over-counting labels on individual complexes when IgE is directly conjugated to organic fluorophores. We verify this observation by measuring pair cross-correlation functions between two distinguishably labeled pools of IgE-FcεRI on the cell surface using both imaging methods. After correcting for over-counting, we observe weak but significant self-clustering of IgE-FcεRI in fluorescence localization measurements, and no residual self-clustering as detected with SEM. We also apply this method to quantify IgE-FcεRI redistribution after deliberate clustering by crosslinking with two distinct trivalent ligands of defined architectures, and we evaluate contributions from both over-counting of labels and redistribution of proteins. PMID:22384026

Proximity-Induced Covalent Labeling of Proteins with a Reactive Fluorophore-Binding Peptide Tag.

PubMed

Sunbul, Murat; Nacheva, Lora; Jäschke, Andres

2015-08-19

Labeling of proteins with fluorescent dyes in live cells enables the investigation of their roles in biological systems by fluorescence microscopy. Because the labeling procedure should not disturb the native function of the protein of interest, it is of high importance to find the optimum labeling method for the problem to be studied. Here, we developed a rapid one-step method to covalently and site-specifically label proteins with a TexasRed fluorophore in vitro and in live bacteria. To this end, a genetically encodable TexasRed fluorophore-binding peptide (TR512) was converted into a reactive tag (ReacTR) by adjoining a cysteine residue which rapidly reacts with N-α-chloroacetamide-conjugated TexasRed fluorophore owing to the proximity effect; ReacTR tag first binds to the TexasRed fluorophore and this interaction brings the nucleophilic cysteine and the electrophilic N-α-chloroacetamide groups in close proximity. Our method has several advantages over existing methods: (i) it utilizes a peptide tag much smaller than fluorescent proteins, the SNAP, CLIP, or HaLo tags; (ii) it allows for labeling of proteins with a small, photostable, red-emitting TexasRed fluorophore; (iii) the probe used is very easy to synthesize; (iv) no enzyme is required to transfer the fluorophore to the peptide tag; and (v) labeling yields a stable covalent product in a very fast reaction.

An Advanced Semimetal-Organic Bi Spheres-g-C3N4 Nanohybrid with SPR-Enhanced Visible-Light Photocatalytic Performance for NO Purification.

PubMed

Dong, Fan; Zhao, Zaiwang; Sun, Yanjuan; Zhang, Yuxin; Yan, Shuai; Wu, Zhongbiao

2015-10-20

To achieve efficient photocatalytic air purification, we constructed an advanced semimetal-organic Bi spheres-g-C3N4 nanohybrid through the in-situ growth of Bi nanospheres on g-C3N4 nanosheets. This Bi-g-C3N4 compound exhibited an exceptionally high and stable visible-light photocatalytic performance for NO removal due to the surface plasmon resonance (SPR) endowed by Bi metal. The SPR property of Bi could conspicuously enhance the visible-light harvesting and the charge separation. The electromagnetic field distribution of Bi spheres involving SPR effect was simulated and reaches its maximum in close proximity to the Bi particle surface. When the Bi metal content was controlled at 25%, the corresponding Bi-g-C3N4 displayed outstanding photocatalytic capability and transcended those of other visible-light photocatalysts. The Bi-g-C3N4 exhibited a high structural stability under repeated photocatalytic runs. A new visible-light-induced SPR-based photocatalysis mechanism with Bi-g-C3N4 was proposed on the basis of the DMPO-ESR spin-trapping. The photoinduced electrons could transfer from g-C3N4 to the Bi metal, as revealed with time-resolved fluorescence spectra. The function of Bi semimetal as a plasmonic cocatalyst for boosting visible light photocatalysis was similar to that of noble metals, which demonstrated a great potential of utilizing the economically feasible Bi element as a substitute for noble metals for the advancement of photocatalysis efficiency.

A new fluorogenic small molecule labeling tool for surface diffusion analysis and advanced fluorescence imaging of β-site amyloid precursor protein (APP)-cleaving enzyme 1 based on silicone rhodamine: SiR-BACE1.

PubMed

Karch, Sandra; Broichhagen, Johannes; Schneider, Julia; Böning, Daniel; Hartmann, Stephanie; Schmid, Benjamin; Tripal, Philipp; Palmisano, Ralf; Alzheimer, Christian; Johnsson, Kai; Huth, Tobias

2018-06-25

β-site APP-cleaving enzyme 1 (BACE1) is a major player in the pathogenesis of Alzheimer's disease. Structural and functional fluorescence microscopy offers a powerful approach to learn about the physiology and pathophysiology of this protease. Up to now, however, common labeling techniques either require genetic manipulation, use large antibodies, or are not compatible with live cell imaging. Fluorescent small molecules that specifically bind to the protein of interest can overcome these limitations. Herein, we introduce SiR-BACE1, a conjugate of the BACE1 inhibitor S-39 and SiR647, as a novel fluorogenic, tag-free, and antibody-free label for BACE1. We present its chemical development, characterize its photo-physical and pharmacologic properties, and evaluate its behavior in solution, in over-expression systems, and in native brain tissue. We demonstrate its applicability in confocal, stimulated emission depletion (STED), and dynamic single molecule microscopy. First functional studies with SiR-BACE1 on the surface mobility of BACE1 revealed a markedly confined diffusion pattern.

Consumption of Bifidobacterium lactis Bi-07 by healthy elderly adults enhances phagocytic activity of monocytes and granulocytes.

PubMed

Maneerat, Sujira; Lehtinen, Markus J; Childs, Caroline E; Forssten, Sofia D; Alhoniemi, Esa; Tiphaine, Milin; Yaqoob, Parveen; Ouwehand, Arthur C; Rastall, Robert A

2013-01-01

Elderly adults have alterations in their gut microbiota and immune functions that are associated with higher susceptibility to infections and metabolic disorders. Probiotics and prebiotics, and their synbiotic combinations are food supplements that have been shown to improve both gut and immune function. The objective of this randomised, double-blind, placebo-controlled, cross-over human clinical trial was to study immune function and the gut microbiota in healthy elderly adults. Volunteers (n 37) consumed prebiotic galacto-oligosaccharides (GOS; 8 g/d), probiotic Bifidobacterium lactis Bi-07 (Bi-07; 10(9) colony-forming units/d), their combination (Bi-07 + GOS) and maltodextrin control (8 g/d) in four 3-week periods separated by 4-week wash-out periods. Immune function was analysed by determining the phagocytic and oxidative burst activity of monocytes and granulocytes, whole-blood response to lipopolysaccharide, plasma chemokine concentrations and salivary IgA levels. Gut microbiota composition and faecal SCFA content were determined using 16S ribosomal RNA fluorescence in situ hybridisation and HPLC, respectively. Primary statistical analyses indicated the presence of carry-over effects and thus measurements from only the first supplementation period were considered valid. Subsequent statistical analysis showed that consumption of Bi-07 improved the phagocytic activity of monocytes (P < 0·001) and granulocytes (P = 0·02). Other parameters were unchanged. We have for the first time shown that the probiotic Bi-07 may provide health benefits to elderly individuals by improving the phagocytic activity of monocytes and granulocytes. The present results also suggest that in the elderly, the effects of some probiotics and prebiotics may last longer than in adults.

“Ultra-high resolution optical trap with single fluorophore sensitivity”

PubMed Central

Comstock, Matthew J; Ha, Taekjip; Chemla, Yann R

2013-01-01

We present a single-molecule instrument that combines a timeshared ultra-high resolution dual optical trap interlaced with a confocal fluorescence microscope. In a demonstration experiment, individual single-fluorophore labeled DNA oligonucleotides were observed to bind and unbind to complementary DNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, coincident angstrom-scale changes in tether extension could be clearly observed. Fluorescence readout allowed us to determine the duplex melting rate as a function of force. The new instrument will enable the simultaneous measurement of angstrom-scale mechanical motion of individual DNA-binding proteins (e.g., single base pair stepping of DNA translocases) along with the detection of fluorescently labeled protein properties (e.g., internal configuration). PMID:21336286

Implementation of Arithmetic and Nonarithmetic Functions on a Label-free and DNA-based Platform

NASA Astrophysics Data System (ADS)

Wang, Kun; He, Mengqi; Wang, Jin; He, Ronghuan; Wang, Jianhua

2016-10-01

A series of complex logic gates were constructed based on graphene oxide and DNA-templated silver nanoclusters to perform both arithmetic and nonarithmetic functions. For the purpose of satisfying the requirements of progressive computational complexity and cost-effectiveness, a label-free and universal platform was developed by integration of various functions, including half adder, half subtractor, multiplexer and demultiplexer. The label-free system avoided laborious modification of biomolecules. The designed DNA-based logic gates can be implemented with readout of near-infrared fluorescence, and exhibit great potential applications in the field of bioimaging as well as disease diagnosis.

Fluorometric determination of Vibrio parahaemolyticus using an F0F1-ATPase-based aptamer and labeled chromatophores.

PubMed

Duan, Nuo; Wu, Shijia; Zhang, Huiling; Zou, Ying; Wang, Zhouping

2018-05-18

An F 0 F 1 -ATPase-based aptasensor is described for the fluorometric determination of Vibrio parahaemolyticus. Chromatophores containing F 0 F 1 -ATPases were first prepared from Rhodospirillum rubrum cells. Then, an aptamer-functionalized chromatophore acts as the capture probe, and a chromatophore labeled with the pH probe fluorescein acts as the signalling probe. In the presence of V. parahaemolyticus, the rotation rate of F 0 F 1 -ATPase is decreased due to the formation of the aptamer-chromatophore complex. This leads to a retarded proton flux out of the chromatophores. As a result, the pH value inside the chromatophores is reduced, and the fluorescence of the pH probe F1300 is accordingly decreased. The relative fluorescence varies linearly over the 15 to 1.5 × 10 6  cfu·mL -1 Vibrio parahaemolyticus concentration range, and the limit of detection is 15 cfu·mL -1 . The method was applied to analyze artificially contaminated salmon samples where it showed excellent perfomance. Graphical abstract In this assay, aptamer functionalized chromatophores act as a capture probe, and the fluoresce in labeled chromatophores as signalling probe. The formation of aptamer-chromatophore complex leads to a retarded proton flux out of the chromatophores. As a result, the pH value inside the chromatophores is reduced, and fluorescence intensity is accordingly decreased.

Temperature optimum of insulin-stimulated 2-deoxy-D-glucose uptake in rat adipocytes. Correlation of cellular transport with membrane spin-label and fluorescence-label data.

PubMed Central

Hyslop, P A; Kuhn, C E; Sauerheber, R D

1984-01-01

The effects of temperature alterations between 22 degrees C and 48 degrees C on basal and insulin-stimulated 2-deoxy-D-[1-14C]glucose uptake were examined in isolated rat adipocytes. A distinct optimum was found near physiological temperature for uptake in the presence of maximally effective insulin concentrations where insulin stimulation and hexose uptake were both conducted at each given assay temperature. Basal uptake was only subtly affected. Control and maximally insulin-stimulated cells incubated at 35 degrees C subsequently exhibited minimal temperature-sensitivity of uptake measured between 30 and 43 degrees C. The data are mostly consistent with the concept that insulin-sensitive glucose transporters are, after stimulation by insulin, functionally similar to basal transporters. Adipocyte plasma membranes were labelled with various spin- and fluorescence-label probes in lipid structural studies. The temperature-dependence of the order parameter S calculated from membranes labelled with 5-nitroxide stearate indicated the presence of a lipid phase change at approx. 33 degrees C. Membranes labelled with the fluorescence label 1,6-diphenylhexa-1,3,5-triene, or the cholesterol-like spin label nitroxide cholestane, reveal sharp transitions at lower temperatures. We suggest that a thermotropic lipid phase separation occurs in the adipocyte membrane that may be correlated with the temperature-dependence of hexose transport and insulin action in the intact cells. PMID:6324752

Fab fragment labeled with ICG-derivative for detecting digestive tract cancer.

PubMed

Yano, Hiromi; Muguruma, Naoki; Ito, Susumu; Aoyagi, Eriko; Kimura, Tetsuo; Imoto, Yoshitaka; Cao, Jianxin; Inoue, Shohei; Sano, Shigeki; Nagao, Yoshimitsu; Kido, Hiroshi

2006-09-01

In previous studies, we generated infrared ray fluorescence-labeled monoclonal antibodies and developed an infrared ray fluorescence endoscope capable of detecting the monoclonal antibodies to establish a novel diagnostic technique for gastrointestinal cancer. Although the whole IgG molecule has commonly been used for preparation of labeled antibodies, labeled IgG displays insufficient sensitivity and specificity, probably resulting from non-specific binding of the Fc fragment to target cells or interference between fluorochromes on the identical labeled antibody, which might be caused by molecular structure. In this in vitro study, we characterized an Fc-free fluorescence-labeled Fab fragment, which was expected to yield more specific binding to target cells than the whole IgG molecule. An anti-mucin antibody and ICG-ATT, an ICG derivative, were used as the labeled antibody and labeling compound, respectively. Paraffin sections of excised gastric cancer tissues were subjected to staining. The labeled whole IgG molecule (ICG-ATT-labeled IgG) and the labeled Fab fragment (ICG-ATT-labeled Fab) were prepared according to a previous report, and the fluorescence properties, antibody activities, and features of fluorescence microscope images obtained from paraffin sections were compared. Both ICG-ATT-labeled Fab and ICG-ATT-labeled IgG were excited by a near infrared ray of 766nm, and maximum emission occurred at 804nm. Antibody activities of ICG-ATT-labeled Fab were shown to be similar to those of unlabeled anti-MUC1 antibody. The fluorescence intensity obtained from paraffin sections of excised gastric cancer tissues revealed a tendency to be greater with ICG-ATT-labeled Fab than with ICG-ATT-labeled IgG. The infrared ray fluorescence-labeled Fab fragment was likely to be more specific than the conventionally labeled antibodies. Fragmentation of antibodies is considered to contribute to improved sensitivity and specificity of labeled antibodies for detection of micro gastrointestinal cancers.

Photonic-plasmonic hybrid single-molecule nanosensor measures the effect of fluorescent labels on DNA-protein dynamics

PubMed Central

Liang, Feng; Guo, Yuzheng; Hou, Shaocong; Quan, Qimin

2017-01-01

Current methods to study molecular interactions require labeling the subject molecules with fluorescent reporters. However, the effect of the fluorescent reporters on molecular dynamics has not been quantified because of a lack of alternative methods. We develop a hybrid photonic-plasmonic antenna-in-a-nanocavity single-molecule biosensor to study DNA-protein dynamics without using fluorescent labels. Our results indicate that the fluorescein and fluorescent protein labels decrease the interaction between a single DNA and a protein due to weakened electrostatic interaction. Although the study is performed on the DNA-XPA system, the conclusion has a general implication that the traditional fluorescent labeling methods might be misestimating the molecular interactions. PMID:28560341

Rapid protein concentration, efficient fluorescence labeling and purification on a micro/nanofluidics chip.

PubMed

Wang, Chen; Ouyang, Jun; Ye, De-Kai; Xu, Jing-Juan; Chen, Hong-Yuan; Xia, Xing-Hua

2012-08-07

Fluorescence analysis has proved to be a powerful detection technique for achieving single molecule analysis. However, it usually requires the labeling of targets with bright fluorescent tags since most chemicals and biomolecules lack fluorescence. Conventional fluorescence labeling methods require a considerable quantity of biomolecule samples, long reaction times and extensive chromatographic purification procedures. Herein, a micro/nanofluidics device integrating a nanochannel in a microfluidics chip has been designed and fabricated, which achieves rapid protein concentration, fluorescence labeling, and efficient purification of product in a miniaturized and continuous manner. As a demonstration, labeling of the proteins bovine serum albumin (BSA) and IgG with fluorescein isothiocyanate (FITC) is presented. Compared to conventional methods, the present micro/nanofluidics device performs about 10(4)-10(6) times faster BSA labeling with 1.6 times higher yields due to the efficient nanoconfinement effect, improved mass, and heat transfer in the chip device. The results demonstrate that the present micro/nanofluidics device promises rapid and facile fluorescence labeling of small amount of reagents such as proteins, nucleic acids and other biomolecules with high efficiency.

Penetration of short fluorescence-labeled peptides into the nucleus in HeLa cells and in vitro specific interaction of the peptides with deoxyribooligonucleotides and DNA.

PubMed

Fedoreyeva, L I; Kireev, I I; Khavinson, V Kh; Vanyushin, B F

2011-11-01

Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.

Bimolecular fluorescence complementation: visualization of molecular interactions in living cells.

PubMed

Kerppola, Tom K

2008-01-01

A variety of experimental methods have been developed for the analysis of protein interactions. The majority of these methods either require disruption of the cells to detect molecular interactions or rely on indirect detection of the protein interaction. The bimolecular fluorescence complementation (BiFC) assay provides a direct approach for the visualization of molecular interactions in living cells and organisms. The BiFC approach is based on the facilitated association between two fragments of a fluorescent protein when the fragments are brought together by an interaction between proteins fused to the fragments. The BiFC approach has been used for visualization of interactions among a variety of structurally diverse interaction partners in many different cell types. It enables detection of transient complexes as well as complexes formed by a subpopulation of the interaction partners. It is essential to include negative controls in each experiment in which the interface between the interaction partners has been mutated or deleted. The BiFC assay has been adapted for simultaneous visualization of multiple protein complexes in the same cell and the competition for shared interaction partners. A ubiquitin-mediated fluorescence complementation assay has also been developed for visualization of the covalent modification of proteins by ubiquitin family peptides. These fluorescence complementation assays have a great potential to illuminate a variety of biological interactions in the future.

ReAsH/FlAsH labeling and image analysis of tetracysteine sensor proteins in cells.

PubMed

Irtegun, Sevgi; Ramdzan, Yasmin M; Mulhern, Terrence D; Hatters, Danny M

2011-08-31

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest, recent developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association. One small tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH (green fluorescent), with high specificity even in live cells. The TC/biarsenical dye system offers far less steric constraints to the host protein than fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions. We recently developed a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington disease. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

Enhanced photocatalytic activity and characterization of magnetic Ag/BiOI/ZnFe2O4 composites for Hg0 removal under fluorescent light irradiation

NASA Astrophysics Data System (ADS)

Li, Chengwei; Zhang, Anchao; Zhang, Lixiang; Song, Jun; Su, Sheng; Sun, Zhijun; Xiang, Jun

2018-03-01

A series of magnetic Ag/BiOI/ZnFe2O4 hybrids synthesized via hydrothermal process, subsequent deposition-precipitation and photoreduction method were employed to remove elemental mercury (Hg0) under fluorescent light irradiation. The effects of Ag content, fluorescent light irradiation, reaction temperature, pH value, flue gas composition, anions and photocatalyst dosage on Hg0 removal were investigated in detail. The as-synthesized photocatalysts were characterized using N2 adsorption-desorption, XRD, SEM, TEM, HRTEM, XPS, VSM, DRS, ESR, PL and photocurrent response. The results showed that the ternary Ag/BiOI/ZnFe2O4 hybrids possessed enhanced visible-light-responsive photocatalytic performances for Hg0 removal. Ag/BiOI/ZnFe2O4 photocatalyst could be easily recovered from the reaction solution by an extra magnet and was stable in the process of Hg0 removal. Lower content of Ag was highly dispersed on the surface of BiOI/ZnFe2O4, while higher content of Ag would result in some aggregations and/or the blockages of micropore. In comparison to BiOI/ZnFe2O4, Ag deposited BiOI/ZnFe2O4 material showed lower recombination rate of electron-hole pairs. The superior Hg0 oxidation removal could correspond to good match of BiOI and ZnFe2O4, excellent fluidity and surface plasmon resonance effect of Ag0 nanoparticles, which led to higher separation efficiency of photogenerated electrons and holes, thereby enhancing the hybrids' photocatalytic activity.

Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based β-lactam probes.

PubMed

Sadhu, Kalyan K; Mizukami, Shin; Watanabe, Shuji; Kikuchi, Kazuya

2011-05-01

Development of protein labeling techniques with small molecules is enthralling because this method brings promises for triumph over the limitations of fluorescent proteins in live cell imaging. This technology deals with the functionalization of proteins with small molecules and is anticipated to facilitate the expansion of various protein assay methods. A new straightforward aggregation and elimination-based technique for a protein labeling system has been developed with a versatile emissive range of fluorophores. These fluorophores have been applied to show their efficiency for protein labeling by exploiting the same basic principle. A genetically modified version of class A type β-lactamase has been used as the tag protein (BL-tag). The strength of the aggregation interaction between a fluorophore and a quencher plays a governing role in the elimination step of the quencher from the probes, which ultimately controls the swiftness of the protein labeling strategy. Modulation in the elimination process can be accomplished by the variation in the nature of the fluorophore. This diversity facilitates the study of the competitive binding order among the synthesized probes toward the BL-tag labeling method. An aggregation and elimination-based BL-tag technique has been explored to develop an order of color labeling from the equimolar mixture of the labeling probe in solutions. The qualitative and quantitative determination of ordering within the probes toward labeling studies has been executed through SDS-PAGE and time-dependent fluorescence intensity enhancement measurements, respectively. The desirable multiple-wavelength fluorescence labeling probes for the BL-tag technology have been developed and demonstrate broad applicability of this labeling technology to live cell imaging with coumarin and fluorescein derivatives by using confocal microscopy.

DNA-stabilized silver nanoclusters and carbon nanoparticles oxide: A sensitive platform for label-free fluorescence turn-on detection of HIV-DNA sequences.

PubMed

Ye, Yu-Dan; Xia, Li; Xu, Dang-Dang; Xing, Xiao-Jing; Pang, Dai-Wen; Tang, Hong-Wu

2016-11-15

Based on the remarkable difference between the interactions of carbon nanoparticles (CNPs) oxide with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and the fact that fluorescence of DNA-stabilized silver nanoclusters (AgNCs) can be quenched by CNPs oxide, DNA-functionalized AgNCs were applied as label-free fluorescence probes and a novel fluorescence resonance energy transfer (FRET) sensor was successfully constructed for the detection of human immunodeficiency virus (HIV) DNA sequences. CNPs oxide were prepared with the oxidation of candle soot, hence it is simple, time-saving and low-cost. The strategy of dual AgNCs probes was applied to improve the detection sensitivity by using dual- probe capturing the same target DNA in a sandwich mode and as the fluorescence donor, and using CNPs oxide as the acceptor. In the presence of target DNA, a dsDNA hybrid forms, leading to the desorption of the ssDNA-AgNCs probes from CNPs oxide, and the recovering of fluorescence of the AgNCs in a HIV-DNA concentration-dependent manner. The results show that HIV-DNA can be detected in the range of 1-50nM with a detection limit of 0.40nM in aqueous buffer. The method is simple, rapid and sensitive with no need of labeled fluorescent probes, and moreover, the design of fluorescent dual-probe makes full use of the excellent fluorescence property of AgNCs and further improves the detection sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

Improved facial nerve identification during parotidectomy with fluorescently labeled peptide.

PubMed

Hussain, Timon; Nguyen, Linda T; Whitney, Michael; Hasselmann, Jonathan; Nguyen, Quyen T

2016-12-01

Additional intraoperative guidance could reduce the risk of iatrogenic injury during parotid gland cancer surgery. We evaluated the intraoperative use of fluorescently labeled nerve binding peptide NP41 to aid facial nerve identification and preservation during parotidectomy in an orthotopic model of murine parotid gland cancer. We also quantified the accuracy of intraoperative nerve detection for surface and buried nerves in the head and neck with NP41 versus white light (WL) alone. Twenty-eight mice underwent parotid gland cancer surgeries with additional fluorescence (FL) guidance versus WL reflectance (WLR) alone. Eight mice were used for additional nerve-imaging experiments. Twenty-eight parotid tumor-bearing mice underwent parotidectomy. Eight mice underwent imaging of both sides of the face after skin removal. Postoperative assessment of facial nerve function measured by automated whisker tracking were compared between FL guidance (n = 13) versus WL alone (n=15). In eight mice, nerve to surrounding tissue contrast was measured under FL versus WLR for all nerve branches detectable in the field of view. Postoperative facial nerve function after parotid gland cancer surgery tended to be better with additional FL guidance. Fluorescent labeling significantly improved nerve to surrounding tissue contrast for both large and smaller buried nerve branches compared to WLR visualization and improved detection sensitivity and specificity. NP41 FL imaging significantly aids the intraoperative identification of nerve braches otherwise nearly invisible to the naked eye. Its application in a murine model of parotid gland cancer surgery tended to improve functional preservation of the facial nerve. NA Laryngoscope, 126:2711-2717, 2016. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

Improved Facial Nerve Identification During Parotidectomy With Fluorescently Labeled Peptide

PubMed Central

Hussain, Timon; Nguyen, Linda T.; Whitney, Michael; Hasselmann, Jonathan; Nguyen, Quyen T.

2016-01-01

Objectives/Hypothesis Additional intraoperative guidance could reduce the risk of iatrogenic injury during parotid gland cancer surgery. We evaluated the intraoperative use of fluorescently labeled nerve binding peptide NP41 to aid facial nerve identification and preservation during parotidectomy in an orthotopic model of murine parotid gland cancer. We also quantified the accuracy of intraoperative nerve detection for surface and buried nerves in the head and neck with NP41 versus white light (WL) alone. Study Design Twenty-eight mice underwent parotid gland cancer surgeries with additional fluorescence (FL) guidance versus WL reflectance (WLR) alone. Eight mice were used for additional nerve-imaging experiments. Methods Twenty-eight parotid tumor-bearing mice underwent parotidectomy. Eight mice underwent imaging of both sides of the face after skin removal. Postoperative assessment of facial nerve function measured by automated whisker tracking were compared between FL guidance (n = 13) versus WL alone (n = 15). In eight mice, nerve to surrounding tissue contrast was measured under FL versus WLR for all nerve branches detectable in the field of view. Results Postoperative facial nerve function after parotid gland cancer surgery tended to be better with additional FL guidance. Fluorescent labeling significantly improved nerve to surrounding tissue contrast for both large and smaller buried nerve branches compared to WLR visualization and improved detection sensitivity and specificity. Conclusions NP41 FL imaging significantly aids the intraoperative identification of nerve braches otherwise nearly invisible to the naked eye. Its application in a murine model of parotid gland cancer surgery tended to improve functional preservation of the facial nerve. PMID:27171862

A novel model for development, organization, and function of gonadotropes in fish pituitary.

PubMed

Golan, Matan; Biran, Jakob; Levavi-Sivan, Berta

2014-01-01

The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are key regulators of the reproductive axis in vertebrates. Despite the high popularity of zebrafish as a model organism for studying reproductive functions, to date no transgenic zebrafish with labeled gonadotropes have been introduced. Using gonadotropin regulatory elements from tilapia, we generated two transgenic zebrafish lines with labeled gonadotropes. The tilapia and zebrafish regulatory sequences were highly divergent but several conserved elements allowed the tilapia promoters to correctly drive the transgenes in zebrafish pituitaries. FSH cells reacted to stimulation with gonadotropin releasing hormone by proliferating and showing increased transgene fluorescence, whereas estrogen exposure caused a decrease in cell number and transgene fluorescence. Transgene fluorescence reflected the expression pattern of the endogenous fshb gene. Ontogenetic expression of the transgenes followed typical patterns, with FSH cells appearing early in development, and LH cells appearing later and increasing dramatically in number with the onset of puberty. Our transgenic lines provide a powerful tool for investigating the development, anatomy, and function of the reproductive axis in lower vertebrates.

Superparamagnetic iron oxide nanoparticle-labeled cells as an effective vehicle for tracking the GFP gene marker using magnetic resonance imaging

PubMed Central

Zhang, Z; Mascheri, N; Dharmakumar, R; Fan, Z; Paunesku, T; Woloschak, G; Li, D

2010-01-01

Background Detection of a gene using magnetic resonance imaging (MRI) is hindered by the magnetic resonance (MR) targeting gene technique. Therefore it may be advantageous to image gene-expressing cells labeled with superparamagnetic iron oxide (SPIO) nanoparticles by MRI. Methods The GFP-R3230Ac (GFP) cell line was incubated for 24 h using SPIO nanoparticles at a concentration of 20 μg Fe/mL. Cell samples were prepared for iron content analysis and cell function evaluation. The labeled cells were imaged using fluorescent microscopy and MRI. Results SPIO was used to label GFP cells effectively, with no effects on cell function and GFP expression. Iron-loaded GFP cells were successfully imaged with both fluorescent microscopy and T2*-weighted MRI. Prussian blue staining showed intracellular iron accumulation in the cells. All cells were labeled (100% labeling efficiency). The average iron content per cell was 4.75±0.11 pg Fe/cell (P<0.05 versus control). Discussion This study demonstrates that the GFP expression of cells is not altered by the SPIO labeling process. SPIO-labeled GFP cells can be visualized by MRI; therefore, GFP, a gene marker, was tracked indirectly with the SPIO-loaded cells using MRI. The technique holds promise for monitoring the temporal and spatial migration of cells with a gene marker and enhancing the understanding of cell- and gene-based therapeutic strategies. PMID:18956269

Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging

PubMed Central

Xiong, Hanqing; Zhou, Zhenqiao; Zhu, Mingqiang; Lv, Xiaohua; Li, Anan; Li, Shiwei; Li, Longhui; Yang, Tao; Wang, Siming; Yang, Zhongqin; Xu, Tonghui; Luo, Qingming; Gong, Hui; Zeng, Shaoqun

2014-01-01

Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding. PMID:24886825

Synthesis and characterization of the fluorescent probes for the labeling of Microthrix parvicella.

PubMed

Li, Songya; Fei, Xuening; Jiao, Xiumei; Lin, Dayong; Zhang, Baolian; Cao, Lingyun

2016-03-01

Although the fluorescent in situ hybridization (FISH) has been widely used to identify the Microthrix parvicella (M. parvicella), there are a few disadvantages and difficulties, such as complicated process, time consuming, etc. In this work, a series of fluorescent probes, which were modified by long-chain alkane with hydrophobic property and based on the property of M. parvicella utilizing long-chain fatty acids (LCFA), for the labeling of M. parvicella in bulking sludge were designed, synthesized, and characterized. The probes were characterized by ultraviolet-visible (UV-Vis) absorption spectra, fluorescence spectra, (1)H NMR spectra, and mass spectra, and the photostability and hydrophobic property of probes were investigated. All the results showed that the probes were quite stable and suitable for the fluorescent labeling. The probes had a large stoke shift of 98-137 nm, which was benefit for the fluorescent labeling. In the fluorescent labeling of M. parvicella by the synthesized probes, the probes had excellent labeling effects. By comparison of the images and the Image Pro Plus 6.0 analysis, the optimal concentration of the probes in the activated sludge sample for labeling was 0.010 mmol/L and the probe 3d had the best labeling. In addition, the effect of the duration time of probes was also investigated, and the results showed that the fluorescent intensity of probes hardly changed in a long period of time and it was suitable for labeling.

The border-to-border distribution method for analysis of cytoplasmic particles and organelles.

PubMed

Yacovone, Shalane K; Ornelles, David A; Lyles, Douglas S

2016-02-01

Comparing the distribution of cytoplasmic particles and organelles between different experimental conditions can be challenging due to the heterogeneous nature of cell morphologies. The border-to-border distribution method was created to enable the quantitative analysis of fluorescently labeled cytoplasmic particles and organelles of multiple cells from images obtained by confocal microscopy. The method consists of four steps: (1) imaging of fluorescently labeled cells, (2) division of the image of the cytoplasm into radial segments, (3) selection of segments of interest, and (4) population analysis of fluorescence intensities at the pixel level either as a function of distance along the selected radial segments or as a function of angle around an annulus. The method was validated using the well-characterized effect of brefeldin A (BFA) on the distribution of the vesicular stomatitis virus G protein, in which intensely labeled Golgi membranes are redistributed within the cytoplasm. Surprisingly, in untreated cells, the distribution of fluorescence in Golgi membrane-containing radial segments was similar to the distribution of fluorescence in other G protein-containing segments, indicating that the presence of Golgi membranes did not shift the distribution of G protein towards the nucleus compared to the distribution of G protein in other regions of the cell. Treatment with BFA caused only a slight shift in the distribution of the brightest G protein-containing segments which had a distribution similar to that in untreated cells. Instead, the major effect of BFA was to alter the annular distribution of G protein in the perinuclear region.

Hessian-based quantitative image analysis of host-pathogen confrontation assays.

PubMed

Cseresnyes, Zoltan; Kraibooj, Kaswara; Figge, Marc Thilo

2018-03-01

Host-fungus interactions have gained a lot of interest in the past few decades, mainly due to an increasing number of fungal infections that are often associated with a high mortality rate in the absence of effective therapies. These interactions can be studied at the genetic level or at the functional level via imaging. Here, we introduce a new image processing method that quantifies the interaction between host cells and fungal invaders, for example, alveolar macrophages and the conidia of Aspergillus fumigatus. The new technique relies on the information content of transmitted light bright field microscopy images, utilizing the Hessian matrix eigenvalues to distinguish between unstained macrophages and the background, as well as between macrophages and fungal conidia. The performance of the new algorithm was measured by comparing the results of our method with that of an alternative approach that was based on fluorescence images from the same dataset. The comparison shows that the new algorithm performs very similarly to the fluorescence-based version. Consequently, the new algorithm is able to segment and characterize unlabeled cells, thus reducing the time and expense that would be spent on the fluorescent labeling in preparation for phagocytosis assays. By extending the proposed method to the label-free segmentation of fungal conidia, we will be able to reduce the need for fluorescence-based imaging even further. Our approach should thus help to minimize the possible side effects of fluorescence labeling on biological functions. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Fluorescent labeling of tetracysteine-tagged proteins in intact cells.

PubMed

Hoffmann, Carsten; Gaietta, Guido; Zürn, Alexander; Adams, Stephen R; Terrillon, Sonia; Ellisman, Mark H; Tsien, Roger Y; Lohse, Martin J

2010-09-01

In this paper, we provide a general protocol for labeling proteins with the membrane-permeant fluorogenic biarsenical dye fluorescein arsenical hairpin binder-ethanedithiol (FlAsH-EDT₂). Generation of the tetracysteine-tagged protein construct by itself is not described, as this is a protein-specific process. This method allows site-selective labeling of proteins in living cells and has been applied to a wide variety of proteins and biological problems. We provide here a generally applicable labeling procedure and discuss the problems that can occur as well as general considerations that must be taken into account when designing and implementing the procedure. The method can even be applied to proteins with expression below 1 pmol mg⁻¹ of protein, such as G protein-coupled receptors, and it can be used to study the intracellular localization of proteins as well as functional interactions in fluorescence resonance energy transfer experiments. The labeling procedure using FlAsH-EDT₂ as described takes 2-3 h, depending on the number of samples to be processed.

A combination of SILAC and nucleotide acyl phosphate labelling reveals unexpected targets of the Rsk inhibitor BI-D1870.

PubMed

Edgar, Alexander J; Trost, Matthias; Watts, Colin; Zaru, Rossana

2014-02-01

Protein kinase inhibitors frequently have interesting effects that cannot be fully ascribed to the intended target kinase(s) but identifying additional targets that might explain the effects is not straightforward. By comparing two different inhibitors of the Rsk (p90 ribosomal S6 kinase) kinases, we found that the increasingly used compound BI-D1870 had biological effects in murine DCs (dendritic cells) that could not be solely ascribed to Rsk or other documented targets. We assessed the ability of BI-D1870 and a second Rsk inhibitor, BIX 02565 to protect enzyme active sites from reaction with biotinylated nucleotide acyl phosphates. Using SILAC (stable isotope labelling by amino acids in cell culture)-labelled DC lysates as a source of enzyme targets, we identify several kinases that interact with BI-D1870 but not with BIX 02565. We confirmed that these kinases, including Slk, Lok and Mst1, are inhibited by BI-D1870 but to a much lesser extent by BIX 02565 and that phosphorylation of some of their substrates is blocked by BI-D1870 in living cells. Our results suggest that the BI-D1870 inhibitor should be used with caution. The SILAC-based methodology we used should be useful for further comparative unbiased profiling of the target spectrum of kinase inhibitors with interesting biological effects under conditions that closely mimic those found in cells. © 2014 The author(s).

Determining Protease Activity In Vivo by Fluorescence Cross-Correlation Analysis

PubMed Central

Kohl, Tobias; Haustein, Elke; Schwille, Petra

2005-01-01

To date, most biochemical approaches to unravel protein function have focused on purified proteins in vitro. Whereas they analyze enzyme performance under assay conditions, they do not necessarily tell us what is relevant within a living cell. Ideally, cellular functions should be examined in situ. In particular, association/dissociation reactions are ubiquitous, but so far there is no standard technique permitting online analysis of these processes in vivo. Featuring single-molecule sensitivity combined with intrinsic averaging, fluorescence correlation spectroscopy is a minimally invasive technique ideally suited to monitor proteins. Moreover, endogenous fluorescence-based assays can be established by genetically encoding fusions of autofluorescent proteins and cellular proteins, thus avoiding the disadvantages of in vitro protein labeling and subsequent delivery to cells. Here, we present an in vivo protease assay as a model system: Green and red autofluorescent proteins were connected by Caspase-3- sensitive and insensitive protein linkers to create double-labeled protease substrates. Then, dual-color fluorescence cross-correlation spectroscopy was employed to study the protease reaction in situ. Allowing assessment of multiple dynamic parameters simultaneously, this method provided internal calibration and improved experimental resolution for quantifying protein stability. This approach, which is easily extended to reversible protein-protein interactions, seems very promising for elucidating intracellular protein functions. PMID:16055538

Bioimaging of Fluorescence-Labeled Mitochondria in Subcutaneously Grafted Murine Melanoma Cells by the “In Vivo Cryotechnique”

PubMed Central

Lei, Ting; Huang, Zheng; Ohno, Nobuhiko; Wu, Bao; Sakoh, Takashi; Saitoh, Yurika; Saiki, Ikuo

2014-01-01

The microenvironments of organs with blood flow affect the metabolic profiles of cancer cells, which are influenced by mitochondrial functions. However, histopathological analyses of these aspects have been hampered by technical artifacts of conventional fixation and dehydration, including ischemia/anoxia. The purpose of this study was to combine the in vivo cryotechnique (IVCT) with fluorescent protein expression, and examine fluorescently labeled mitochondria in grafted melanoma tumors. The intensity of fluorescent proteins was maintained well in cultured B16-BL6 cells after cryotechniques followed by freeze-substitution (FS). In the subcutaneous tumors of mitochondria-targeted DsRed2 (mitoDsRed)-expressing cells, a higher number of cancer cells were found surrounding the widely opened blood vessels that contained numerous erythrocytes. Such blood vessels were immunostained positively for immunoglobulin M and ensheathed by basement membranes. MitoDsRed fluorescence was detected in scattering melanoma cells using the IVCT-FS method, and the total mitoDsRed volume in individual cancer cells was significantly decreased with the expression of markers of hypoxia. MitoDsRed was frequently distributed throughout the cytoplasm and in processes extending along basement membranes. IVCT combined with fluorescent protein expression is a useful tool to examine the behavior of fluorescently labeled cells and organelles. We propose that the mitochondrial volume is dynamically regulated in the hypoxic microenvironment and that mitochondrial distribution is modulated by cancer cell interactions with basement membranes. PMID:24394469

Detection of Thrombin Based on Fluorescence Energy Transfer between Semiconducting Polymer Dots and BHQ-Labelled Aptamers.

PubMed

Liu, Yizhang; Jiang, Xuekai; Cao, Wenfeng; Sun, Junyong; Gao, Feng

2018-02-14

Carboxyl-functionalized semiconducting polymer dots (Pdots) were synthesized as an energy donor by the nanoprecipitation method. A black hole quenching dye (BHQ-labelled thrombin aptamers) was used as the energy acceptor, and fluorescence resonance energy transfer between the aptamers and Pdots was used for fluorescence quenching of the Pdots. The addition of thrombin restored the fluorescence intensity. Under the optimized experimental conditions, the fluorescence of the system was restored to the maximum when the concentration of thrombin reached 130 nM, with a linear range of 0-50 nM (R² = 0.990) and a detection limit of 0.33 nM. This sensor was less disturbed by impurities, showing good specificity and signal response to thrombin, with good application in actual samples. The detection of human serum showed good linearity in the range of 0-30 nM (R² = 0.997), with a detection limit of 0.56 nM and a recovery rate of 96.2-104.1%, indicating that this fluorescence sensor can be used for the detection of thrombin content in human serum.

Direct fluorescent labeling for efficient biological assessment of inhalable particles.

PubMed

Poudel, Bijay Kumar; Park, Jae Hong; Lim, Jiseok; Byeon, Jeong Hoon

2017-10-01

Labeling of aerosol particles with a radioactive, magnetic, or optical tracer has been employed to confirm particle localization in cell compartments, which has provided useful evidence for correlating toxic effects of inhaled particles. However, labeling requires several physicochemical steps to identify functionalities of the inner or outer surfaces of particles, and moreover, these steps can cause changes in size, surface charge, and bioactivity of the particles, resulting in misinterpretations regarding their toxic effects. This study addresses this challenging issue with a goal of introducing an efficient strategy for constantly supplying labeled aerosol particles in a single-pass configuration without any pre- or post-physicochemical batch treatments of aerosol particles. Carbon black (CB, simulating combustion-generated soot) or calcium carbonate (CC, simulating brake-wear fragments) particles were constantly produced via spark ablation or bubble bursting, respectively. These minute particles were incorporated with fluorescein isothiocyanate-poly(ethylene glycol) 2-aminoethyl ether acetic acid solution at the orifice of a collison atomizer to fabricate hybrid droplets. The droplets successively entered a diffusion dryer containing 254-nm UV irradiation; therefore, the droplets were dynamically stiffened by UV to form fluorescent probes on particles during solvent extraction in the dryer. Particle size distributions, morphologies, and surface charges before and after labeling were measured to confirm fluorescence labeling without significant changes in the properties. In vitro assays, including confocal imaging, were conducted to confirm the feasibility of the labeling approach without inducing significant differences in bioactivity compared with untreated CB or CC particles.

Versatile hydrogel-based nanocrystal microreactors towards uniform fluorescent photonic crystal supraballs

NASA Astrophysics Data System (ADS)

Zhang, Jing; Tian, Yu; Ling, Lu-Ting; Yin, Su-Na; Wang, Cai-Feng; Chen, Su

2014-12-01

Versatile hydrogel-based nanocrystal (NC) microreactors were designed in this work for the construction of uniform fluorescence colloidal photonic crystal (CPC) supraballs. The hydrogel-based microspheres with sizes ranging from 150 to 300 nm were prepared by seeded copolymerization of acrylic acid and 2-hydroxyethyl methacrylate with micrometer-sized PS seed particles. As an independent NC microreactor, the as-synthesized hydrogel microsphere can effectively capture the guest cadmium ions due to the abundant carboxyl groups inside. Followed by the introduction of chalcogenides, in situ generation of higher-uptake NCs with sizes less than 5 nm was finally realized. Additionally, with the aid of the microfluidic device, the as-obtained NC-latex hybrids can be further self-assembled to bi-functional CPC supraballs bearing brilliant structural colors and uniform fluorescence. This research offers an alternative way to finely bind CPCs with NCs, which will facilitate progress in fields of self-assembled functional colloids and photonic materials.

Long term imaging of living brain cancer cells

NASA Astrophysics Data System (ADS)

Farias, Patricia M. A.; Galembeck, André; Milani, Raquel; Andrade, Arnaldo C. D. S.; Stingl, Andreas

2018-02-01

QDs synthesized in aqueous medium and functionalized with polyethylene glycol were used as fluorescent probes. They label and monitor living healthy and cancer brain glial cells in culture. Physical-chemical characterization was performed. Toxicological studies were performed by in vivo short and long-term inhalation in animal models. Healthy and cancer glial living cells were incubated in culture media with highly controlled QDs. Specific features of glial cancer cells were enhanced by QD labelling. Cytoplasmic labelling pattern was clearly distinct for healthy and cancer cells. Labelled cells kept their normal activity for same period as non-labelled control samples.

Live Imaging of Endogenous PSD-95 Using ENABLED: A Conditional Strategy to Fluorescently Label Endogenous Proteins

PubMed Central

Fortin, Dale A.; Tillo, Shane E.; Yang, Guang; Rah, Jong-Cheol; Melander, Joshua B.; Bai, Suxia; Soler-Cedeño, Omar; Qin, Maozhen; Zemelman, Boris V.; Guo, Caiying

2014-01-01

Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo. PMID:25505322

Bi-enzyme functionlized hollow PtCo nanochains as labels for an electrochemical aptasensor.

PubMed

Bai, Lijuan; Yuan, Ruo; Chai, Yaqin; Yuan, Yali; Zhuo, Ying; Mao, Li

2011-07-15

In this work, a new signal amplification strategy based on hollow PtCo nanochains (HPtCoNCs) functionalized by bi-enzyme-horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme) and glucose oxidase (GOD), as well as ferrocene-labeled secondary thrombin aptamer (Fc-TBA 2), is developed to construct a highly sensitive electrochemical aptasensor. The HRP-DNAzyme contains a special G-quadruplex structure with an intercalated hemin. With the surface area enlarged by HPtCoNCs, the amount of immobilized Fc-TBA 2, hemin and GOD can be enhanced. Under the enzyme catalysis of GOD, d-glucose is rapidly oxidized into gluconic acid accompanying with the generation of H₂O₂, which is further electrocatalyzed by Pt nanoparticles and HPR-DNAzyme to improve the electrochemical signal of Fc. With several amplification factors mentioned above, a wide linear ranged from 0.001 to 30 nM is acquired with a relatively low detection limit of 0.39 pM for thrombin. The present work demonstrates that using HPtCoNCs as labels is a promising way to amplify the analysis signal and improve the sensitivity of aptasensors. Copyright © 2011 Elsevier B.V. All rights reserved.

Novel infectivity-enhanced oncolytic adenovirus with a capsid-incorporated dual-imaging moiety for monitoring virotherapy in ovarian cancer.

PubMed

Kimball, Kristopher J; Rivera, Angel A; Zinn, Kurt R; Icyuz, Mert; Saini, Vaibhav; Li, Jing; Zhu, Zeng B; Siegal, Gene P; Douglas, Joanne T; Curiel, David T; Alvarez, Ronald D; Borovjagin, Anton V

2009-01-01

We sought to develop a cancer-targeted, infectivity-enhanced oncolytic adenovirus that embodies a capsid-labeling fusion for noninvasive dual-modality imaging of ovarian cancer virotherapy. A functional fusion protein composed of fluorescent and nuclear imaging tags was genetically incorporated into the capsid of an infectivity-enhanced conditionally replicative adenovirus. Incorporation of herpes simplex virus thymidine kinase (HSV-tk) and monomeric red fluorescent protein 1 (mRFP1) into the viral capsid and its genomic stability were verified by molecular analyses. Replication and oncolysis were evaluated in ovarian cancer cells. Fusion functionality was confirmed by in vitro gamma camera and fluorescent microscopy imaging. Comparison of tk-mRFP virus to single-modality controls revealed similar replication efficiency and oncolytic potency. Molecular fusion did not abolish enzymatic activity of HSV-tk as the virus effectively phosphorylated thymidine both ex vivo and in vitro. In vitro fluorescence imaging demonstrated a strong correlation between the intensity of fluorescent signal and cytopathic effect in infected ovarian cancer cells, suggesting that fluorescence can be used to monitor viral replication. We have in vitro validated a new infectivity-enhanced oncolytic adenovirus with a dual-imaging modality-labeled capsid, optimized for ovarian cancer virotherapy. The new agent could provide incremental gains toward climbing the barriers for achieving conditionally replicated adenovirus efficacy in human trials.

Effect of silver nanoparticles on the fluorescence of Pb2+ and compositional dependence of Sm3+ fluorescence in borate glasses

NASA Astrophysics Data System (ADS)

Olumoroti, Akinloluwa T.

Borate glasses have been widely studied due to their good optical and mechanical properties. Lead and bismuth (PbO/Bi2O 3:B2O3) borate glasses belong to a family of heavy metal oxide (HMO) glasses which are well known to be chemically durable, stable against atmospheric moisture, have low melting temperatures and good corrosion resistance. The first part of this work deals with lead borate glasses with silver nanoparticles (NPs) introduced into the glass matrix. Transmission electron microscopy characterization is done to verify the nucleation of NPs. Fluorescence and optical absorption experiments are then carried out after different heat treatment duration to investigate the influence of silver NPs on the optical properties of lead (Pb2+) by comparing with a glass sample without silver NPs. Optical absorption experiments show that a well-defined surface plasmon resonance (SPR) peak due to Ag NPs can be observed only for samples that were annealed for 36 hrs. Pb2+ fluorescence spectra reveal that the presence of silver NPs creates new emission centers for Pb2+ ions by altering their chemical environment. The second part of the work involves the use of samarium (a rare earth ion) as a dopant in lead and bismuth borate glasses. The concentration of samarium (Sm3+) is fixed and the base glass composition is varied. The goal is to investigate the compositional dependence of optical properties of samarium in the base glass (PbO/Bi2O3:B 2O3). Optical absorption spectra have been collected and the oscillator strength of each transition - including the hypersensitive - is obtained. The Optical absorption edge is found to shift toward lower energies with increasing PbO/Bi2O3 concentration. Both the oscillator strength and the peak position of the hypersensitive transition show significant variation with glass composition. Strong interaction between Sm3+ ions and Pb2+/Bi3+ ions can also be seen from the variations in the fluorescence emission properties of Sm3+ as a function of base glass composition. Studying the variation of these optical properties will help to create the optimum rare-earth ion-host configuration for possible technological applications. This is the thrust of our future investigations of these glass systems. Keywords: Borate glasses, nanoparticles, fluorescence, transmission electron microscopy, optical absorption, surface plasmon resonance, rare-earth (RE) ions, oscillator strength, hypersensitive transition (HST).

Improved Tumor Targeting and Longer Retention Time of NIR Fluorescent Probes Using Bioorthogonal Chemistry.

PubMed

Zhang, Xianghan; Wang, Bo; Zhao, Na; Tian, Zuhong; Dai, Yunpeng; Nie, Yongzhan; Tian, Jie; Wang, Zhongliang; Chen, Xiaoyuan

2017-01-01

The traditional labeling method for targeted NIR fluorescence probes requires directly covalent-bonded conjugation of targeting domains and fluorophores in vitro . Although this strategy works well, it is not sufficient for detecting or treating cancers in vivo , due to steric hindrance effects that relatively large fluorophore molecules exert on the configurations and physiological functions of specific targeting domains. The copper-free, "click-chemistry"-assisted assembly of small molecules in living systems may enhance tumor accumulation of fluorescence probes by improving the binding affinities of the targeting factors. Here, we employed a vascular homing peptide, GEBP11, as a targeting factor for gastric tumors, and we demonstrate its effectiveness for in vivo imaging via click-chemistry-mediated conjugation with fluorescence molecules in tumor xenograft mouse models. This strategy showed higher binding affinities than those of the traditional conjugation method, and our results showed that the tumor accumulation of click-chemistry-mediated probes are 11-fold higher than that of directly labeled probes. The tracking life was prolonged by 12-fold, and uptake of the probes into the kidney was reduced by 6.5-fold. For lesion tumors of different sizes, click-chemistry-mediated probes can achieve sufficient signal-to-background ratios (3.5-5) for in vivo detection, and with diagnostic sensitivity approximately 3.5 times that of traditional labeling probes. The click-chemistry-assisted detection strategy utilizes the advantages of "small molecule" probes while not perturbing their physiological functions; this enables tumor detection with high sensitivity and specific selectivity.

Non-linear optical measurements using a scanned, Bessel beam

NASA Astrophysics Data System (ADS)

Collier, Bradley B.; Awasthi, Samir; Lieu, Deborah K.; Chan, James W.

2015-03-01

Oftentimes cells are removed from the body for disease diagnosis or cellular research. This typically requires fluorescent labeling followed by sorting with a flow cytometer; however, possible disruption of cellular function or even cell death due to the presence of the label can occur. This may be acceptable for ex vivo applications, but as cells are more frequently moving from the lab to the body, label-free methods of cell sorting are needed to eliminate these issues. This is especially true of the growing field of stem cell research where specialized cells are needed for treatments. Because differentiation processes are not completely efficient, cells must be sorted to eliminate any unwanted cells (i.e. un-differentiated or differentiated into an unwanted cell type). In order to perform label-free measurements, non-linear optics (NLO) have been increasingly utilized for single cell analysis because of their ability to not disrupt cellular function. An optical system was developed for the measurement of NLO in a microfluidic channel similar to a flow cytometer. In order to improve the excitation efficiency of NLO, a scanned Bessel beam was utilized to create a light-sheet across the channel. The system was tested by monitoring twophoton fluorescence from polystyrene microbeads of different sizes. Fluorescence intensity obtained from light-sheet measurements were significantly greater than measurements made using a static Gaussian beam. In addition, the increase in intensity from larger sized beads was more evident for the light-sheet system.

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

PubMed Central

Irtegun, Sevgi; Ramdzan, Yasmin M.; Mulhern, Terrence D.; Hatters, Danny M.

2011-01-01

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH (green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations). PMID:21897361

Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Dukes, Madeline J; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

On chip preconcentration and fluorescence labeling of model proteins by use of monolithic columns: device fabrication, optimization, and automation.

PubMed

Yang, Rui; Pagaduan, Jayson V; Yu, Ming; Woolley, Adam T

2015-01-01

Microfluidic systems with monolithic columns have been developed for preconcentration and on-chip labeling of model proteins. Monoliths were prepared in microchannels by photopolymerization, and their properties were optimized by varying the composition and concentration of the monomers to improve flow and extraction. On-chip labeling of proteins was achieved by driving solutions through the monolith by use of voltage then incubating fluorescent dye with protein retained on the monolith. Subsequently, the labeled proteins were eluted, by applying voltages to reservoirs on the microdevice, and then detected, by monitoring laser-induced fluorescence. Monoliths prepared from octyl methacrylate combine the best protein retention with the possibility of separate elution of unattached fluorescent label with 50% acetonitrile. Finally, automated on-chip extraction and fluorescence labeling of a model protein were successfully demonstrated. This method involves facile sample pretreatment, and therefore has potential for production of integrated bioanalysis microchips.

Quantum dot coating of baculoviral vectors enables visualization of transduced cells and tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Ying; Lo, Seong Loong; Zheng, Yuangang

2013-04-26

Highlights: •The use of quantum dot (QD)-labeled viral vectors for in vivo imaging is not well investigated. •A new method to label enveloped baculovirus with glutathione-capped CdTe QDs is developed. •The labeling enables the identification of transduced, cultured cells based on fluorescence. •The labeling also allows evaluation of viral transduction in a real-time manner in living mice. •The method has the potential to assess viral vector-based gene therapy protocols in future. -- Abstract: Imaging of transduced cells and tissues is valuable in developing gene transfer vectors and evaluating gene therapy efficacy. We report here a simple method to use brightmore » and photostable quantum dots to label baculovirus, an emerging gene therapy vector. The labeling was achieved through the non-covalent interaction of glutathione-capped CdTe quantum dots with the virus envelope, without the use of chemical conjugation. The quantum dot labeling was nondestructive to viral transduction function and enabled the identification of baculoviral vector-transduced, living cells based on red fluorescence. When the labeled baculoviral vectors were injected intravenously or intraventricularly for in vivo delivery of a transgene into mice, quantum dot fluorescence signals allow us monitor whether or not the injected tissues were transduced. More importantly, using a dual-color whole-body imaging technology, we demonstrated that in vivo viral transduction could be evaluated in a real-time manner in living mice. Thus, our method of labeling a read-to-use gene delivery vector with quantum dots could be useful towards the improvement of vector design and will have the potential to assess baculovirus-based gene therapy protocols in future.« less

Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

PubMed Central

Lindhoud, Simon; Westphal, Adrie H.; van Mierlo, Carlo P. M.; Visser, Antonie J. W. G.; Borst, Jan Willem

2014-01-01

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway. PMID:25535076

Specific in vivo labeling with GFP retroviruses, lentiviruses, and adenoviruses for imaging

NASA Astrophysics Data System (ADS)

Hoffman, Robert M.; Kishimoto, Hiroyuki; Fujiwara, Toshiyoshi

2008-02-01

Fluorescent proteins have revolutionized the field of imaging. Our laboratory pioneered in vivo imaging with fluorescent proteins. Fluorescent proteins have enabled imaging at the subcellular level in mice. We review here the use of different vectors carrying fluorescent proteins to selectively label normal and tumor tissue in vivo. We show that a GFP retrovirus and telomerase-driven GFP adenovirus can selectively label tumors in mice. We also show that a GFP lentivirus can selectively label the liver in mice. The practical application of these results are discussed.

An accurate proteomic quantification method: fluorescence labeling absolute quantification (FLAQ) using multidimensional liquid chromatography and tandem mass spectrometry.

PubMed

Liu, Junyan; Liu, Yang; Gao, Mingxia; Zhang, Xiangmin

2012-08-01

A facile proteomic quantification method, fluorescent labeling absolute quantification (FLAQ), was developed. Instead of using MS for quantification, the FLAQ method is a chromatography-based quantification in combination with MS for identification. Multidimensional liquid chromatography (MDLC) with laser-induced fluorescence (LIF) detection with high accuracy and tandem MS system were employed for FLAQ. Several requirements should be met for fluorescent labeling in MS identification: Labeling completeness, minimum side-reactions, simple MS spectra, and no extra tandem MS fragmentations for structure elucidations. A fluorescence dye, 5-iodoacetamidofluorescein, was finally chosen to label proteins on all cysteine residues. The fluorescent dye was compatible with the process of the trypsin digestion and MALDI MS identification. Quantitative labeling was achieved with optimization of reacting conditions. A synthesized peptide and model proteins, BSA (35 cysteines), OVA (five cysteines), were used for verifying the completeness of labeling. Proteins were separated through MDLC and quantified based on fluorescent intensities, followed by MS identification. High accuracy (RSD% < 1.58) and wide linearity of quantification (1-10(5) ) were achieved by LIF detection. The limit of quantitation for the model protein was as low as 0.34 amol. Parts of proteins in human liver proteome were quantified and demonstrated using FLAQ. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single-molecule imaging at high fluorophore concentrations by local activation of dye

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geertsema, Hylkje J.; Mangel, Walter F.; Schulte, Aartje C.

Single-molecule fluorescence microscopy is a powerful approach to observe biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual, labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Then, making use ofmore » short-distance energy-transfer mechanisms, the fluorescence from only those proteins bound to their substrate are selectively activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 (IFI16) with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease (pVIc-AVP) on DNA in the presence of a background of hundreds of nM Cy5 fluorophore.« less

Single-molecule imaging at high fluorophore concentrations by local activation of dye

DOE PAGES

Geertsema, Hylkje J.; Mangel, Walter F.; Schulte, Aartje C.; ...

2015-02-17

Single-molecule fluorescence microscopy is a powerful approach to observe biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual, labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Then, making use ofmore » short-distance energy-transfer mechanisms, the fluorescence from only those proteins bound to their substrate are selectively activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 (IFI16) with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease (pVIc-AVP) on DNA in the presence of a background of hundreds of nM Cy5 fluorophore.« less

Analysis of the photophysical properties of zearalenone using density functional theory

USDA-ARS?s Scientific Manuscript database

The intrinsic photophysical properties of the resorcylic acid moiety of zearalenone offer a convenient label free method to determine zearalenone levels in contaminated agricultural products. Density functional theory and steady-state fluorescence methods were applied to investigate the role of stru...

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging.

PubMed

Zhang, Nan; Membreno, Edward; Raj, Susan; Zhang, Hongjie; Khan, Liakot A; Gobel, Verena

2017-10-03

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay.

PubMed

Hudry, Bruno; Viala, Séverine; Graba, Yacine; Merabet, Samir

2011-01-28

Protein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC) technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels. Using a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners. Our results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development.

Enhancement of Single Molecule Fluorescence Signals by Colloidal Silver Nanoparticles in Studies of Protein Translation

PubMed Central

Bharill, Shashank; Chen, Chunlai; Stevens, Benjamin; Kaur, Jaskiran; Smilansky, Zeev; Mandecki, Wlodek; Gryczynski, Ignacy; Gryczynski, Zygmunt; Cooperman, Barry S.; Goldman, Yale E.

2011-01-01

Metal enhanced fluorescence (MEF) increased total photon emission of Cy3- and Cy5-labeled ribosomal initiation complexes near 50 nm silver particles 4- and 5.5-fold respectively. Fluorescence intensity fluctuations above shot noise, at 0.1 – 5 Hz, were greater on silver particles. Overall signal to noise ratio was similar or slightly improved near the particles. Proximity to silver particles did not compromise ribosome function, as measured by codon-dependent binding of fluorescent tRNA, dynamics of fluorescence resonance energy transfer between adjacent tRNAs in the ribosome, and tRNA translocation induced by elongation factor G. PMID:21158483

Enhancement of single-molecule fluorescence signals by colloidal silver nanoparticles in studies of protein translation.

PubMed

Bharill, Shashank; Chen, Chunlai; Stevens, Benjamin; Kaur, Jaskiran; Smilansky, Zeev; Mandecki, Wlodek; Gryczynski, Ignacy; Gryczynski, Zygmunt; Cooperman, Barry S; Goldman, Yale E

2011-01-25

Metal-enhanced fluorescence (MEF) increased total photon emission of Cy3- and Cy5-labeled ribosomal initiation complexes near 50 nm silver particles 4- and 5.5-fold, respectively. Fluorescence intensity fluctuations above shot noise, at 0.1-5 Hz, were greater on silver particles. Overall signal-to-noise ratio was similar or slightly improved near the particles. Proximity to silver particles did not compromise ribosome function, as measured by codon-dependent binding of fluorescent tRNA, dynamics of fluorescence resonance energy transfer between adjacent tRNAs in the ribosome, and tRNA translocation induced by elongation factor G.

Label-free imaging of developing vasculature in zebrafish with phase variance optical coherence microscopy

NASA Astrophysics Data System (ADS)

Chen, Yu; Fingler, Jeff; Trinh, Le A.; Fraser, Scott E.

2016-03-01

A phase variance optical coherence microscope (pvOCM) has been created to visualize blood flow in the vasculature of zebrafish embryos, without using exogenous labels. The pvOCM imaging system has axial and lateral resolutions of 2 μm in tissue, and imaging depth of more than 100 μm. Imaging of 2-5 days post-fertilization zebrafish embryos identified the detailed structures of somites, spinal cord, gut and notochord based on intensity contrast. Visualization of the blood flow in the aorta, veins and intersegmental vessels was achieved with phase variance contrast. The pvOCM vasculature images were confirmed with corresponding fluorescence microscopy of a zebrafish transgene that labels the vasculature with green fluorescent protein. The pvOCM images also revealed functional information of the blood flow activities that is crucial for the study of vascular development.

Expression of fluorescently tagged connexins: a novel approach to rescue function of oligomeric DsRed-tagged proteins.

PubMed

Lauf, U; Lopez, P; Falk, M M

2001-06-01

A novel, brilliantly red fluorescent protein, DsRed has become available recently opening up a wide variety of experimental opportunities for double labeling and fluorescence resonance electron transfer experiments in combination with green fluorescent protein (GFP). Unlike in the case of GFP, proteins tagged with DsRed were often found to aggregate within the cell. Here we report a simple method that allows rescuing the function of an oligomeric protein tagged with DsRed. We demonstrate the feasibility of this approach on the subunit proteins of an oligomeric membrane channel, gap junction connexins. Additionally, DsRed fluorescence was easily detected 12-16 h post transfection, much earlier than previously reported, and could readily be differentiated from co-expressed GFP. Thus, this approach can eliminate the major drawbacks of this highly attractive autofluorescent protein.

Photoacoustic-fluorescence in vitro flow cytometry for quantification of absorption, scattering and fluorescence properties of the cells

NASA Astrophysics Data System (ADS)

Nedosekin, D. A.; Sarimollaoglu, M.; Foster, S.; Galanzha, E. I.; Zharov, V. P.

2013-03-01

Fluorescence flow cytometry is a well-established analytical tool that provides quantification of multiple biological parameters of cells at molecular levels, including their functional states, morphology, composition, proliferation, and protein expression. However, only the fluorescence and scattering parameters of the cells or labels are available for detection. Cell pigmentation, presence of non-fluorescent dyes or nanoparticles cannot be reliably quantified. Herewith, we present a novel photoacoustic (PA) flow cytometry design for simple integration of absorbance measurements into schematics of conventional in vitro flow cytometers. The integrated system allow simultaneous measurements of light absorbance, scattering and of multicolor fluorescence from single cells in the flow at rates up to 2 m/s. We compared various combinations of excitation laser sources for multicolor detection, including simultaneous excitation of PA and fluorescence using a single 500 kHz pulsed nanosecond laser. Multichannel detection scheme allows simultaneous detection of up to 8 labels, including 4 fluorescent tags and 4 PA colors. In vitro PA-fluorescence flow cytometer was used for studies of nanoparticles uptake and for the analysis of cell line pigmentation, including genetically encoded melanin expression in breast cancer cell line. We demonstrate that this system can be used for direct nanotoxicity studies with simultaneous quantification of nanoparticles content and assessment of cell viability using a conventional fluorescent apoptosis assays.

Selective Chemical Labeling of Proteins with Small Fluorescent Molecules Based on Metal-Chelation Methodology

PubMed Central

Soh, Nobuaki

2008-01-01

Site-specific chemical labeling utilizing small fluorescent molecules is a powerful and attractive technique for in vivo and in vitro analysis of cellular proteins, which can circumvent some problems in genetic encoding labeling by large fluorescent proteins. In particular, affinity labeling based on metal-chelation, advantageous due to the high selectivity/simplicity and the small tag-size, is promising, as well as enzymatic covalent labeling, thereby a variety of novel methods have been studied in recent years. This review describes the advances in chemical labeling of proteins, especially highlighting the metal-chelation methodology. PMID:27879749

Dynamical optical imaging monocytes/macrophages migration and activation in contact hypersensitivity (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zhang, Zhihong

2017-02-01

Inflammatory monocytes/macrophages (Mon/Mφ) play an important role in cutaneous allergic inflammation. However, their migration and activation in dermatitis and how they accelerate the inflammatory reaction are largely unknown. Optical molecular imaging is the most promising tool for investigating the function and motility of immune cells in vivo. We have developed a multi-scale optical imaging approach to evaluate the spatio-temporal dynamic behavior and properties of immune cells from the whole field of organs to the cellular level at the inflammatory site in delayed type hypersensitivity reaction. Here, we developed some multi-color labeling mouse models based on the endogenous labeling with fluorescent proteins and the exogenous labeling with fluorescent dyes. We investigated the cell movement, cell interaction and function of immunocytes (e.g. Mon/Mφ, DC, T cells and neutrophils) in the skin allergy inflammation (e.g., contact hypersensitivity) by using intravital microscopy. The long-term imaging data showed that after inflammatory Mon/Mφ transendothelial migration in dermis, they migrating in interstitial space of dermis. Depletion of blood monocyte with clodronate liposome extremely reduced the inflammatory reaction. Our finding provided further insight into inflammatory Mon/Mφ mediating the inflammatory cascade through functional migration in allergic contact dermatitis.

Pharmacokinetics of lidocaine and bupivacaine following subarachnoid administration in surgical patients: simultaneous investigation of absorption and disposition kinetics using stable isotopes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burm, A.G.; Van Kleef, J.W.; Vermeulen, N.P.

1988-10-01

The pharmacokinetics of lidocaine and bupivacaine following subarachnoid administration were studied in 12 surgical patients using a stable isotope method. After subarachnoid administration of the agent to be evaluated, a deuterium-labelled analogue was administered intravenously. Blood samples were collected for 24 h. Plasma concentrations of the unlabelled and the deuterium-labelled local anesthetics were determined using a combination of capillary gas chromatography and mass fragmentography. Bi-exponential functions were fitted to the plasma concentration-time data of the deuterium-labelled local anesthetics. The progression of the absorption was evaluated using deconvolution. Mono- and bi-exponential functions were then fitted to the fraction absorbed versus timemore » data. The distribution and elimination half-lives of the deuterium-labelled analogues were 25 +/- 13 min (mean +/- SD) and 121 +/- 31 min for lidocaine and 19 +/- 10 min and 131 +/- 33 min for bupivacaine. The volumes of the central compartment and steady-state volumes of distribution were: lidocaine 57 +/- 10 l and 105 +/- 25 l, bupivacaine 25 +/- 6 l and 63 +/- 22 l. Total plasma clearance values averaged 0.97 +/- 0.21 l/min for lidocaine and 0.56 +/- 0.14 l/min for bupivacaine. The absorption of lidocaine could be described by a single first order absorption process, characterized by a half-life of 71 +/- 17 min in five out of six patients. The absorption of bupivacaine could be described adequately assuming two parallel first order absorption processes in all six patients. The half-lives, characterizing the fast and slow absorption processes of bupivacaine, were 50 +/- 27 min and 408 +/- 275 min, respectively. The fractions of the dose, absorbed in the fast and slow processes, were 0.35 +/- 0.17 and 0.61 +/- 0.16, respectively.« less

A multifunctional probe based on the use of labeled aptamer and magnetic nanoparticles for fluorometric determination of adenosine 5'-triphosphate.

PubMed

Liu, Xiaojie; Lin, Bixia; Yu, Ying; Cao, Yujuan; Guo, Manli

2018-04-02

A multifunctional fluorescent probe is synthesized for the determination of adenosine 5'-triphosphate (ATP). The 6-carboxyfluorescein-labeled aptamer (FAM-aptamer) was bound to the surface of magnetite nanoparticles coated with polydopamine (Fe 3 O 4 @PDA) by π-π stacking interaction to form the multifunctional probe. The probe has three functions including recognition, magnetic separation, and yielding a fluorescent signal. In the presence of ATP, FAM-aptamer on the surface of the probe binds to ATP and returns to the solution. Thus, the fluorescence of the supernatant is enhanced and can be related to the concentration of ATP. Fluorescence intensities were measured at excitation/emission wavelengths of 494/526 nm. Response is linear in the 0.1-100 μM ATP concentration range, and the detection limit is 89 nM. The probe was applied to the quantitation of ATP in spiked human urine and serum samples, with recoveries ranging between 94.8 and 102%. Graphical abstract A multifunctional fluorescent probe based on the use of FAM-aptamer and Fe 3 O 4 @PDA is described for the determination of ATP in spiked human urine and serum samples. FAM-aptamer: 6-carboxyfluorescein-labeled aptamer; Fe 3 O 4 @PDA: magnetite nanoparticles coated with polydopamine. ATP: adenosine 5'-triphosphate.

Multiphoton microscopy for the in-situ investigation of cellular processes and integrity in cryopreservation.

PubMed

Doerr, Daniel; Stark, Martin; Ehrhart, Friederike; Zimmermann, Heiko; Stracke, Frank

2009-08-01

In this study we demonstrate a new noninvasive imaging method to monitor freezing processes in biological samples and to investigate life in the frozen state. It combines a laser scanning microscope with a computer-controlled cryostage. Nearinfrared (NIR) femtosecond laser pulses evoke the fluorescence of endogenous fluorophores and fluorescent labels due to multiphoton absorption.The inherent optical nonlinearity of multiphoton absorption allows 3D fluorescence imaging for optical tomography of frozen biological material in-situ. As an example for functional imaging we use fluorescence lifetime imaging (FLIM) to create images with chemical and physical contrast.

Selective labeling of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein

NASA Astrophysics Data System (ADS)

Watanabe, Wataru; Shimada, Tomoko; Matsunaga, Sachihiro; Kurihara, Daisuke; Arimura, Shin-ichi; Tsutsumi, Nobuhiro; Fukui, Kiichi; Itoh, Kazuyoshi

2008-02-01

We present space-selective labeling of organelles by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. Two-photon excitation of photoconvertible fluorescent-protein, Kaede, enables space-selective labeling of organelles. We alter the fluorescence of target mitochondria in a tobacco BY-2 cell from green to red by focusing femtosecond laser pulses with a wavelength of 750 nm.

Chemical reactivation of fluorescein isothiocyanate immunofluorescence-labeled resin-embedded samples

NASA Astrophysics Data System (ADS)

Li, Longhui; Rao, Gong; Lv, Xiaohua; Chen, Ruixi; Cheng, Xiaofeng; Wang, Xiaojun; Zeng, Shaoqun; Liu, Xiuli

2018-02-01

Resin embedding is widely used and facilitates microscopic imaging of biological tissues. In contrast, quenching of fluorescence during embedding process hinders the application of resin embedding for imaging of fluorescence-labeled samples. For samples expressing fluorescent proteins, it has been demonstrated that the weakened fluorescence could be recovered by reactivating the fluorophore with alkaline buffer. We extended this idea to immunofluorescence-labeling technology. We showed that the fluorescence of pH-sensitive fluorescein isothiocyanate (FITC) was quenched after resin embedding but reactivated after treating by alkaline buffer. We observed 138.5% fluorescence preservation ratio of reactivated state, sixfold compared with the quenched state in embedding resin, which indicated its application for fluorescence imaging of high signal-to-background ratio. Furthermore, we analyzed the chemical reactivation mechanism of FITC fluorophore. This work would show a way for high-resolution imaging of immunofluorescence-labeled samples embedded in resin.

Folate receptor targeted, rare-earth oxide nanocrystals for bi-modal fluorescence and magnetic imaging of cancer cells.

PubMed

Setua, Sonali; Menon, Deepthy; Asok, Adersh; Nair, Shantikumar; Koyakutty, Manzoor

2010-02-01

Targeted cancer imaging using rare-earth oxide nanocrystals, free from heavy metals (Cd, Se, Te, Hg and Pb), showing bright red-fluorescence and magnetic resonance imaging (MRI) is presented. Y(2)O(3) nanocrystals (YO NC) doped in situ with fluorescent (Eu(3+)) and paramagnetic (Gd(3+)) impurities and conjugated with a potential cancer targeting ligand, folic acid (FA), were prepared using an all-aqueous wet-chemical process. Structural, optical and magnetic properties of these multifunctional nanocrystals were investigated by X-ray diffraction, electron microscopy, photoluminescence and magnetization studies. Highly monodisperse nanocrystals of size approximately 20 nm with cubic bixbyite crystal structure showed bright red-fluorescence when doped with Eu(3+). Co-doping with Gd(3+) and mild air drying resulted significantly enhanced fluorescence quantum efficiency of approximately 60% together with paramagnetic functionality, enabling T(1)-weighted MR contrast with approximately 5 times higher spin-lattice relaxivity compared to the clinically used Gd(3+) contrast agent. Cytotoxicity and reactive oxygen stress studies show no toxicity by YO NC in both normal and cancer cells up to higher doses of 500 microm and longer incubation time, 48h. Cancer targeting capability of FA conjugated NCs was demonstrated on folate receptor positive (FR+) human nasopharyngeal carcinoma cells (KB) with FR depressed KB (FRd) and FR negative (FR-) lung cancer cells A549 as controls. Fluorescence microscopy and flow-cytometry data show highly specific binding and cellular uptake of large concentration of FA conjugated NCs on FR+ve cells compared to the controls. Thus, the present study reveals, unique bi-modal contrast imaging capability, non-toxicity and cancer targeting capability of multiple impurities doped rare-earth oxide nanocrystals that can find promising application in molecular imaging.

Electron Detachment Dissociation (EDD) of Fluorescently Labeled Sialylated Oligosaccharides

PubMed Central

Zhou, Wen; Håkansson, Kristina

2012-01-01

We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared to IRMPD. Neutral losses and satellite ions such as C – 2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared to 2-AA labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag. PMID:22120881

Fluorescently labeled dengue viruses as probes to identify antigen-specific memory B cells by multiparametric flow cytometry

PubMed Central

Woda, Marcia; Mathew, Anuja

2015-01-01

Low frequencies of memory B cells in the peripheral blood make it challenging to measure the functional and phenotypic characteristics of this antigen experienced subset of B cells without in vitro culture. To date, reagents are lacking to measure ex vivo frequencies of dengue virus (DENV)-specific memory B cells. We wanted to explore the possibility of using fluorescently labeled DENV as probes to detect antigen-specific memory B cells in the peripheral blood of DENV immune individuals. Alexa Fluor dye-labeled DENV yielded viable virus that could be stored at −80°C for long periods of time. Using a careful gating strategy and methods to decrease non-specific binding, we were able to identify a small frequency of B cells from dengue immune individuals that bound labeled DENV. Sorted DENV+ B cells from immune, but not naïve donors secreted antibodies that bound intact virions after in vitro stimulation. Overall, Alexa Fluor dye labeled -DENV are useful reagents to enable the detection and characterization of memory B cells in DENV immune individuals. PMID:25497702

Fluorescent labeling of tetracysteine-tagged proteins in intact cells

PubMed Central

Hoffmann, Carsten; Gaietta, Guido; Zürn, Alexander; Adams, Stephen R; Terrillon, Sonia; Ellisman, Mark H; Tsien, Roger Y; Lohse, Martin J

2011-01-01

In this paper, we provide a general protocol for labeling proteins with the membrane-permeant fluorogenic biarsenical dye fluorescein arsenical hairpin binder–ethanedithiol (FlAsH-EDT2). Generation of the tetracysteine-tagged protein construct by itself is not described, as this is a protein-specific process. This method allows site-selective labeling of proteins in living cells and has been applied to a wide variety of proteins and biological problems. We provide here a generally applicable labeling procedure and discuss the problems that can occur as well as general considerations that must be taken into account when designing and implementing the procedure. The method can even be applied to proteins with expression below 1 pmol mg−1 of protein, such as G protein–coupled receptors, and it can be used to study the intracellular localization of proteins as well as functional interactions in fluorescence resonance energy transfer experiments. The labeling procedure using FlAsH-EDT2 as described takes 2–3 h, depending on the number of samples to be processed. PMID:20885379

Shaping Small Bioactive Molecules to Untangle Their Biological Function: A Focus on Fluorescent Plant Hormones.

PubMed

Lace, Beatrice; Prandi, Cristina

2016-08-01

Modern biology overlaps with chemistry in explaining the structure and function of all cellular processes at the molecular level. Plant hormone research is perfectly located at the interface between these two disciplines, taking advantage of synthetic and computational chemistry as a tool to decipher the complex biological mechanisms regulating the action of plant hormones. These small signaling molecules regulate a wide range of developmental processes, adapting plant growth to ever changing environmental conditions. The synthesis of small bioactive molecules mimicking the activity of endogenous hormones allows us to unveil many molecular features of their functioning, giving rise to a new field, plant chemical biology. In this framework, fluorescence labeling of plant hormones is emerging as a successful strategy to track the fate of these challenging molecules inside living organisms. Thanks to the increasing availability of new fluorescent probes as well as advanced and innovative imaging technologies, we are now in a position to investigate many of the dynamic mechanisms through which plant hormones exert their action. Such a deep and detailed comprehension is mandatory for the development of new green technologies for practical applications. In this review, we summarize the results obtained so far concerning the fluorescent labeling of plant hormones, highlighting the basic steps leading to the design and synthesis of these compelling molecular tools and their applications. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Enhanced detection of fluorescence quenching in labeled cells

DOEpatents

Crissman, H.A.; Steinkamp, J.A.

1987-11-30

A method is provided for quantifying BrdU labeled DNA in cells. The BrdU is substituted onto the DNA and the DNA is stained with a first fluorochrome having a fluorescence which is quenchable by BrdU. The first fluorochrome is preferably a thymidine base halogen analogue, such as a Hoechst fluorochrome. The DNA is then stained with a second fluorochrome having a fluorescence which is substantially uneffected by BrdU. The second fluorochrome may be selected from the group consisting of mithramycin, chromomycin A3, olivomycin, propidium iodide and ethidium bromine. The fluorescence from the first and second fluorochromes is then measured to obtain first and second output signals, respectively. The first output signal is subtracted from the second output signal to obtain a difference signal which is functionally related to the quantity of BrdU incorporated into DNA. The technique is particularly useful for quantifying the synthesis of DNA during the S-phase of the cell cycle. 2 figs.

Enhanced detection of fluorescence quenching in labeled cells

DOEpatents

Crissman, Harry A.; Steinkamp, John A.

1992-01-01

A method is provided for quantifying BrdU labeled DNA in cells. The BrdU is incorporated into the DNA and the DNA is stained with a first fluorochrome having a fluorescence which is quenchable by BrdU. The first fluorochrome is preferably a thymidine base halogen analogue, such as a Hoechst fluorochrome. The DNA is then stained with a second fluorochrome having a fluorescence that is substantially uneffected by BrdU. The second fluorochrome may be selected from the group consisting of mithramycin, chromomycin A3, olivomycin, propidium iodide and ethidium bromine. The fluorescence from the first and second fluorochromes is then measured to obtain first and second output signals, respectively. The first output signal is substracted from the second output signal to obtain a difference signal which is functionally related to the quantity of BrdU incorporated into DNA. The technique is particularly useful for quantifying the synthesis of DNA during the S-phase of the cell cycle.

A combination of positive dielectrophoresis driven on-line enrichment and aptamer-fluorescent silica nanoparticle label for rapid and sensitive detection of Staphylococcus aureus.

PubMed

Shangguan, Jingfang; Li, Yuhong; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Zou, Zhen; Shi, Hui

2015-07-07

Staphylococcus aureus (S. aureus) is an important human pathogen that causes several diseases ranging from superficial skin infections to life-threatening diseases. Here, a method combining positive dielectrophoresis (pDEP) driven on-line enrichment and aptamer-fluorescent silica nanoparticle label has been developed for the rapid and sensitive detection of S. aureus in microfluidic channels. An aptamer, having high affinity to S. aureus, is used as the molecular recognition tool and immobilized onto chloropropyl functionalized fluorescent silica nanoparticles through a click chemistry approach to obtain S. aureus aptamer-nanoparticle bioconjugates (Apt(S.aureus)/FNPs). The pDEP driven on-line enrichment technology was used for accumulating the Apt(S.aureus)/FNP labeled S. aureus. After incubating with S. aureus, the mixture of Apt(S.aureus)/FNP labeled S. aureus and Apt(S.aureus)/FNPs was directly introduced into the pDEP-based microfluidic system. By applying an AC voltage in a pDEP frequency region, the Apt(S.aureus)/FNP labelled S. aureus moved to the electrodes and accumulated in the electrode gap, while the free Apt(S.aureus)/FNPs flowed away. The signal that came from the Apt(S.aureus)/FNP labelled S. aureus in the focused detection areas was then detected. Profiting from the specificity of aptamer, signal amplification of FNP label and pDEP on-line enrichment, this assay can detect as low as 93 and 270 cfu mL(-1)S. aureus in deionized water and spiked water samples, respectively, with higher sensitivities than our previously reported Apt(S.aureus)/FNP based flow cytometry. Moreover, without the need for separation and washing steps usually required for FNP label involved bioassays, the total assay time including sample pretreatment was within 2 h.

Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

PubMed Central

Okamura, Yukio; Kondo, Satoshi; Sase, Ichiro; Suga, Takayuki; Mise, Kazuyuki; Furusawa, Iwao; Kawakami, Shigeki; Watanabe, Yuichiro

2000-01-01

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method. PMID:11121494

Protein specific fluorescent microspheres for labelling a protein

NASA Technical Reports Server (NTRS)

Rembaum, Alan (Inventor)

1982-01-01

Highly fluorescent, stable and biocompatible microspheres are obtained by copolymerizing an acrylic monomer containing a covalent bonding group such as hydroxyl, amine or carboxyl, for example, hydroxyethylmethacrylate, with an addition polymerizable fluorescent comonomer such as dansyl allyl amine. A lectin or antibody is bound to the covalent site to provide cell specificity. When the microspheres are added to a cell suspension the marked microspheres will specifically label a cell membrane by binding to a specific receptor site thereon. The labeled membrane can then be detected by fluorescence of the fluorescent monomer.

Far-Red Fluorescent Lipid-Polymer Probes for an Efficient Labeling of Enveloped Viruses.

PubMed

Lacour, William; Adjili, Salim; Blaising, Julie; Favier, Arnaud; Monier, Karine; Mezhoud, Sarra; Ladavière, Catherine; Place, Christophe; Pécheur, Eve-Isabelle; Charreyre, Marie-Thérèse

2016-08-01

Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

GMars-T Enabling Multimodal Subdiffraction Structural and Functional Fluorescence Imaging in Live Cells.

PubMed

Wang, Sheng; Chen, Xuanze; Chang, Lei; Ding, Miao; Xue, Ruiying; Duan, Haifeng; Sun, Yujie

2018-06-05

Fluorescent probes with multimodal and multilevel imaging capabilities are highly valuable as imaging with such probes not only can obtain new layers of information but also enable cross-validation of results under different experimental conditions. In recent years, the development of genetically encoded reversibly photoswitchable fluorescent proteins (RSFPs) has greatly promoted the application of various kinds of live-cell nanoscopy approaches, including reversible saturable optical fluorescence transitions (RESOLFT) and stochastic optical fluctuation imaging (SOFI). However, these two classes of live-cell nanoscopy approaches require different optical characteristics of specific RSFPs. In this work, we developed GMars-T, a monomeric bright green RSFP which can satisfy both RESOLFT and photochromic SOFI (pcSOFI) imaging in live cells. We further generated biosensor based on bimolecular fluorescence complementation (BiFC) of GMars-T which offers high specificity and sensitivity in detecting and visualizing various protein-protein interactions (PPIs) in different subcellular compartments under physiological conditions (e.g., 37 °C) in live mammalian cells. Thus, the newly developed GMars-T can serve as both structural imaging probe with multimodal super-resolution imaging capability and functional imaging probe for reporting PPIs with high specificity and sensitivity based on its derived biosensor.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

PubMed Central

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M.; Specht, Christian G.; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-01

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling. PMID:26711992

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

PubMed

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M; Specht, Christian G; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-19

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Live imaging of endogenous PSD-95 using ENABLED: a conditional strategy to fluorescently label endogenous proteins.

PubMed

Fortin, Dale A; Tillo, Shane E; Yang, Guang; Rah, Jong-Cheol; Melander, Joshua B; Bai, Suxia; Soler-Cedeño, Omar; Qin, Maozhen; Zemelman, Boris V; Guo, Caiying; Mao, Tianyi; Zhong, Haining

2014-12-10

Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo. Copyright © 2014 the authors 0270-6474/14/3416698-15$15.00/0.

Multi-photon excited luminescence of magnetic FePt core-shell nanoparticles.

PubMed

Seemann, K M; Kuhn, B

2014-07-01

We present magnetic FePt nanoparticles with a hydrophilic, inert, and biocompatible silico-tungsten oxide shell. The particles can be functionalized, optically detected, and optically manipulated. To show the functionalization the fluorescent dye NOPS was bound to the FePt core-shell nanoparticles with propyl-triethoxy-silane linkers and fluorescence of the labeled particles were observed in ethanol (EtOH). In aqueous dispersion the NOPS fluorescence is quenched making them invisible using 1-photon excitation. However, we observe bright luminescence of labeled and even unlabeled magnetic core-shell nanoparticles with multi-photon excitation. Luminescence can be detected in the near ultraviolet and the full visible spectral range by near infrared multi-photon excitation. For optical manipulation, we were able to drag clusters of particles, and maybe also single particles, by a focused laser beam that acts as optical tweezers by inducing an electric dipole in the insulated metal nanoparticles. In a first application, we show that the luminescence of the core-shell nanoparticles is bright enough for in vivo multi-photon imaging in the mouse neocortex down to cortical layer 5.

High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes.

PubMed

Wittig, Ilka; Karas, Michael; Schägger, Hermann

2007-07-01

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.

Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

PubMed Central

Warren, Sean C.; Margineanu, Anca; Alibhai, Dominic; Kelly, Douglas J.; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Katan, Matilda

2013-01-01

Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment. PMID:23940626

Synthesis, characterization, and protein labeling of difunctional magnetic nanoparticles modified with thiazole orange dye

NASA Astrophysics Data System (ADS)

Fei, Xuening; Zhu, Huifang; Zhou, Jianguo; Yu, Lu

2014-03-01

A dual functional nanoparticle was designed and synthesized by encapsulating magnetic core inside silica particles and subsequently a thiazole orange (TO) dye derivative was modified on the surface of the nanoparticles. The obtained particles were characterized by Fourier transform infrared spectroscope, Uv-Vis spectrophotometer, fluorescence spectrophotometer, transmission electron microscope, dynamic light scattering, etc. The size of preliminary magnetic particles is ca. 7 nm, but after coating a silica layer and dye, the size of particles is increased to ca. 60 nm. The hydrodynamic diameter, water dispersibility, and zeta potential were also determined. The hydrodynamic diameter of particles with silica and dye is 65.2 and 70.5 nm, respectively, with positive zeta potential (25.1, 38.5 mV). Furthermore magnetic properties of the particles were measured and the experimental results suggested that it could meet the requirement of application as magnetic resonance imaging agent. Finally to verify the availability of the particles as fluorescent labeling, protein labeling experiment was performed using bovine serum albumin (BSA) protein and the results showed that the dual functional particle has higher affinity with BSA than TO molecule itself.

Inhibition of Oncogenic functionality of STAT3 Protein by Membrane Anchoring

NASA Astrophysics Data System (ADS)

Liu, Baoxu; Fletcher, Steven; Gunning, Patrick; Gradinaru, Claudiu

2009-03-01

Signal Transducer and Activator of Transcription 3 (STAT3) protein plays an important role in oncogenic processes. A novel molecular therapeutic approach to inhibit the oncogenic functionality of STAT3 is to design a prenylated small peptide sequence which could sequester STAT3 to the plasma membrane. We have also developed a novel fluorescein derivative label (F-NAc), which is much more photostable compared to the popular fluorescein label FITC. Remarkably, the new dye shows fluorescent properties that are invariant over a wide pH range, which is advantageous for our application. We have shown that F-NAc is suitable for single-molecule measurements and its properties are not affected by ligation to biomolecules. The membrane localization via high-affinity prenylated small-molecule binding agents is studied by encapsulating FNAc-labeled STAT3 and inhibitors within a liposome model cell system. The dynamics of the interaction between the protein and the prenylated ligands is investigated at single molecule level. The efficiency and stability of the STAT3 anchoring in lipid membranes are addressed via quantitative confocal imaging and single-molecule spectroscopy using a custom-built multiparameter fluorescence microscope.

Single-molecule fluorescence study of the inhibition of the oncogenic functionality of STAT3

NASA Astrophysics Data System (ADS)

Liu, Baoxu; Badali, Daniel; Fletcher, Steven; Avadisian, Miriam; Gunning, Patrick; Gradinaru, Claudiu

2009-06-01

Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein plays an important role in the onset of cancers such as leukemia and lymphoma. In this study, we aim to test the effectiveness of a novel peptide drug designed to tether STAT3 to the phospholipid bilayer of the cell membrane and thus inhibit unwanted transcription. As a first step, STAT3 proteins were successfully labelled with tetramethylrhodamine (TMR), a fluorescent dye with suitable photostability for single molecule studies. The effectiveness of labelling was determined using fluorescence correlation spectroscopy in a custom built confocal microscope, from which diffusion times and hydrodynamic radii of individual proteins were determined. A newly developed fluorescein derivative label (F-NAc) has been designed to be incorporated into the structure of the peptide drug so that peptide-STAT3 interactions can be examined. This dye is spectrally characterized and is found to be well suited for its application to this project, as well as other single-molecule studies. The membrane localization via high-affinity cholesterol-bound small-molecule binding agents can be demonstrated by encapsulating TMR-labeled STAT3 and inhibitors within a vesicle model cell system. To this end, unilaminar lipid vesicles were examined for size and encapsulation ability. Preliminary results of the efficiency and stability of the STAT3 anchoring in lipid membranes obtained via quantitative confocal imaging and single-molecule spectroscopy using a custom-built multiparameter fluorescence microscope are reported here.

Rainbow Vectors for Broad-Range Bacterial Fluorescence Labeling.

PubMed

Barbier, Mariette; Damron, F Heath

2016-01-01

Since their discovery, fluorescent proteins have been widely used to study protein function, localization or interaction, promoter activity and regulation, drug discovery or for non-invasive imaging. They have been extensively modified to improve brightness, stability, and oligomerization state. However, only a few studies have focused on understanding the dynamics of fluorescent proteins expression in bacteria. In this work, we developed a set plasmids encoding 12 fluorescent proteins for bacterial labeling to facilitate the study of pathogen-host interactions. These broad-spectrum plasmids can be used with a wide variety of Gram-negative microorganisms including Escherichia coli, Pseudomonas aeruginosa, Burkholderia cepacia, Bordetella bronchiseptica, Shigella flexneri or Klebsiella pneumoniae. For comparison, fluorescent protein expression and physical characteristics in Escherichia coli were analyzed using fluorescence microscopy, flow cytometry and in vivo imaging. Fluorescent proteins derived from the Aequorea Victoria family showed high photobleaching, while proteins form the Discosoma sp. and the Fungia coccina family were more photostable for microscopy applications. Only E2-Crimson, mCherry and mKeima were successfully detected for in vivo applications. Overall, E2-Crimson was the fastest maturing protein tested in E. coli with the best overall performance in the study parameters. This study provides a unified comparison and comprehensive characterization of fluorescent protein photostability, maturation and toxicity, and offers general recommendations on the optimal fluorescent proteins for in vitro and in vivo applications.

Evaluation of rapid readout biological indicators for 132 degrees C gravity and 132 degrees C vacuum-assisted steam sterilization cycles using a new automated fluorescent reader.

PubMed

Alfa, Michelle J; Olson, Nancy; DeGagne, Pat; Jackson, Michele

2002-07-01

The primary objective of this study was to evaluate fluorescent readout results of Attest 1291 Biological Indicators (BIs) (3M Health Care, St. Paul, MN) and Attest 1296 BI test packs (containing Attest 1292 BIs) using full and fractional cycles compared with the growth data when prolonged incubation (7 days) was included. Gravity displacement and vacuum-assisted steam sterilization cycles were evaluated. A secondary objective of this study was to evaluate the new automated rapid fluorescent reader (Attest 290 Auto Reader). The rapid readout BIs for gravity displacement and vacuum-assisted steam autoclave cycles at 132 degrees C were processed using full (4 minutes) and four fractional cycles that provided 30% to 80% positive results for growth after 24 hours of incubation (48 hours of incubation for Attest 1292 BIs from the Attest 1296 test packs). Sixty of each type of BI were tested for each cycle (300 of each BI type in total). For all full steam sterilization cycles, results of the rapid fluorescent readout and the 24-hour, 48-hour, and 7-day growth tests were negative for all Attest 1291 and 1292 BIs tested. For all fractional cycles, the 24- and 48-hour growth results for the Attest 1291 and 1292 BIs, respectively, were the same as the 7-day growth results. The fractional cycle data indicated that fluorescent rapid readout was a more sensitive indicator than growth. There were rare (0.9%) false-negative results for BIs under fractional cycle conditions and these all correlated with short fractional cycle exposure times. The fluorescent rapid readout results of the 1291 BIs and 1296 BI test packs reliably predict both 24- and 48-hour and 7-day growth. These data support the value of rapid readout BIs for sterilizer monitoring for both the vacuum-assisted and the gravity displacement steam sterilization cycles. The new automated reader requires less manipulation of the BI and makes monitoring user friendly and less prone to user errors.

Single Cell Fluorescence Imaging Using Metal Plasmon-Coupled Probe

PubMed Central

Zhang, Jian; Fu, Yi; Lakowicz, Joseph R.

2009-01-01

This work constitutes the first fluorescent imaging of cells using metal plasmon-coupled probes (PCPs) at single cell resolution. N-(2-Mercapto-propionyl)glycine-coated silver nanoparticles were synthesized by reduction of silver nitrate using sodium borohyride and then succinimidylated via ligand exchange. Alexa Fluor 647-labeled concanavalin A (con A) was chemically bound to the silver particles to make the fluorescent metal plasmon-coupled probes. The fluorescence images were collected using a scanning confocal microscopy. The fluorescence intensity was observed to enhance 7-fold when binding the labeled con A on a single silver particle. PCPs were conjugated on HEK 293 A cells. Imaging results demonstrate that cells labeled by PCPs were 20-fold brighter than those by free labeled con A. PMID:17375898

Synthesis of Bipartite Tetracysteine PNA Probes for DNA In Situ Fluorescent Labeling.

PubMed

Fang, Ge-Min; Seitz, Oliver

2017-12-24

"Label-free" fluorescent probes that avoid additional steps or building blocks for conjugation of fluorescent dyes with oligonucleotides can significantly reduce the time and cost of parallel bioanalysis of a large number of nucleic acid samples. A method for the synthesis of "label-free" bicysteine-modified PNA probes using solid-phase synthesis and procedures for sequence-specific DNA in situ fluorescent labeling is described here. The concept is based on the adjacent alignment of two bicysteine-modified peptide nucleic acids on a DNA target to form a structurally optimized bipartite tetracysteine motif, which induces a sequence-specific fluorogenic reaction with commercially available biarsenic dyes, even in complex media such as cell lysate. This unit will help researchers to quickly synthesize bipartite tetracysteine PNA probes and carry out low-cost DNA in situ fluorescent labeling experiments. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Fluorescently labeled bevacizumab in human breast cancer: defining the classification threshold

NASA Astrophysics Data System (ADS)

Koch, Maximilian; de Jong, Johannes S.; Glatz, Jürgen; Symvoulidis, Panagiotis; Lamberts, Laetitia E.; Adams, Arthur L. L.; Kranendonk, Mariëtte E. G.; Terwisscha van Scheltinga, Anton G. T.; Aichler, Michaela; Jansen, Liesbeth; de Vries, Jakob; Lub-de Hooge, Marjolijn N.; Schröder, Carolien P.; Jorritsma-Smit, Annelies; Linssen, Matthijs D.; de Boer, Esther; van der Vegt, Bert; Nagengast, Wouter B.; Elias, Sjoerd G.; Oliveira, Sabrina; Witkamp, Arjen J.; Mali, Willem P. Th. M.; Van der Wall, Elsken; Garcia-Allende, P. Beatriz; van Diest, Paul J.; de Vries, Elisabeth G. E.; Walch, Axel; van Dam, Gooitzen M.; Ntziachristos, Vasilis

2017-07-01

In-vivo fluorescently labelled drug (bevacizumab) breast cancer specimen where obtained from patients. We propose a new structured method to determine the optimal classification threshold in targeted fluorescence intra-operative imaging.

Siloxane nanoprobes for labeling and dual modality imaging of neural stem cells

PubMed Central

Addington, Caroline P.; Cusick, Alex; Shankar, Rohini Vidya; Agarwal, Shubhangi; Stabenfeldt, Sarah E.; Kodibagkar, Vikram D.

2015-01-01

Cell therapy represents a promising therapeutic for a myriad of medical conditions, including cancer, traumatic brain injury, and cardiovascular disease among others. A thorough understanding of the efficacy and cellular dynamics of these therapies necessitates the ability to non-invasively track cells in vivo. Magnetic resonance imaging (MRI) provides a platform to track cells as a non-invasive modality with superior resolution and soft tissue contrast. We recently reported a new nanoprobe platform for cell labeling and imaging using fluorophore doped siloxane core nanoemulsions as dual modality (1H MRI/Fluorescence), dual-functional (oximetry/detection) nanoprobes. Here, we successfully demonstrate the labeling, dual-modality imaging, and oximetry of neural progenitor/stem cells (NPSCs) in vitro using this platform. Labeling at a concentration of 10 μl/104 cells with a 40%v/v polydimethylsiloxane core nanoemulsion, doped with rhodamine, had minimal effect on viability, no effect on migration, proliferation and differentiation of NPSCs and allowed for unambiguous visualization of labeled NPSCs by 1H MR and fluorescence and local pO2 reporting by labeled NPSCs. This new approach for cell labeling with a positive contrast 1H MR probe has the potential to improve mechanistic knowledge of current therapies, and guide the design of future cell therapies due to its clinical translatability. PMID:26597417

Generation of the additional fluorescence radiation in the elastomeric shields used in computer tomography (CT).

PubMed

Szajerski, P; Zaborski, M; Bem, H; Baryn, W; Kusiak, E

Two commercially available (EP, Z) and eight new elastomeric composites (M1-M4, G1-G4, of thickness ≈1 mm) containing mixtures of differing proportions of heavy metal additives (Bi, W, Gd and Sb) have been synthesised and examined as protective shields. The intensity of the X-ray fluorescence radiation generated in the typical elastomeric shields for CT, containing Bi and other heavy metal additives influence on the practical shielding properties. A method for assessing the radiation shielding properties of elastomeric composites used in CT examination procedures via X-ray spectrometry has been proposed. To measure the radiation reduction ability of the protective shields, the dose reduction factor (DRF) has been determined. The lead equivalents for the examined composites were within the ranges of 0.046-0.128 and 0.048-0.130 mm for 122.1 and 136.5 keV photons, respectively. The proposed method, unlike to the common approach, includes a dose contribution from the induced X-ray fluorescence radiation of the heavy metal elements in the protective shields. The results clearly indicate that among the examined compositions, the highest values DRF have been achieved with preparations containing Bi+W, Bi+W+Gd and Bi+W+Sb mixtures with gradually decreasing content of heavy metal additives in the following order: Bi, W, Gd and Sb. The respective values of DRF obtained for the investigated composites were 21, 28 and 27 % dose reduction for a 1 mm thick shield and 39 and ~50 % for a 2 mm thick layer (M1-M4).

Specifically and wash-free labeling of SNAP-tag fused proteins with a hybrid sensor to monitor local micro-viscosity.

PubMed

Wang, Chao; Song, Xinbo; Chen, Lingcheng; Xiao, Yi

2017-05-15

Viscosity, as one of the major factors of intracellular microenvironment, influences the function of proteins. To detect local micro-viscosity of a protein, it is a precondition to apply a viscosity sensor for specifically target to proteins. However, all the reported small-molecule probes are just suitable for sensing/imaging of macro-viscosity in biological fluids of entire cells or organelles. To this end, we developed a hybrid sensor BDP-V BG by connecting a viscosity-sensitive boron-dipyrromethene (BODIPY) molecular rotor (BDP-V) to O 6 -benzylguanine (BG) for specific detection of local micro-viscosity of SNAP-tag fused proteins. We measured and calculated the reaction efficiency between the sensor and SNAP-tag protein in vitro to confirm the high labeling specificity. We also found that the labeling reaction results in a 53-fold fluorescence enhancement for the rotor, which qualifies it as a wash-free sensor with ignorable background fluorescence. The high sensitivity of protein labeled sensor (BDP-V-SNAP) to the changes of local viscosity was evaluated by detecting the enhancement of fluorescence lifetimes. Further, with the sensor BDP-V BG, we achieved high specific labeling of cells expressing two SNAP-tag fused proteins (nuclear histone H2B and mitochondrial COX8A). Two-photon excited fluorescence lifetime imaging revealed that, the micro-viscosities nearby the SNAP-tag fused two proteins are distinct. The different changes of local micro-viscosity of SNAP-tag fused histone protein in apoptosis induced by three nucleus-targeted drugs were also characterized for the first time. Copyright © 2016 Elsevier B.V. All rights reserved.

Intrinsically Labeled Fluorescent Oligonucleotide Probes on Quantum Dots for Transduction of Nucleic Acid Hybridization.

PubMed

Shahmuradyan, Anna; Krull, Ulrich J

2016-03-15

Quantum dots (QDs) have been widely used in chemical and biosensing due to their unique photoelectrical properties and are well suited as donors in fluorescence resonance energy transfer (FRET). Selective hybridization interactions of oligonucleotides on QDs have been determined by FRET. Typically, the QD-FRET constructs have made use of labeled targets or have implemented labeled sandwich format assays to introduce dyes in proximity to the QDs for the FRET process. The intention of this new work is to explore a method to incorporate the acceptor dye into the probe molecule. Thiazole orange (TO) derivatives are fluorescent intercalating dyes that have been used for detection of double-stranded nucleic acids. One such dye system has been reported in which single-stranded oligonucleotide probes were doubly labeled with adjacent thiazole orange derivatives. In the absence of the fully complementary (FC) oligonucleotide target, the dyes form an H-aggregate, which results in quenching of fluorescence emission due to excitonic interactions between the dyes. The hybridization of the FC target to the probe provides for dissociation of the aggregate as the dyes intercalate into the double stranded duplex, resulting in increased fluorescence. This work reports investigation of the dependence of the ratiometric signal on the type of linkage used to conjugate the dyes to the probe, the location of the dye along the length of the probe, and the distance between adjacent dye molecules. The limit of detection for 34mer and 90mer targets was found to be identical and was 10 nM (2 pmol), similar to analogous QD-FRET using labeled oligonucleotide target. The detection system could discriminate a one base pair mismatch (1BPM) target and was functional without substantial compromise of the signal in 75% serum. The 1BPM was found to reduce background signal, indicating that the structure of the mismatch affected the environment of the intercalating dyes.

Horseradish peroxidase-labeled oligonucleotides and fluorescent tyramides for rapid detection of chromosome-specific repeat sequences.

PubMed

van Gijlswijk, R P; Wiegant, J; Vervenne, R; Lasan, R; Tanke, H J; Raap, A K

1996-01-01

We present a sensitive and rapid fluorescence in situ hybridization (FISH) strategy for detecting chromosome-specific repeat sequences. It uses horseradish peroxidase (HRP)-labeled oligonucleotide sequences in combination with fluorescent tyramide-based detection. After in situ hybridization, the HRP conjugated to the oligonucleotide probe is used to deposit fluorescently labeled tyramide molecules at the site of hybridization. The method features full chemical synthesis of probes, strong FISH signals, and short processing periods, as well as multicolor capabilities.

16 CFR 305.15 - Labeling for lighting products.

Code of Federal Regulations, 2011 CFR

2011-01-01

...) Package labeling. For purposes of labeling under this section, packaging for such fluorescent lamp... individually or in small numbers. The encircled capital letter “E” on packages containing fluorescent lamp... ink, on the surface of the package on which printing or a label normally appears. If the package...

DESIGN, SYNTHESIS, AND APPLICATION OF THE TRIMETHOPRIM-BASED CHEMICAL TAG FOR LIVE CELL IMAGING

PubMed Central

Jing, Chaoran; Cornish, Virginia W.

2013-01-01

Over the past decade chemical tags have been developed to complement the use of fluorescent proteins in live cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon-output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E.coli dihydrofolatereductase and the antibiotic trimethoprim and subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live cell imaging. Alternative protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. PMID:23839994

The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

PubMed

Lu, Ting; Lin, Zongwei; Ren, Jianwei; Yao, Peng; Wang, Xiaowei; Wang, Zhe; Zhang, Qunye

2016-01-01

MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs. Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it. These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay

PubMed Central

2011-01-01

Background Protein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC) technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels. Results Using a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners. Conclusion Our results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development. PMID:21276241

A selective and label-free strategy for rapid screening of telomere-binding Ligands via fluorescence regulation of DNA/silver nanocluster

NASA Astrophysics Data System (ADS)

Cheng, Rui; Xu, Jing; Zhang, Xiafei; Shi, Zhilu; Zhang, Qi; Jin, Yan

2017-03-01

Herein, the conformational switch of G-rich oligonucleotide (GDNA) demonstrated the obvious functional switch of GDNA which was found to significantly affect the fluorescence of the in-situ synthesized DNA/silver nanocluster (DNA-AgNC) in homogeneous solution. We envisioned that the allosteric interaction between GDNA and DNA-AgNC would be possible to be used for screening telomere-binding ligands. A unimolecular probe (12C5TG) is ingeniously designed consisting of three contiguous DNA elements: G-rich telomeric DNA (GDNA) as molecular recognition sequence, T-rich DNA as linker and C-rich DNA as template of DNA-AgNC. The quantum yield and stability of 12C5TG-AgNC is greatly improved because the nearby deoxyguanosines tended to protect DNA/AgNC against oxidation. However, in the presence of ligands, the formation of G-quadruplex obviously quenched the fluorescence of DNA-AgNC. By taking full advantage of intramolecular allosteric effect, telomere-binding ligands were selectively and label-free screened by using deoxyguanines and G-quadruplex as natural fluorescence enhancer and quencher of DNA-AgNC respectively. Therefore, the functional switching of G-rich structure offers a cost-effective, facile and reliable way to screen drugs, which holds a great potential in bioanalysis as well.

Nanosizing a Metal-Organic Framework Enzyme Carrier for Accelerating Nerve Agent Hydrolysis

DTIC Science & Technology

2016-10-05

Previously, biodegradable liposome nano- carriers have been shown to be effective at providing functionally significant amounts of highly purified enzymes in...AlexaFluor-647 dye was purchased from Life Technologies (Thermo Fisher Scientific). Methyl 6-(pinacolboryl)-2-naphthoate was synthesized using a published...Hitachi) and PXRD (Smartlab, Rigaku). Labeling OPAA with Fluorescent Dye . AlexaFluor-647-labeled OPAA (OPAA647) was prepared by reacting OPAA (0.5

LIFEGUARD proteins support plant colonization by biotrophic powdery mildew fungi.

PubMed

Weis, Corina; Hückelhoven, Ralph; Eichmann, Ruth

2013-09-01

Pathogenic microbes manipulate eukaryotic cells during invasion and target plant proteins to achieve host susceptibility. BAX INHIBITOR-1 (BI-1) is an endoplasmic reticulum-resident cell death suppressor in plants and animals and is required for full susceptibility of barley to the barley powdery mildew fungus Blumeria graminis f.sp. hordei. LIFEGUARD (LFG) proteins resemble BI-1 proteins in terms of predicted membrane topology and cell-death-inhibiting function in metazoans, but display clear sequence-specific distinctions. This work shows that barley (Hordeum vulgare L.) and Arabidopsis thaliana genomes harbour five LFG genes, HvLFGa-HvLFGe and AtLFG1-AtLFG5, whose functions are largely uncharacterized. As observed for HvBI-1, single-cell overexpression of HvLFGa supports penetration success of B. graminis f.sp. hordei into barley epidermal cells, while transient-induced gene silencing restricts it. In penetrated barley epidermal cells, a green fluorescent protein-tagged HvLFGa protein accumulates at the site of fungal entry, around fungal haustoria and in endosomal or vacuolar membranes. The data further suggest a role of LFG proteins in plant-powdery mildew interactions in both monocot and dicot plants, because stable overexpression or knockdown of AtLFG1 or AtLFG2 also support or delay development of the powdery mildew fungus Erysiphe cruciferarum on the respective Arabidopsis mutants. Together, this work has identified new modulators of plant-powdery mildew interactions, and the data further support functional similarities between BI-1 and LFG proteins beyond cell death regulation.

LIFEGUARD proteins support plant colonization by biotrophic powdery mildew fungi

PubMed Central

Weis, Corina; Hückelhoven, Ralph; Eichmann, Ruth

2013-01-01

Pathogenic microbes manipulate eukaryotic cells during invasion and target plant proteins to achieve host susceptibility. BAX INHIBITOR-1 (BI-1) is an endoplasmic reticulum-resident cell death suppressor in plants and animals and is required for full susceptibility of barley to the barley powdery mildew fungus Blumeria graminis f.sp. hordei. LIFEGUARD (LFG) proteins resemble BI-1 proteins in terms of predicted membrane topology and cell-death-inhibiting function in metazoans, but display clear sequence-specific distinctions. This work shows that barley (Hordeum vulgare L.) and Arabidopsis thaliana genomes harbour five LFG genes, HvLFGa–HvLFGe and AtLFG1–AtLFG5, whose functions are largely uncharacterized. As observed for HvBI-1, single-cell overexpression of HvLFGa supports penetration success of B. graminis f.sp. hordei into barley epidermal cells, while transient-induced gene silencing restricts it. In penetrated barley epidermal cells, a green fluorescent protein-tagged HvLFGa protein accumulates at the site of fungal entry, around fungal haustoria and in endosomal or vacuolar membranes. The data further suggest a role of LFG proteins in plant–powdery mildew interactions in both monocot and dicot plants, because stable overexpression or knockdown of AtLFG1 or AtLFG2 also support or delay development of the powdery mildew fungus Erysiphe cruciferarum on the respective Arabidopsis mutants. Together, this work has identified new modulators of plant–powdery mildew interactions, and the data further support functional similarities between BI-1 and LFG proteins beyond cell death regulation. PMID:23888068

A label-free fluorescent aptamer sensor based on regulation of malachite green fluorescence

PubMed Central

Xu, Weichen; Lu, Yi

2009-01-01

We report a label-free fluorescent aptamer sensor for adenosine based on the regulation of malachite green (MG) fluorescence, with comparable sensitivity and selectivity to other labeled adenosine aptamer-based sensors. The sensor consists of free MG, an aptamer strand containing an adenosine aptamer next to an MG aptamer, and a bridging strand that partially hybridizes to the aptamer strand. Such a hybridization prevents MG from binding to MG aptamer, resulting in low fluorescence of MG in the absence of adenosine. Addition of adenosine causes the adenosine aptamer to bind adenosine, weakening the hybridization of the aptamer strand with the bridging strand, making it possible for MG to bind to the aptamer strand and exhibits high fluorescence intensity. Since this design is based purely on nucleic acid hybridization, it can be generally applied to other aptamers for the label-free detection of a broad range of analytes. PMID:20017558

Interrogating Surface Functional Group Heterogeneity of Activated Thermoplastics Using Super-Resolution Fluorescence Microscopy.

PubMed

ONeil, Colleen E; Jackson, Joshua M; Shim, Sang-Hee; Soper, Steven A

2016-04-05

We present a novel approach for characterizing surfaces utilizing super-resolution fluorescence microscopy with subdiffraction limit spatial resolution. Thermoplastic surfaces were activated by UV/O3 or O2 plasma treatment under various conditions to generate pendant surface-confined carboxylic acids (-COOH). These surface functional groups were then labeled with a photoswitchable dye and interrogated using single-molecule, localization-based, super-resolution fluorescence microscopy to elucidate the surface heterogeneity of these functional groups across the activated surface. Data indicated nonuniform distributions of these functional groups for both COC and PMMA thermoplastics with the degree of heterogeneity being dose dependent. In addition, COC demonstrated relative higher surface density of functional groups compared to PMMA for both UV/O3 and O2 plasma treatment. The spatial distribution of -COOH groups secured from super-resolution imaging were used to simulate nonuniform patterns of electroosmotic flow in thermoplastic nanochannels. Simulations were compared to single-particle tracking of fluorescent nanoparticles within thermoplastic nanoslits to demonstrate the effects of surface functional group heterogeneity on the electrokinetic transport process.

Permeabilization Activated Reduction in Fluorescence (PARF): a novel method to measure kinetics of protein interactions with intracellular structures

PubMed Central

Singh, Pali P.; Hawthorne, Jenci L.; Davis, Christie A.; Quintero, Omar A.

2016-01-01

Understanding kinetic information is fundamental in understanding biological function. Advanced imaging technologies have fostered the development of kinetic analyses in cells. We have developed Permeabilization Activated Reduction in Fluorescence (PARF) analysis for determination of apparent t1/2 and immobile fraction, describing the dissociation of a protein of interest from intracellular structures. To create conditions where dissociation events are observable, cells expressing a fluorescently-tagged protein are permeabilized with digitonin, diluting the unbound protein into the extracellular media. As the media volume is much larger than the cytosolic volume, the concentration of the unbound pool decreases drastically, shifting the system out of equilibrium--favoring dissociation events. Loss of bound protein is observed as loss of fluorescence from intracellular structures and can be fit to an exponential decay. We compared PARF dissociation kinetics with previously published equilibrium kinetics as determined by FRAP. PARF dissociation rates agreed with the equilibrium-based FRAP analysis predictions of the magnitude of those rates. When used to investigate binding kinetics of a panel of cytoskeletal proteins, PARF analysis revealed that filament stabilization resulted in slower fluorescence loss. Additionally, commonly used “general” F-actin labels display differences in kinetic properties, suggesting that not all fluorescently-tagged actin labels interact with the actin network in the same way. We also observed differential dissociation kinetics for GFP-VASP depending on which cellular structure was being labeled. These results demonstrate that PARF analysis of non-equilibrium systems reveals kinetic information without the infrastructure investment required for other quantitative approaches such as FRAP, photoactivation, or in vitro reconstitution assays. PMID:27126922

Near-infrared squaraine dyes for fluorescence enhanced surface assay

PubMed Central

Matveeva, Evgenia G.; Terpetschnig, Ewald A.; Stevens, Megan; Patsenker, Leonid; Kolosova, Olga S.; Gryczynski, Zygmunt; Gryczynski, Ignacy

2009-01-01

Commercially available, near-infrared fluorescent squaraine dyes (Seta-635 and Seta-670) were covalently bound to antibodies and employed insurface enhanced immunoassay. From fluorescence intensity and lifetime changes determined for a surface which had been coated with silver nanoparticles as well as a non-coated glass surface, both labelled compounds exhibited a 15 to 20-fold enhancement of fluorescence on the silver coated surface compared to that achieved on the non-coated surface. In addition, the fluorescence lifetime changes drastically for both labels in the case of silver-coated surfaces. The fluorescence signal enhancement obtained for the two dyes was greater than that previously recorded for Rhodamine Red-X and AlexaFluor-647 labels. PMID:20046935

Electron detachment dissociation of fluorescently labeled sialylated oligosaccharides.

PubMed

Zhou, Wen; Håkansson, Kristina

2011-12-01

We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared with infrared multiphoton dissociation. Neutral losses and satellite ions such as C-2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA-labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared with 2-AA-labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous neuron- and astrocyte-specific fluorescent marking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schulze, Wiebke; Hayata-Takano, Atsuko; Kamo, Toshihiko

2015-03-27

Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignmentmore » of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains. - Highlights: • We develop a method for the specific fluorescent labeling of neurons and astrocytes. • Neuron-specific labeling is achieved using Scg10 and synapsin promoters. • Astrocyte-specific labeling is generated using the minimal GFAP promoter. • Nuclear localization of fluorescent proteins is achieved with histone 2B protein.« less

Chemical biology-based approaches on fluorescent labeling of proteins in live cells.

PubMed

Jung, Deokho; Min, Kyoungmi; Jung, Juyeon; Jang, Wonhee; Kwon, Youngeun

2013-05-01

Recently, significant advances have been made in live cell imaging owing to the rapid development of selective labeling of proteins in vivo. Green fluorescent protein (GFP) was the first example of fluorescent reporters genetically introduced to protein of interest (POI). While GFP and various types of engineered fluorescent proteins (FPs) have been actively used for live cell imaging for many years, the size and the limited windows of fluorescent spectra of GFP and its variants set limits on possible applications. In order to complement FP-based labeling methods, alternative approaches that allow incorporation of synthetic fluorescent probes to target POIs were developed. Synthetic fluorescent probes are smaller than fluorescent proteins, often have improved photochemical properties, and offer a larger variety of colors. These synthetic probes can be introduced to POIs selectively by numerous approaches that can be largely categorized into chemical recognition-based labeling, which utilizes metal-chelating peptide tags and fluorophore-carrying metal complexes, and biological recognition-based labeling, such as (1) specific non-covalent binding between an enzyme tag and its fluorophore-carrying substrate, (2) self-modification of protein tags using substrate variants conjugated to fluorophores, (3) enzymatic reaction to generate a covalent binding between a small molecule substrate and a peptide tag, and (4) split-intein-based C-terminal labeling of target proteins. The chemical recognition-based labeling reaction often suffers from compromised selectivity of metal-ligand interaction in the cytosolic environment, consequently producing high background signals. Use of protein-substrate interactions or enzyme-mediated reactions generally shows improved specificity but each method has its limitations. Some examples are the presence of large linker protein, restriction on the choice of introducible probes due to the substrate specificity of enzymes, and competitive reaction mediated by an endogenous analogue of the introduced protein tag. These limitations have been addressed, in part, by the split-intein-based labeling approach, which introduces fluorescent probes with a minimal size (~4 amino acids) peptide tag. In this review, the advantages and the limitations of each labeling method are discussed.

A low-cost method for visible fluorescence imaging.

PubMed

Tarver, Crissy L; Pusey, Marc

2017-12-01

A wide variety of crystallization solutions are screened to establish conditions that promote the growth of a diffraction-quality crystal. Screening these conditions requires the assessment of many crystallization plates for the presence of crystals. Automated systems for screening and imaging are very expensive. A simple approach to imaging trace fluorescently labeled protein crystals in crystallization plates has been devised, and can be implemented at a cost as low as $50. The proteins β-lactoglobulin B, trypsin and purified concanavalin A (ConA) were trace fluorescently labeled using three different fluorescent probes: Cascade Yellow (CY), Carboxyrhodamine 6G (CR) and Pacific Blue (PB). A crystallization screening plate was set up using β-lactoglobulin B labeled with CR, trypsin labeled with CY, ConA labeled with each probe, and a mixture consisting of 50% PB-labeled ConA and 50% CR-labeled ConA. The wells of these plates were imaged using a commercially available macro-imaging lens attachment for smart devices that have a camera. Several types of macro lens attachments were tested with smartphones and tablets. Images with the highest quality were obtained with an iPhone 6S and an AUKEY Ora 10× macro lens. Depending upon the fluorescent probe employed and its Stokes shift, a light-emitting diode or a laser diode was used for excitation. An emission filter was used for the imaging of protein crystals labeled with CR and crystals with two-color fluorescence. This approach can also be used with microscopy systems commonly used to observe crystallization plates.

Embedding and Chemical Reactivation of Green Fluorescent Protein in the Whole Mouse Brain for Optical Micro-Imaging

PubMed Central

Gang, Yadong; Zhou, Hongfu; Jia, Yao; Liu, Ling; Liu, Xiuli; Rao, Gong; Li, Longhui; Wang, Xiaojun; Lv, Xiaohua; Xiong, Hanqing; Yang, Zhongqin; Luo, Qingming; Gong, Hui; Zeng, Shaoqun

2017-01-01

Resin embedding has been widely applied to fixing biological tissues for sectioning and imaging, but has long been regarded as incompatible with green fluorescent protein (GFP) labeled sample because it reduces fluorescence. Recently, it has been reported that resin-embedded GFP-labeled brain tissue can be imaged with high resolution. In this protocol, we describe an optimized protocol for resin embedding and chemical reactivation of fluorescent protein labeled mouse brain, we have used mice as experiment model, but the protocol should be applied to other species. This method involves whole brain embedding and chemical reactivation of the fluorescent signal in resin-embedded tissue. The whole brain embedding process takes a total of 7 days. The duration of chemical reactivation is ~2 min for penetrating 4 μm below the surface in the resin-embedded brain. This protocol provides an efficient way to prepare fluorescent protein labeled sample for high-resolution optical imaging. This kind of sample was demonstrated to be imaged by various optical micro-imaging methods. Fine structures labeled with GFP across a whole brain can be detected. PMID:28352214

Selective dye-labeling of newly synthesized proteins in bacterial cells.

PubMed

Beatty, Kimberly E; Xie, Fang; Wang, Qian; Tirrell, David A

2005-10-19

We describe fluorescence labeling of newly synthesized proteins in Escherichia coli cells by means of Cu(I)-catalyzed cycloaddition between alkynyl amino acid side chains and the fluorogenic dye 3-azido-7-hydroxycoumarin. The method involves co-translational labeling of proteins by the non-natural amino acids homopropargylglycine (Hpg) or ethynylphenylalanine (Eth) followed by treatment with the dye. As a demonstration, the model protein barstar was expressed and treated overnight with Cu(I) and 3-azido-7-hydroxycoumarin. Examination of treated cells by confocal microscopy revealed that strong fluorescence enhancement was observed only for alkynyl-barstar treated with Cu(I) and the reactive dye. The cellular fluorescence was punctate, and gel electrophoresis confirmed that labeled barstar was localized in inclusion bodies. Other proteins showed little fluorescence. Examination of treated cells by fluorimetry demonstrated that cultures supplemented with Eth or Hpg showed an 8- to 14-fold enhancement in fluorescence intensity after labeling. Addition of a protein synthesis inhibitor reduced the emission intensity to levels slightly above background, confirming selective labeling of newly synthesized proteins in the bacterial cell.

Dual-Labeled Near-Infrared/99mTc Imaging Probes Using PAMAM-Coated Silica Nanoparticles for the Imaging of HER2-Expressing Cancer Cells

PubMed Central

Yamaguchi, Haruka; Tsuchimochi, Makoto; Hayama, Kazuhide; Kawase, Tomoyuki; Tsubokawa, Norio

2016-01-01

We sought to develop dual-modality imaging probes using functionalized silica nanoparticles to target human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer cells and achieve efficient target imaging of HER2-expressing tumors. Polyamidoamine-based functionalized silica nanoparticles (PCSNs) for multimodal imaging were synthesized with near-infrared (NIR) fluorescence (indocyanine green (ICG)) and technetium-99m (99mTc) radioactivity. Anti-HER2 antibodies were bound to the labeled PCSNs. These dual-imaging probes were tested to image HER2-overexpressing breast carcinoma cells. In vivo imaging was also examined in breast tumor xenograft models in mice. SK-BR3 (HER2 positive) cells were imaged with stronger NIR fluorescent signals than that in MDA-MB231 (HER2 negative) cells. The increased radioactivity of the SK-BR3 cells was also confirmed by phosphor imaging. NIR images showed strong fluorescent signals in the SK-BR3 tumor model compared to muscle tissues and the MDA-MB231 tumor model. Automatic well counting results showed increased radioactivity in the SK-BR3 xenograft tumors. We developed functionalized silica nanoparticles loaded with 99mTc and ICG for the targeting and imaging of HER2-expressing cells. The dual-imaging probes efficiently imaged HER2-overexpressing cells. Although further studies are needed to produce efficient isotope labeling, the results suggest that the multifunctional silica nanoparticles are a promising vehicle for imaging specific components of the cell membrane in a dual-modality manner. PMID:27399687

Dual-Labeled Near-Infrared/(99m)Tc Imaging Probes Using PAMAM-Coated Silica Nanoparticles for the Imaging of HER2-Expressing Cancer Cells.

PubMed

Yamaguchi, Haruka; Tsuchimochi, Makoto; Hayama, Kazuhide; Kawase, Tomoyuki; Tsubokawa, Norio

2016-07-07

We sought to develop dual-modality imaging probes using functionalized silica nanoparticles to target human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer cells and achieve efficient target imaging of HER2-expressing tumors. Polyamidoamine-based functionalized silica nanoparticles (PCSNs) for multimodal imaging were synthesized with near-infrared (NIR) fluorescence (indocyanine green (ICG)) and technetium-99m ((99m)Tc) radioactivity. Anti-HER2 antibodies were bound to the labeled PCSNs. These dual-imaging probes were tested to image HER2-overexpressing breast carcinoma cells. In vivo imaging was also examined in breast tumor xenograft models in mice. SK-BR3 (HER2 positive) cells were imaged with stronger NIR fluorescent signals than that in MDA-MB231 (HER2 negative) cells. The increased radioactivity of the SK-BR3 cells was also confirmed by phosphor imaging. NIR images showed strong fluorescent signals in the SK-BR3 tumor model compared to muscle tissues and the MDA-MB231 tumor model. Automatic well counting results showed increased radioactivity in the SK-BR3 xenograft tumors. We developed functionalized silica nanoparticles loaded with (99m)Tc and ICG for the targeting and imaging of HER2-expressing cells. The dual-imaging probes efficiently imaged HER2-overexpressing cells. Although further studies are needed to produce efficient isotope labeling, the results suggest that the multifunctional silica nanoparticles are a promising vehicle for imaging specific components of the cell membrane in a dual-modality manner.

Fluorescence labeling of colloidal core-shell particles with defined isoelectric points for in vitro studies.

PubMed

Daberkow, Timo; Meder, Fabian; Treccani, Laura; Schowalter, Marco; Rosenauer, Andreas; Rezwan, Kurosch

2012-02-01

In the light of in vitro nanotoxicological studies fluorescence labeling has become standard for particle localization within the cell environment. However, fluorescent labeling is also known to significantly alter the particle surface chemistry and therefore potentially affect the outcome of cell studies. Hence, fluorescent labeling is ideally carried out without changing, for example, the isoelectric point. A simple and straightforward method for obtaining fluorescently labeled spherical metal oxide particles with well-defined isoelectric points and a narrow size distribution is presented in this study. Spherical amorphous silica (SiO2, 161 nm diameter) particles were used as the substrate material and were coated with silica, alumina (Al2O3), titania (TiO2), or zirconia (ZrO2) using sol-gel chemistry. Fluorescent labeling was achieved by directly embedding rhodamine 6G dye in the coating matrix without affecting the isoelectric point of the metal oxide coatings. The coating quality was confirmed by high resolution transmission electron microscopy, energy filtered transmission electron microscopy and electrochemical characterization. The coatings were proven to be stable for at least 240 h under different pH conditions. The well-defined fluorescent particles can be directly used for biomedical investigations, e.g. elucidation of particle-cell interactions in vitro. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Bi-fluorescence imaging for estimating accurately the nuclear condition of Rhizoctonia spp.

USDA-ARS?s Scientific Manuscript database

Aims: To simplify the determination of the nuclear condition of the pathogenic Rhizoctonia, which currently needs to be performed either using two fluorescent dyes, thus is more costly and time-consuming, or using only one fluorescent dye, and thus less accurate. Methods and Results: A red primary ...

Novel image processing method study for a label-free optical biosensor

NASA Astrophysics Data System (ADS)

Yang, Chenhao; Wei, Li'an; Yang, Rusong; Feng, Ying

2015-10-01

Optical biosensor is generally divided into labeled type and label-free type, the former mainly contains fluorescence labeled method and radioactive-labeled method, while fluorescence-labeled method is more mature in the application. The mainly image processing methods of fluorescent-labeled biosensor includes smooth filtering, artificial gridding and constant thresholding. Since some fluorescent molecules may influence the biological reaction, label-free methods have been the main developing direction of optical biosensors nowadays. The using of wider field of view and larger angle of incidence light path which could effectively improve the sensitivity of the label-free biosensor also brought more difficulties in image processing, comparing with the fluorescent-labeled biosensor. Otsu's method is widely applied in machine vision, etc, which choose the threshold to minimize the intraclass variance of the thresholded black and white pixels. It's capacity-constrained with the asymmetrical distribution of images as a global threshold segmentation. In order to solve the irregularity of light intensity on the transducer, we improved the algorithm. In this paper, we present a new image processing algorithm based on a reflectance modulation biosensor platform, which mainly comprises the design of sliding normalization algorithm for image rectification and utilizing the improved otsu's method for image segmentation, in order to implement automatic recognition of target areas. Finally we used adaptive gridding method extracting the target parameters for analysis. Those methods could improve the efficiency of image processing, reduce human intervention, enhance the reliability of experiments and laid the foundation for the realization of high throughput of label-free optical biosensors.

Subcellular localization of proteins in the anaerobic sulfate reducer Desulfovibrio vulgaris via SNAP-tag labeling and photoconversion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gorur, A.; Leung, C. M.; Jorgens, D.

2010-06-01

Systems Biology studies the temporal and spatial 3D distribution of macromolecular complexes with the aim that such knowledge will allow more accurate modeling of biological function and will allow mathematical prediction of cellular behavior. However, in order to accomplish accurate modeling precise knowledge of spatial 3D organization and distribution inside cells is necessary. And while a number of macromolecular complexes may be identified by its 3D structure and molecular characteristics alone, the overwhelming number of proteins will need to be localized using a reporter tag. GFP and its derivatives (XFPs) have been traditionally employed for subcelllar localization using photoconversion approaches,more » but this approach cannot be taken for obligate anaerobic bacteria, where the intolerance towards oxygen prevents XFP approaches. As part of the GTL-funded PCAP project (now ENIGMA) genetic tools have been developed for the anaerobe sulfate reducer Desulfovibrio vulgaris that allow the high-throughput generation of tagged-protein mutant strains, with a focus on the commercially available SNAP-tag cell system (New England Biolabs, Ipswich, MA), which is based on a modified O6-alkylguanine-DNA alkyltransferase (AGT) tag, that has a dead-end reaction with a modified O6-benzylguanine (BG) derivative and has been shown to function under anaerobic conditions. After initial challenges with respect to variability, robustness and specificity of the labeling signal we have optimized the labeling. Over the last year, as a result of the optimized labeling protocol, we now obtain robust labeling of 20 out of 31 SNAP strains. Labeling for 13 strains were confirmed at least five times. We have also successfully performed photoconversion on 5 of these 13 strains, with distinct labeling patterns for different strains. For example, DsrC robustly localizes to the periplasmic portion of the inner membrane, where as a DNA-binding protein localizes to the center of the cell, where the chromosome is located. Two other proteins - Thiosulfate reductase and ATP binding protein were found to be cytoplasmically distributed, whereas a molybdenum transporter was found to locate to the cell periphery. We judge labeling outcome by (1) SDS gel electrophoresis, followed by direct fluorescence imaging of the gel to address specificity of labeling/confirm expected molecular weight, and subsequent Coomassie analysis to ensure comparable protein levels (2) fluorescence intensity of culture by plate reader for statistical sampling (after adjustment for respective cell numbers) and (3) fluorescence microscopy for addressing cell-to-cell signal variation and potential localization patterns. All three assays were usually found to be consistent with one another. While we have been able to improve the efficacy of photoconversion by drastically reducing (eliminating) non-specific binding with our altered labeling protocol, we are currently working on reducing non-specific photoconversion reaction arising occasionally in non-labeled cells. In addition, we have confirmed the presence of SNAP tagged constructs in three recently cloned E.coli strains under promotor control, and are in the process of utilizing them for evaluating the sensitivity of the photoconversion protocol. Fluorescent Activated Cell Sorting was successfully applied to labeled E.coli cells containing SNAP tagged AtpA protein. Different batches of sorted cells, representing low and high labeling intensity, were re-grown and re-labeled and displayed a labeling efficiency similar to the starter culture, supporting the notion that cell-to-cell differences in labeling reflect difference in protein expression, rather then genetic differences.« less

Ballistic delivery of dyes for structural and functional studies of the nervous system

PubMed Central

Gan, Wen-Biao; Grutzendler, Jaime; Wong, Rachel O.; Lichtman, Jeff W.

2010-01-01

This chapter describes a detail protocol for rapid labeling of cells in a variety of preparations by means of particle-mediated ballistic (gene gun) delivery of fluorescent dyes. This method has been used for rapid labeling of cells with either lipid or water-soluble dyes in a variety of preparations. In particular, carbocyanine lipophilic dyes such as DiI have been used to obtain Golgi-like labeling of neurons and glia in fixed and live cell cultures, brain slices, as well as fixed post-mortem human brain. Water-soluble calcium indicators such as calcium green-1 dextran have been used to image calcium dynamics in living brain slices and retinal explants. This ballistic labeling technique is thus useful for studying the structure and function of neurons and glia in both living and fixed specimens. PMID:20147144

Regulation of WNT Signaling at the Neuromuscular Junction by the Immunoglobulin Superfamily Protein RIG-3 in Caenorhabditis elegans

PubMed Central

Pandey, Pratima; Bhardwaj, Ashwani; Babu, Kavita

2017-01-01

Perturbations in synaptic function could affect the normal behavior of an animal, making it important to understand the regulatory mechanisms of synaptic signaling. Previous work has shown that in Caenorhabditis elegans an immunoglobulin superfamily protein, RIG-3, functions in presynaptic neurons to maintain normal acetylcholine receptor levels at the neuromuscular junction (NMJ). In this study, we elucidate the molecular and functional mechanism of RIG-3. We demonstrate by genetic and BiFC (Bi-molecular Fluorescence Complementation) assays that presynaptic RIG-3 functions by directly interacting with the immunoglobulin domain of the nonconventional Wnt receptor, ROR receptor tyrosine kinase (RTK), CAM-1, which functions in postsynaptic body-wall muscles. This interaction in turn inhibits Wnt/LIN-44 signaling through the ROR/CAM-1 receptor, and allows for maintenance of normal acetylcholine receptor, AChR/ACR-16, levels at the neuromuscular synapse. Further, this work reveals that RIG-3 and ROR/CAM-1 function through the β-catenin/HMP-2 at the NMJ. Taken together, our results demonstrate that RIG-3 functions as an inhibitory molecule of the Wnt/LIN-44 signaling pathway through the RTK, CAM-1. PMID:28515212

Fluorescently labeled dengue viruses as probes to identify antigen-specific memory B cells by multiparametric flow cytometry.

PubMed

Woda, Marcia; Mathew, Anuja

2015-01-01

Low frequencies of memory B cells in the peripheral blood make it challenging to measure the functional and phenotypic characteristics of this antigen experienced subset of B cells without in vitro culture. To date, reagents are lacking to measure ex vivo frequencies of dengue virus (DENV)-specific memory B cells. We wanted to explore the possibility of using fluorescently labeled DENV as probes to detect antigen-specific memory B cells in the peripheral blood of DENV immune individuals. Alexa Fluor dye-labeled DENV yielded viable virus that could be stored at -80°C for long periods of time. Using a careful gating strategy and methods to decrease non-specific binding, we were able to identify a small frequency of B cells from dengue immune individuals that bound labeled DENV. Sorted DENV(+) B cells from immune, but not naïve donors secreted antibodies that bound DENV after in vitro stimulation. Overall, Alexa Fluor dye-labeled DENVs are useful reagents to enable the detection and characterization of memory B cells in DENV immune individuals. Copyright © 2014 Elsevier B.V. All rights reserved.

Visualization of the Interaction between the Precursors of VPg, the Viral Protein Linked to the Genome of Turnip Mosaic Virus, and the Translation Eukaryotic Initiation Factor iso 4E In Planta▿

PubMed Central

Beauchemin, Chantal; Boutet, Nathalie; Laliberté, Jean-François

2007-01-01

The RNA genome of Turnip mosaic virus is covalently linked at its 5′ end to a viral protein known as VPg. This protein binds to the translation eukaryotic initiation factor iso 4E [eIF(iso)4E]. This interaction has been shown to be important for virus infection, although its exact biological function(s) has not been elucidated. In this study, we investigated the subcellular site of the VPg-eIF(iso)4E interaction using bimolecular fluorescence complementation (BiFC). As a first step, eIF(iso)4E, 6K-VPg-Pro, and VPg-Pro were expressed as full-length green fluorescent protein (GFP) fusions in Nicotiana benthamiana, and their subcellular localizations were visualized by confocal microscopy. eIF(iso)4E was predominantly associated with the endoplasmic reticulum (ER), and VPg-Pro was observed in the nucleus and possibly the nucleolus, while 6K-VPg-Pro-GFP induced the formation of cytoplasmic vesicles budding from the ER. In BiFC experiments, reconstituted green fluorescence was observed throughout the nucleus, with a preferential accumulation in subnuclear structures when the GFP split fragments were fused to VPg-Pro and eIF(iso)4E. On the other hand, the interaction of 6K-VPg-Pro with eIF(iso)4E was observed in cytoplasmic vesicles embedded in the ER. These data suggest that the association of VPg with the translation factor might be needed for two different functions, depending of the VPg precursor involved in the interaction. VPg-Pro interaction with eIF(iso)4E may be involved in perturbing normal cellular functions, while 6K-VPg-Pro interaction with the translation factor may be needed for viral RNA translation and/or replication. PMID:17079311

Introducing Ratiometric Fluorescence to MnO2 Nanosheet-Based Biosensing: A Simple, Label-Free Ratiometric Fluorescent Sensor Programmed by Cascade Logic Circuit for Ultrasensitive GSH Detection.

PubMed

Fan, Daoqing; Shang, Changshuai; Gu, Wenling; Wang, Erkang; Dong, Shaojun

2017-08-09

Glutathione (GSH) plays crucial roles in various biological functions, the level alterations of which have been linked to varieties of diseases. Herein, we for the first time expanded the application of oxidase-like property of MnO 2 nanosheet (MnO 2 NS) to fluorescent substrates of peroxidase. Different from previously reported fluorescent quenching phenomena, we found that MnO 2 NS could not only largely quench the fluorescence of highly fluorescent Scopoletin (SC) but also surprisingly enhance that of nonfluorescent Amplex Red (AR) via oxidation reaction. If MnO 2 NS is premixed with GSH, it will be reduced to Mn 2+ and lose the oxidase-like property, accompanied by subsequent increase in SC's fluorescence and decrease in AR's. On the basis of the above mechanism, we construct the first MnO 2 NS-based ratiometric fluorescent sensor for ultrasensitive and selective detection of GSH. Notably, this ratiometric sensor is programmed by the cascade logic circuit (an INHIBIT gate cascade with a 1 to 2 decoder). And a linear relationship between ratiometric fluorescent intensities of the two substrates and logarithmic values of GSH's concentrations is obtained. The detection limit of GSH is as low as 6.7 nM, which is much lower than previous ratiometric fluorescent sensors, and the lowest MnO 2 NS-based fluorescent GSH sensor reported so far. Furthermore, this sensor is simple, label-free, and low-cost; it also presents excellent applicability in human serum samples.

Post-transcriptional labeling by using Suzuki-Miyaura cross-coupling generates functional RNA probes.

PubMed

Walunj, Manisha B; Tanpure, Arun A; Srivatsan, Seergazhi G

2018-06-20

Pd-catalyzed C-C bond formation, an important vertebra in the spine of synthetic chemistry, is emerging as a valuable chemoselective transformation for post-synthetic functionalization of biomacromolecules. While methods are available for labeling protein and DNA, development of an analogous procedure to label RNA by cross-coupling reactions remains a major challenge. Herein, we describe a new Pd-mediated RNA oligonucleotide (ON) labeling method that involves post-transcriptional functionalization of iodouridine-labeled RNA transcripts by using Suzuki-Miyaura cross-coupling reaction. 5-Iodouridine triphosphate (IUTP) is efficiently incorporated into RNA ONs at one or more sites by T7 RNA polymerase. Further, using a catalytic system made of Pd(OAc)2 and 2-aminopyrimidine-4,6-diol (ADHP) or dimethylamino-substituted ADHP (DMADHP), we established a modular method to functionalize iodouridine-labeled RNA ONs in the presence of various boronic acid and ester substrates under very mild conditions (37°C and pH 8.5). This method is highly chemoselective, and offers direct access to RNA ONs labeled with commonly used fluorescent and affinity tags and new fluorogenic environment-sensitive nucleoside probes in a ligand-controlled stereoselective fashion. Taken together, this simple approach of generating functional RNA ON probes by Suzuki-Miyaura coupling will be a very important addition to the resources and tools available for analyzing RNA motifs.

Lack of evidence for the presence of emerging HoBi-like viruses in North American fetal bovine serum lots.

USDA-ARS?s Scientific Manuscript database

The detection of HoBi-like virus in fetal bovine serum (FBS) labeled as United States of America (USA) origin, but packaged in Europe, raised concerns that HoBi-like virus may have entered the USA. In this study, 90 lots of FBS originating in North America (NA) were screened for pestivirus antigen ...

PEGylation of Concanavalin A to decrease nonspecific interactions in a fluorescent glucose sensor

NASA Astrophysics Data System (ADS)

Abraham, Alexander A.; Cummins, Brian M.; Locke, Andrea K.; Grunlan, Melissa A.; Coté, Gerard L.

2014-02-01

The ability of people with diabetes to both monitor and regulate blood sugar levels is limited by the conventional "finger-prick" test that provides intermittent, single point measurements. Toward the development of a continuous glucose monitoring (CGM) system, the lectin, Concanavalin A (ConA), has been utilized as a component in a Förster resonance energy transfer (FRET), competitive glucose binding assay. Recently, to avoid reversibility problems associated with ConA aggregation, a suitable competing ligand labeled with 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) has been engineered. However, its ability to function as part of a glucose sensing assay is compromised due to the negative charge (at physiological pH) of native ConA that gives rise to non-specific binding with other ConA groups as well as with electrostatically charged assay-delivery carriers. To minimize these undesirable interactions, we have conjugated ConA with monomethoxy-poly(ethylene glycol) (mPEG) (i.e. "PEGylation"). In this preliminary research, fluorescently-labeled ConA was successfully PEGylated with mPEG-Nhydroxylsuccinimide( succinimidyl carbonate) (mPEG-NHS(SC)). The FRET response of APTS-labeled competing ligand (donor) conveyed an increase in the fluorescence intensity with increasing glucose concentrations.

Micro-Droplet Detection Method for Measuring the Concentration of Alkaline Phosphatase-Labeled Nanoparticles in Fluorescence Microscopy

PubMed Central

Li, Rufeng; Wang, Yibei; Xu, Hong; Fei, Baowei; Qin, Binjie

2017-01-01

This paper developed and evaluated a quantitative image analysis method to measure the concentration of the nanoparticles on which alkaline phosphatase (AP) was immobilized. These AP-labeled nanoparticles are widely used as signal markers for tagging biomolecules at nanometer and sub-nanometer scales. The AP-labeled nanoparticle concentration measurement can then be directly used to quantitatively analyze the biomolecular concentration. Micro-droplets are mono-dispersed micro-reactors that can be used to encapsulate and detect AP-labeled nanoparticles. Micro-droplets include both empty micro-droplets and fluorescent micro-droplets, while fluorescent micro-droplets are generated from the fluorescence reaction between the APs adhering to a single nanoparticle and corresponding fluorogenic substrates within droplets. By detecting micro-droplets and calculating the proportion of fluorescent micro-droplets to the overall micro-droplets, we can calculate the AP-labeled nanoparticle concentration. The proposed micro-droplet detection method includes the following steps: (1) Gaussian filtering to remove the noise of overall fluorescent targets, (2) a contrast-limited, adaptive histogram equalization processing to enhance the contrast of weakly luminescent micro-droplets, (3) an red maximizing inter-class variance thresholding method (OTSU) to segment the enhanced image for getting the binary map of the overall micro-droplets, (4) a circular Hough transform (CHT) method to detect overall micro-droplets and (5) an intensity-mean-based thresholding segmentation method to extract the fluorescent micro-droplets. The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073. The detection method can be successfully applied to measure AP-labeled nanoparticle concentration in fluorescence microscopy. PMID:29160812

Micro-Droplet Detection Method for Measuring the Concentration of Alkaline Phosphatase-Labeled Nanoparticles in Fluorescence Microscopy.

PubMed

Li, Rufeng; Wang, Yibei; Xu, Hong; Fei, Baowei; Qin, Binjie

2017-11-21

This paper developed and evaluated a quantitative image analysis method to measure the concentration of the nanoparticles on which alkaline phosphatase (AP) was immobilized. These AP-labeled nanoparticles are widely used as signal markers for tagging biomolecules at nanometer and sub-nanometer scales. The AP-labeled nanoparticle concentration measurement can then be directly used to quantitatively analyze the biomolecular concentration. Micro-droplets are mono-dispersed micro-reactors that can be used to encapsulate and detect AP-labeled nanoparticles. Micro-droplets include both empty micro-droplets and fluorescent micro-droplets, while fluorescent micro-droplets are generated from the fluorescence reaction between the APs adhering to a single nanoparticle and corresponding fluorogenic substrates within droplets. By detecting micro-droplets and calculating the proportion of fluorescent micro-droplets to the overall micro-droplets, we can calculate the AP-labeled nanoparticle concentration. The proposed micro-droplet detection method includes the following steps: (1) Gaussian filtering to remove the noise of overall fluorescent targets, (2) a contrast-limited, adaptive histogram equalization processing to enhance the contrast of weakly luminescent micro-droplets, (3) an red maximizing inter-class variance thresholding method (OTSU) to segment the enhanced image for getting the binary map of the overall micro-droplets, (4) a circular Hough transform (CHT) method to detect overall micro-droplets and (5) an intensity-mean-based thresholding segmentation method to extract the fluorescent micro-droplets. The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073. The detection method can be successfully applied to measure AP-labeled nanoparticle concentration in fluorescence microscopy.

Correlated Fluorescence-Atomic Force Microscopy Studies of the Clathrin Mediated Endocytosis in SKMEL Cells

NASA Astrophysics Data System (ADS)

Smith, Steve; Hor, Amy; Luu, Anh; Kang, Lin; Scott, Brandon; Bailey, Elizabeth; Hoppe, Adam

Clathrin-mediated endocytosis is one of the central pathways for cargo transport into cells, and plays a major role in the maintenance of cellular functions, such as intercellular signaling, nutrient intake, and turnover of plasma membrane in cells. The clathrin-mediated endocytosis process involves invagination and formation of clathrin-coated vesicles. However, the biophysical mechanisms of vesicle formation are still debated. We investigate clathrin vesicle formation mechanisms through the utilization of tapping-mode atomic force microscopy for high resolution topographical imaging in neutral buffer solution of unroofed cells exposing the inner membrane, combined with fluorescence imaging to definitively label intracellular constituents with specific fluorescent fusion proteins (actin filaments labeled with green phalloidin-antibody and clathrin coated vesicles with the fusion protein Tq2) in SKMEL (Human Melanoma) cells. Results from our work are compared against dynamical polarized total internal fluorescence (TIRF), super-resolution photo-activated localization microscopy (PALM) and transmission electron microscopy (TEM) to draw conclusions regarding the prominent model of vesicle formation in clathrin-mediated endocytosis. Funding provided by NSF MPS/DMR/BMAT award # 1206908.

Efficient Site-Specific Labeling of Proteins via Cysteines

PubMed Central

Kim, Younggyu; Ho, Sam O.; Gassman, Natalie R.; Korlann, You; Landorf, Elizabeth V.; Collart, Frank R.; Weiss, Shimon

2011-01-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70–90%, and specificities are better than ~95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis. PMID:18275130

Efficient site-specific labeling of proteins via cysteines.

PubMed

Kim, Younggyu; Ho, Sam O; Gassman, Natalie R; Korlann, You; Landorf, Elizabeth V; Collart, Frank R; Weiss, Shimon

2008-03-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than approximately 95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.

DNA-Based Self-Assembly of Fluorescent Nanodiamonds.

PubMed

Zhang, Tao; Neumann, Andre; Lindlau, Jessica; Wu, Yuzhou; Pramanik, Goutam; Naydenov, Boris; Jelezko, Fedor; Schüder, Florian; Huber, Sebastian; Huber, Marinus; Stehr, Florian; Högele, Alexander; Weil, Tanja; Liedl, Tim

2015-08-12

As a step toward deterministic and scalable assembly of ordered spin arrays we here demonstrate a bottom-up approach to position fluorescent nanodiamonds (NDs) with nanometer precision on DNA origami structures. We have realized a reliable and broadly applicable surface modification strategy that results in DNA-functionalized and perfectly dispersed NDs that were then self-assembled in predefined geometries. With optical studies we show that the fluorescence properties of the nitrogen-vacancy color centers in NDs are preserved during surface modification and DNA assembly. As this method allows the nanoscale arrangement of fluorescent NDs together with other optically active components in complex geometries, applications based on self-assembled spin lattices or plasmon-enhanced spin sensors as well as improved fluorescent labeling for bioimaging could be envisioned.

In Silico Labeling: Predicting Fluorescent Labels in Unlabeled Images.

PubMed

Christiansen, Eric M; Yang, Samuel J; Ando, D Michael; Javaherian, Ashkan; Skibinski, Gaia; Lipnick, Scott; Mount, Elliot; O'Neil, Alison; Shah, Kevan; Lee, Alicia K; Goyal, Piyush; Fedus, William; Poplin, Ryan; Esteva, Andre; Berndl, Marc; Rubin, Lee L; Nelson, Philip; Finkbeiner, Steven

2018-04-19

Microscopy is a central method in life sciences. Many popular methods, such as antibody labeling, are used to add physical fluorescent labels to specific cellular constituents. However, these approaches have significant drawbacks, including inconsistency; limitations in the number of simultaneous labels because of spectral overlap; and necessary perturbations of the experiment, such as fixing the cells, to generate the measurement. Here, we show that a computational machine-learning approach, which we call "in silico labeling" (ISL), reliably predicts some fluorescent labels from transmitted-light images of unlabeled fixed or live biological samples. ISL predicts a range of labels, such as those for nuclei, cell type (e.g., neural), and cell state (e.g., cell death). Because prediction happens in silico, the method is consistent, is not limited by spectral overlap, and does not disturb the experiment. ISL generates biological measurements that would otherwise be problematic or impossible to acquire. Copyright © 2018 Elsevier Inc. All rights reserved.

Synthesis and preliminary biological evaluations of fluorescent or 149Promethium labeled Trastuzumab-polyethylenimine

DOE PAGES

Fitzsimmons, Jonathan; Nayak, Tapan; Cutler, Cathy; ...

2015-12-30

Radioimmunotherapy utilize a targeting antibody coupled to a therapeutic isotope to target and treat a tumor or disease. In this study we examine the synthesis and cell binding of a polymer scaffold containing a radiotherapeutic isotope and a targeting antibody. Methods: The multistep synthesis of a fluorescent or 149Promethium-labeled Trastuzumab-polyethyleneimine (PEI), Trastuzumab, or PEI is described. In vitro uptake, internalization and/or the binding affinity to the Her2/neu expressing human breast adenocarcinoma SKBr3 cells was investigated with the labeled compounds. Fluorescent-labeled Trastuzumab-PEI was internalized more into cells at 2 and 18 h than fluorescent-labeled Trastuzumab or PEI. The fluorescent-labeled Trastuzumab wasmore » concentrated on the cell surface at 2 and 18 h and the labeled PEI had minimal uptake. DOTA-PEI was prepared and contained an average of 16 chelates per PEI; the compound was radio-labeled with 149Promethium and conjugated to Trastuzumab. The purified 149Pm-DOTA-PEI-Trastuzumab had a radiochemical purity of 96.7% and a specific activity of 0.118 TBq/g. The compound demonstrated a dissociation constant for the Her2/neu receptor of 20.30 ± 6.91 nM. In conclusion, the results indicate the DOTA-PEI-Trastuzumab compound has potential as a targeted therapeutic carrier, and future in vivo studies should be performed.« less

In vivo Labeling of Constellations of Functionally Identified Neurons for Targeted in vitro Recordings

PubMed Central

Lien, Anthony D.; Scanziani, Massimo

2011-01-01

Relating the functional properties of neurons in an intact organism with their cellular and synaptic characteristics is necessary for a mechanistic understanding of brain function. However, while the functional properties of cortical neurons (e.g., tuning to sensory stimuli) are necessarily determined in vivo, detailed cellular and synaptic analysis relies on in vitro techniques. Here we describe an approach that combines in vivo calcium imaging (for functional characterization) with photo-activation of fluorescent proteins (for neuron labeling), thereby allowing targeted in vitro recording of multiple neurons with known functional properties. We expressed photo-activatable GFP rendered non-diffusible through fusion with a histone protein (H2B–PAGFP) in the mouse visual cortex to rapidly photo-label constellations of neurons in vivo at cellular and sub-cellular resolution using two-photon excitation. This photo-labeling method was compatible with two-photon calcium imaging of neuronal responses to visual stimuli, allowing us to label constellations of neurons with specific functional properties. Photo-labeled neurons were easily identified in vitro in acute brain slices and could be targeted for whole-cell recording. We also demonstrate that in vitro and in vivo image stacks of the same photo-labeled neurons could be registered to one another, allowing the exact in vivo response properties of individual neurons recorded in vitro to be known. The ability to perform in vitro recordings from neurons with known functional properties opens up exciting new possibilities for dissecting the cellular, synaptic, and circuit mechanisms that underlie neuronal function in vivo. PMID:22144948

Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis.

PubMed

Vedula, Pavan; Cruz, Lissette A; Gutierrez, Natasha; Davis, Justin; Ayee, Brian; Abramczyk, Rachel; Rodriguez, Alexis J

2016-06-30

Quantifying multi-molecular complex assembly in specific cytoplasmic compartments is crucial to understand how cells use assembly/disassembly of these complexes to control function. Currently, biophysical methods like Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy provide quantitative measurements of direct protein-protein interactions, while traditional biochemical approaches such as sub-cellular fractionation and immunoprecipitation remain the main approaches used to study multi-protein complex assembly/disassembly dynamics. In this article, we validate and quantify multi-protein adherens junction complex assembly in situ using light microscopy and Fluorescence Covariance Analysis. Utilizing specific fluorescently-labeled protein pairs, we quantified various stages of adherens junction complex assembly, the multiprotein complex regulating epithelial tissue structure and function following de novo cell-cell contact. We demonstrate: minimal cadherin-catenin complex assembly in the perinuclear cytoplasm and subsequent localization to the cell-cell contact zone, assembly of adherens junction complexes, acto-myosin tension-mediated anchoring, and adherens junction maturation following de novo cell-cell contact. Finally applying Fluorescence Covariance Analysis in live cells expressing fluorescently tagged adherens junction complex proteins, we also quantified adherens junction complex assembly dynamics during epithelial monolayer formation.

Factors Affecting the Quantification of Biomolecular Interactions by Fluorescence Cross-Correlation Spectroscopy

PubMed Central

Foo, Yong Hwee; Naredi-Rainer, Nikolaus; Lamb, Don C.; Ahmed, Sohail; Wohland, Thorsten

2012-01-01

Fluorescence cross-correlation spectroscopy (FCCS) is used to determine interactions and dissociation constants (Kds) of biomolecules. The determination of a Kd depends on the accurate measurement of the auto- and cross-correlation function (ACF and CCF) amplitudes. In the case of complete binding, the ratio of the CCF/ACF amplitudes is expected to be 1. However, measurements performed on tandem fluorescent proteins (FPs), in which two different FPs are linked, yield CCF/ACF amplitude ratios of ∼0.5 or less for different FCCS schemes. We use single wavelength FCCS and pulsed interleaved excitation FCCS to measure various tandem FPs constituted of different red and green FPs and determine the causes for this suboptimal ratio. The main causes for the reduced CCF/ACF amplitude ratio are differences in observation volumes for the different labels, the existence of dark FPs due to maturation problems, photobleaching, and to a lesser extent Förster (or fluorescence) resonance energy transfer between the labels. We deduce the fraction of nonfluorescent proteins for EGFP, mRFP, and mCherry as well as the differences in observation volumes. We use this information to correct FCCS measurements of the interaction of Cdc42, a small Rho-GTPase, with its effector IQGAP1 in live cell measurements to obtain a label-independent value for the Kd. PMID:22404940

Label-Free Carbon-Dots-Based Ratiometric Fluorescence pH Nanoprobes for Intracellular pH Sensing.

PubMed

Shangguan, Jingfang; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Liu, Jinquan; Tang, Jinlu; Yang, Xue; Huang, Jin

2016-08-02

Measuring pH in living cells is of great importance for better understanding cellular functions as well as providing pivotal assistance for early diagnosis of diseases. In this work, we report the first use of a novel kind of label-free carbon dots for intracellular ratiometric fluorescence pH sensing. By simple one-pot hydrothermal treatment of citric acid and basic fuchsin, the carbon dots showing dual emission bands at 475 and 545 nm under single-wavelength excitation were synthesized. It is demonstrated that the fluorescence intensities of the as-synthesized carbon dots at the two emissions are pH-sensitive simultaneously. The intensity ratio (I475 nm/I545 nm) is linear against pH values from 5.2 to 8.8 in buffer solution, affording the capability as ratiometric probes for intracellular pH sensing. It also displays that the carbon dots show excellent reversibility and photostability in pH measurements. With this nanoprobe, quantitative fluorescence imaging using the ratio of two emissions (I475 nm/I545 nm) for the detection of intracellular pH were successfully applied in HeLa cells. In contrast to most of the reported nanomaterials-based ratiometric pH sensors which rely on the attachment of additional dyes, these carbon-dots-based ratiometric probes are low in toxicity, easy to synthesize, and free from labels.

Enhancing Peripheral Nerve Regeneration with a Novel Drug-Delivering Nerve Conduit

DTIC Science & Technology

2015-10-01

data with the release of fluorescently labeled dextran that indicate the new device is sealed and able to release therapeutics in a controlled manner...accurately predict the release of fluorescently labeled dextran , with a similar diffusion coefficient to NGF, over a period of approximately 40 days...enables the release of NGF from the reservoir into the inner chamber. 7 Figure 2. Fluorescently labeled dextran (blue) was released from our PLGA nerve

Synthesis of γ-Phosphate-Labeled and Doubly Labeled Adenosine Triphosphate Analogs.

PubMed

Hacker, Stephan M; Welter, Moritz; Marx, Andreas

2015-03-09

This unit describes the synthesis of γ-phosphate-labeled and doubly labeled adenosine triphosphate (ATP) analogs and their characterization using the phosphodiesterase I from Crotalus adamanteus (snake venom phosphodiesterase; SVPD). In the key step of the synthesis, ATP or an ATP analog, bearing a linker containing a trifluoroacetamide group attached to the nucleoside, are modified with an azide-containing linker at the terminal phosphate using an alkylation reaction. Subsequently, different labels are introduced to the linkers by transformation of one functional group to an amine and coupling to an N-hydroxysuccinimide ester. Specifically, the Staudinger reaction of the azide is employed as a straightforward means to obtain an amine in the presence of various labels. Furthermore, the fluorescence characteristics of a fluorogenic, doubly labeled ATP analog are investigated following enzymatic cleavage by SVPD. Copyright © 2015 John Wiley & Sons, Inc.

Microbial detection in microfluidic devices through dual staining of quantum dots-labeled immunoassay and RNA hybridization.

PubMed

Zhang, Qing; Zhu, Liang; Feng, Hanhua; Ang, Simon; Chau, Fook Siong; Liu, Wen-Tso

2006-01-18

This paper reported the development of a microfludic device for the rapid detection of viable and nonviable microbial cells through dual labeling by fluorescent in situ hybridization (FISH) and quantum dots (QDs)-labeled immunofluorescent assay (IFA). The coin sized device consists of a microchannel and filtering pillars (gap=1-2 microm) and was demonstrated to effectively trap and concentrate microbial cells (i.e. Giardia lamblia). After sample injection, FISH probe solution and QDs-labeled antibody solution were sequentially pumped into the device to accelerate the fluorescent labeling reactions at optimized flow rates (i.e. 1 and 20 microL/min, respectively). After 2 min washing for each assay, the whole process could be finished within 30 min, with minimum consumption of labeling reagents and superior fluorescent signal intensity. The choice of QDs 525 for IFA resulted in bright and stable fluorescent signal, with minimum interference with the Cy3 signal from FISH detection.

Effect of Surface Chemistry on the Fluorescence of Detonation Nanodiamonds.

PubMed

Reineck, Philipp; Lau, Desmond W M; Wilson, Emma R; Fox, Kate; Field, Matthew R; Deeleepojananan, Cholaphan; Mochalin, Vadym N; Gibson, Brant C

2017-11-28

Detonation nanodiamonds (DNDs) have unique physical and chemical properties that make them invaluable in many applications. However, DNDs are generally assumed to show weak fluorescence, if any, unless chemically modified with organic molecules. We demonstrate that detonation nanodiamonds exhibit significant and excitation-wavelength-dependent fluorescence from the visible to the near-infrared spectral region above 800 nm, even without the engraftment of organic molecules to their surfaces. We show that this fluorescence depends on the surface functionality of the DND particles. The investigated functionalized DNDs, produced from the same purified DND as well as the as-received polyfunctional starting material, are hydrogen, hydroxyl, carboxyl, ethylenediamine, and octadecylamine-terminated. All DNDs are investigated in solution and on a silicon wafer substrate and compared to fluorescent high-pressure high-temperature nanodiamonds. The brightest fluorescence is observed from octadecylamine-functionalized particles and is more than 100 times brighter than the least fluorescent particles, carboxylated DNDs. The majority of photons emitted by all particle types likely originates from non-diamond carbon. However, we locally find bright and photostable fluorescence from nitrogen-vacancy centers in diamond in hydrogenated, hydroxylated, and carboxylated detonation nanodiamonds. Our results contribute to understanding the effects of surface chemistry on the fluorescence of DNDs and enable the exploration of the fluorescent properties of DNDs for applications in theranostics as nontoxic fluorescent labels, sensors, nanoscale tracers, and many others where chemically stable and brightly fluorescent nanoparticles with tailorable surface chemistry are needed.

Hydrophilic interaction liquid chromatography of anthranilic acid-labelled oligosaccharides with a 4-aminobenzoic acid ethyl ester-labelled dextran hydrolysate internal standard.

PubMed

Neville, David C A; Alonzi, Dominic S; Butters, Terry D

2012-04-13

Hydrophilic interaction liquid chromatography (HILIC) of fluorescently labelled oligosaccharides is used in many laboratories to analyse complex oligosaccharide mixtures. Separations are routinely performed using a TSK gel-Amide 80 HPLC column, and retention times of different oligosaccharide species are converted to glucose unit (GU) values that are determined with reference to an external standard. However, if retention times were to be compared with an internal standard, consistent and more accurate GU values would be obtained. We present a method to perform internal standard-calibrated HILIC of fluorescently labelled oligosaccharides. The method relies on co-injection of 4-aminobenzoic acid ethyl ester (4-ABEE)-labelled internal standard and detection by UV absorption, with 2-AA (2-aminobenzoic acid)-labelled oligosaccharides. 4-ABEE is a UV chromophore and a fluorophore, but there is no overlap of the fluorescent spectrum of 4-ABEE with the commonly used fluorescent reagents. The dual nature of 4-ABEE allows for accurate calculation of the delay between UV and fluorescent signals when determining the GU values of individual oligosaccharides. The GU values obtained are inherently more accurate as slight differences in gradients that can influence retention are negated by use of an internal standard. Therefore, this paper provides the first method for determination of HPLC-derived GU values of fluorescently labelled oligosaccharides using an internal calibrant. Copyright © 2012 Elsevier B.V. All rights reserved.

ZrO2 nanoparticles labeled via a native protein corona: detection by fluorescence microscopy and Raman microspectroscopy in rat lungs.

PubMed

Silge, Anja; Bräutigam, Katharina; Bocklitz, Thomas; Rösch, Petra; Vennemann, Antje; Schmitz, Inge; Popp, Jürgen; Wiemann, Martin

2015-08-07

ZrO2 nanoparticles are frequently used in composite materials such as dental fillers from where they may be released and inhaled upon polishing and grinding. Since the overall distribution of ZrO2 NP inside the lung parenchyma can hardly be observed by routine histology, here a labeling with a fluorphore was used secondary to the adsorption of serum proteins. Particles were then intratracheally instilled into rat lungs. After 3 h fluorescent structures consisted of agglomerates scattered throughout the lung parenchyma, which were mainly concentrated in alveolar macrophages after 3 d. A detection method based on Raman microspectroscopy was established to investigate the chemical composition of those fluorescent structures in detail. Raman measurements were arranged such that no spectral interference with the protein-bound fluorescence label was evident. Applying chemometrical methods, Raman signals of the ZrO2 nanomaterial were co-localized with the fluorescence label, indicating the stability of the nanomaterial-protein-dye complex inside the rat lung. The combination of Raman microspectroscopy and adsorptive fluorescence labeling may, therefore, become a useful tool for studying the localization of protein-coated nanomaterials in cells and tissues.

Permeabilization of the plasma membrane of L1210 mouse leukemia cells using lithotripter shock waves.

PubMed

Gambihler, S; Delius, M; Ellwart, J W

1994-09-01

Permeabilization of L1210 cells by lithotripter shock waves in vitro was monitored by evaluating the accumulation of fluorescein-labeled dextrans with a relative molecular mass ranging from 3,900-2,000,000. Incubation with labeled dextran alone caused a dose- and time-dependent increase in cellular fluorescence as determined by flow cytometry, with a vesicular distribution pattern in the cells consistent with endocytotic uptake. Shock wave exposure prior to incubation with labeled dextran revealed similar fluorescence intensities compared to incubation with labeled dextran alone. When cells were exposed to shock waves in the presence of labeled dextran, mean cellular fluorescence was further increased, indicating additional internalization of the probe. Confocal laser scanning microscopy confirmed intracellular fluorescence of labeled dextran with a diffuse distribution pattern. Fluorescence-activated cell sorting with subsequent determination of proliferation revealed that permeabilized cells were viable and able to proliferate. Permeabilization of the membrane of L1210 cells by shock waves in vitro allowed loading of dextrans with a relative molecular mass up to 2,000,000. Permeabilization of tumor cells by shock waves provides a useful tool for introducing molecules into cells which might be of interest for drug targeting in tumor therapy in vivo.

The MARVEL transmembrane motif of occludin mediates oligomerization and targeting to the basolateral surface in epithelia.

PubMed

Yaffe, Yakey; Shepshelovitch, Jeanne; Nevo-Yassaf, Inbar; Yeheskel, Adva; Shmerling, Hedva; Kwiatek, Joanna M; Gaus, Katharina; Pasmanik-Chor, Metsada; Hirschberg, Koret

2012-08-01

Occludin (Ocln), a MARVEL-motif-containing protein, is found in all tight junctions. MARVEL motifs are comprised of four transmembrane helices associated with the localization to or formation of diverse membrane subdomains by interacting with the proximal lipid environment. The functions of the Ocln MARVEL motif are unknown. Bioinformatics sequence- and structure-based analyses demonstrated that the MARVEL domain of Ocln family proteins has distinct evolutionarily conserved sequence features that are consistent with its basolateral membrane localization. Live-cell microscopy, fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) were used to analyze the intracellular distribution and self-association of fluorescent-protein-tagged full-length human Ocln or the Ocln MARVEL motif excluding the cytosolic C- and N-termini (amino acids 60-269, FP-MARVEL-Ocln). FP-MARVEL-Ocln efficiently arrived at the plasma membrane (PM) and was sorted to the basolateral PM in filter-grown polarized MDCK cells. A series of conserved aromatic amino acids within the MARVEL domain were found to be associated with Ocln dimerization using BiFC. FP-MARVEL-Ocln inhibited membrane pore growth during Triton-X-100-induced solubilization and was shown to increase the membrane-ordered state using Laurdan, a lipid dye. These data demonstrate that the Ocln MARVEL domain mediates self-association and correct sorting to the basolateral membrane.

Enhanced labeling of microalgae cellular lipids by application of an electric field generated by alternating current.

PubMed

Su, Li-Chien; Hsu, Yi-Hsiang; Wang, Hsiang-Yu

2012-05-01

An alternating current was used to generate an electric field to enhance the fluorescent labeling of microalgae cellular lipids with Nile red and LipidTOX. The decay of the fluorescence intensity of Chlorella vulgaris cells in 0 V/cm was more than 50% after 10 min, and the intensity variation was as high as 7% in 20s. At 2000 V/cm, the decay rate decreased to 1.22% per minute and the intensity fluctuation was less than 1% for LipidTOX-labeled cells. For Spirulina sp. cells at 0 V/cm, the fluorescence intensity increased by 10% after 10 min, whereas at 2000 V/cm, labeling was more rapid and fluorescence intensity doubled. These results show that applying an electric field can improve the quality of fluorescence detection by alleviating decay and fluctuation or by enhancing signal intensity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Correlative fluorescence and scanning transmission electron microscopy of quantum dot-labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Bandmann, Vera; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment. © 2014 Elsevier Inc. All rights reserved.

Using Fluorescent Viruses for Detecting Bacteria in Water

NASA Technical Reports Server (NTRS)

Tabacco, Mary Beth; Qian, Xiaohua; Russo, Jaimie A.

2009-01-01

A method of detecting water-borne pathogenic bacteria is based partly on established molecular-recognition and fluorescent-labeling concepts, according to which bacteria of a species of interest are labeled with fluorescent reporter molecules and the bacteria can then be detected by fluorescence spectroscopy. The novelty of the present method lies in the use of bacteriophages (viruses that infect bacteria) to deliver the fluorescent reporter molecules to the bacteria of the species of interest.

Fluorescent silica nanoparticles containing covalently bound dyes for reporter, marker, and sensor applications

NASA Astrophysics Data System (ADS)

Patonay, Gabor; Henary, Maged; Chapman, Gala; Emer, Kyle; Crow, Sidney

2016-03-01

Silica nanoparticles have proven to be useful in many bioanalytical and medical applications and have been used in numerous applications during the last decade. Combining the properties of silica nanoparticles and fluorescent dyes that may be used as chemical probes or labels can be relatively easy by simply soaking porous silica nanoparticles in a solution of the dye of interest. Under proper conditions the entrapped dye can stay inside the silica nanoparticle for several hours resulting in a useful probe. In spite of the relative durability of these probes, leaching can still occur. A much better approach is to synthesize silica nanoparticles that have the fluorescent dye covalently attached to the backbone structure of the silica nanoparticle. This can be achieved by using appropriately modified tetraethyl orthosilicate (TEOS) analogues during the silica nanoparticle synthesis. The molar ratio of TEOS and modified TEOS will determine the fluorescent dye load in the silica nanoparticle. Dependent on the chemical stability of the reporting dye either reverse micellar (RM) or Stöber method can be used for silica nanoparticle synthesis. If dye stability allows RM procedure is preferred as it results in a much easier control of the silica nanoparticle reaction itself. Also controlling the size and uniformity of the silica nanoparticles are much easier using RM method. Dependent on the functional groups present in the reporting dye used in preparation of the modified TEOS, the silica nanoparticles can be utilized in many applications such as pH sensor, metal ion sensors, labels, etc. In addition surface activated silica nanoparticles with reactive moieties are also excellent reporters or they can be used as bright fluorescent labels. Many different fluorescent dyes can be used to synthesize silica nanoparticles including visible and NIR dyes. Several bioanalytical applications are discussed including studying amoeba phagocytosis.

Application of meta- and para- phenylenediamine as enhanced oxime ligation catalysts for protein labeling, PEGylation, immobilization and release

PubMed Central

Mahmoodi, Mohammad M.; Rashidian, Mohammad; Zhang, Yi; Distefano, Mark D.

2015-01-01

Meta- and para- phenylenediamines have recently been shown to catalyze oxime and hydrazone ligation reactions at rates much faster than aniline, a commonly used catalyst. Here, it is demonstrated how these new catalysts can be used in a generally applicable procedure for fluorescent labeling, PEGylation, immobilization and release of aldehyde and ketone functionalized proteins. The chemical orthogonality of phenylenediamine-catalyzed oxime ligation versus copper catalyzed click reaction has also been harnessed for simultaneous dual labeling of bifunctional proteins containing both aldehyde and alkyne groups in high yield. PMID:25640893

The cell wall of Arabidopsis thaliana influences actin network dynamics.

PubMed

Tolmie, Frances; Poulet, Axel; McKenna, Joseph; Sassmann, Stefan; Graumann, Katja; Deeks, Michael; Runions, John

2017-07-20

In plant cells, molecular connections link the cell wall-plasma membrane-actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)-fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP-Lifeact-labelled network that appeared much more sparse with thick bundles that underwent 'simple movement', in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, 'pairwise image subtraction', was applied to measurement of the more rapidly remodelling actin network labelled with GFP-FABD2, while the second, 'cumulative fluorescence intensity', was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP-Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall-plasma membrane-actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Novel Fitc-Labeled Igy Antibody: Fluorescence Imaging Toxoplasma Gondii In Vitro.

PubMed

Sert, Mehtap; Cakir Koc, Rabia; Budama Kilinc, Yasemin

2017-04-12

Toxoplasmosis is caused by T. gondii and can create serious health problems in humans and also worldwide economic harm. Because of the clinical and veterinary importance of toxoplasmosis, its timely and accurate diagnosis has a major impact on disease-fighting strategies. T. gondii surface antigen 1 (SAG1), an immunodominant-specific antigen, is often used as a diagnostic tool. Therefore, the aim of this study was the optimization of novel fluorescein isothiocyanate (FITC) labeling of the SAG1-specific IgY antibody to show the potential for immunofluorescence imaging of T. gondii in vitro. The specificity of IgY antibodies was controlled by an enzyme-linked immunosorbent assay (ELISA), and the concentration of the IgY antibody was detected using a spectrophotometer. The optimum incubation time and FITC concentration were determined with a fluorescence spectrometer. The obtained FITC-labeled IgY was used for marking T. gondii tachyzoites, which were cultured in vitro and viewed using light microscopy. The interaction of the fluorescence-labeled antibody and the T. gondii tachyzoites was examined with a fluorescence microscope. In this study, for the first time, a FITC-labeled anti-SAG1 IgY antibody was developed according to ELISA, fluorescence spectrometer, and fluorescence imaging of cell culture. In the future, the obtained FITC-labeled T. gondii tachyzoites' specific IgY antibodies may be used as diagnostic tools for the detection of T. gondii infections in different samples.

Fluorescent speckle microscopy of microtubules: how low can you go?

PubMed

Waterman-Storer, C M; Salmon, E D

1999-12-01

Fluorescent speckle microscopy (FSM) is a new technique for visualizing the movement, assembly, and turnover of macromolecular assemblies like the cytoskeleton in living cells. In this method, contrast is created by coassembly of a small fraction of fluorescent subunits in a pool of unlabeled subunits. Random variation in association creates a nonuniform "fluorescent speckle" pattern. Fluorescent speckle movements in time-lapse recordings stand out to the eye and can be measured. Because fluorescent speckles represent fiduciary marks on the polymer lattice, FSM provides the opportunity for the first time to see the 2- and 3-dimensional trajectories of lattice movements within large arrays of polymers as well as identifying sites of assembly and disassembly of individual polymers. The technique works with either microinjection of fluorescently labeled subunits or expression of subunits ligated to green fluorescent protein (GFP). We have found for microtubules assembled in vitro that speckles containing one fluorophore can be detected and recorded using a conventional wide-field epi-fluorescence light microscope and digital imaging with a low noise cooled CCD camera. In living cells, optimal speckle contrast occurs at fractions of labeled tubulin of approximately 0.1-0.5% where the fluorescence of each speckle corresponds to one to seven fluorophores per resolvable unit (approximately 0.27 microm) in the microscope. This small fraction of labeled subunits significantly reduces out-of-focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells.

Development of bimolecular fluorescence complementation using rsEGFP2 for detection and super-resolution imaging of protein-protein interactions in live cells

PubMed Central

Wang, Sheng; Ding, Miao; Chen, Xuanze; Chang, Lei; Sun, Yujie

2017-01-01

Direct visualization of protein-protein interactions (PPIs) at high spatial and temporal resolution in live cells is crucial for understanding the intricate and dynamic behaviors of signaling protein complexes. Recently, bimolecular fluorescence complementation (BiFC) assays have been combined with super-resolution imaging techniques including PALM and SOFI to visualize PPIs at the nanometer spatial resolution. RESOLFT nanoscopy has been proven as a powerful live-cell super-resolution imaging technique. With regard to the detection and visualization of PPIs in live cells with high temporal and spatial resolution, here we developed a BiFC assay using split rsEGFP2, a highly photostable and reversibly photoswitchable fluorescent protein previously developed for RESOLFT nanoscopy. Combined with parallelized RESOLFT microscopy, we demonstrated the high spatiotemporal resolving capability of a rsEGFP2-based BiFC assay by detecting and visualizing specifically the heterodimerization interactions between Bcl-xL and Bak as well as the dynamics of the complex on mitochondria membrane in live cells. PMID:28663931

16 CFR 305.15 - Labeling for lighting products.

Code of Federal Regulations, 2013 CFR

2013-01-01

... surface of the fluorescent lamp ballast. (3) Package labeling. For purposes of labeling under this section...” on packages containing fluorescent lamp ballasts or the luminaires into which they are incorporated must appear conspicuously, in color-contrasting ink, on the surface of the package on which printing or...

16 CFR 305.15 - Labeling for lighting products.

Code of Federal Regulations, 2010 CFR

2010-01-01

... surface of the fluorescent lamp ballast. (3) Package labeling. For purposes of labeling under this section...” on packages containing fluorescent lamp ballasts or the luminaires into which they are incorporated must appear conspicuously, in color-contrasting ink, on the surface of the package on which printing or...

16 CFR 305.15 - Labeling for lighting products.

Code of Federal Regulations, 2014 CFR

2014-01-01

... surface of the fluorescent lamp ballast. (3) Package labeling. For purposes of labeling under this section...” on packages containing fluorescent lamp ballasts or the luminaires into which they are incorporated must appear conspicuously, in color-contrasting ink, on the surface of the package on which printing or...

16 CFR 305.15 - Labeling for lighting products.

Code of Federal Regulations, 2012 CFR

2012-01-01

... surface of the fluorescent lamp ballast. (3) Package labeling. For purposes of labeling under this section...” on packages containing fluorescent lamp ballasts or the luminaires into which they are incorporated must appear conspicuously, in color-contrasting ink, on the surface of the package on which printing or...

Flip-Flop of Phospholipids in Proteoliposomes Reconstituted from Detergent Extract of Chloroplast Membranes: Kinetics and Phospholipid Specificity

PubMed Central

Rajasekharan, Archita; Gummadi, Sathyanarayana N.

2011-01-01

Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6±1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents. PMID:22174798

Flip-flop of phospholipids in proteoliposomes reconstituted from detergent extract of chloroplast membranes: kinetics and phospholipid specificity.

PubMed

Rajasekharan, Archita; Gummadi, Sathyanarayana N

2011-01-01

Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6 ± 1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents.

Vectors for fluorescent protein tagging in Phytophthora: tools for functional genomics and cell biology.

PubMed

Ah-Fong, Audrey M V; Judelson, Howard S

2011-09-01

Fluorescent tagging has become the strategy of choice for examining the subcellular localisation of proteins. To develop a versatile community resource for this method in oomycetes, plasmids were constructed that allow the expression of either of four spectrally distinct proteins [cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and mCherry], alone or fused at their N- or C-termini, to sequences of interest. Equivalent sets of plasmids were made using neomycin or hygromycin phosphotransferases (nptII, hpt) as selectable markers, to facilitate double-labelling and aid work in diverse species. The fluorescent proteins and drug-resistance markers were fused to transcriptional regulatory sequences from the oomycete Bremia lactucae, which are known to function in diverse oomycetes, although the promoter in the fluorescence cassette (ham34) can be replaced easily by a promoter of interest. The function of each plasmid was confirmed in Phytophthora infestans. Moreover, fusion proteins were generated using targeting sequences for the endoplasmic reticulum, Golgi, mitochondria, nuclei, and peroxisomes. Studies of the distribution of the fusions in mycelia and sporangia provided insight into cellular organisation at different stages of development. This toolbox of vectors should advance studies of gene function and cell biology in Phytophthora and other oomycetes. Copyright © 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Near-infrared fluorescence probes for enzymes based on binding affinity modulation of squarylium dye scaffold.

PubMed

Oushiki, Daihi; Kojima, Hirotatsu; Takahashi, Yuki; Komatsu, Toru; Terai, Takuya; Hanaoka, Kenjiro; Nishikawa, Makiya; Takakura, Yoshinobu; Nagano, Tetsuo

2012-05-15

We present a novel design strategy for near-infrared (NIR) fluorescence probes utilizing dye-protein interaction as a trigger for fluorescence enhancement. The design principle involves modification of a polymethine dye with cleavable functional groups that reduce the dye's protein-binding affinity. When these functional groups are removed by specific interaction with the target enzymes, the dye's protein affinity is restored, protein binding occurs, and the dye's fluorescence is strongly enhanced. To validate this strategy, we first designed and synthesized an alkaline phosphatase (ALP) sensor by introducing phosphate into the squarylium dye scaffold; this sensor was able to detect ALP-labeled secondary antibodies in Western blotting analysis. Second, we synthesized a probe for β-galactosidase (widely used as a reporter of gene expression) by means of β-galactosyl substitution of the squarylium scaffold; this sensor was able to visualize β-galactosidase activity both in vitro and in vivo. Our strategy should be applicable to obtain NIR fluorescence probes for a wide range of target enzymes.

Enzymatic production of single-molecule FISH and RNA capture probes.

PubMed

Gaspar, Imre; Wippich, Frank; Ephrussi, Anne

2017-10-01

Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed. © 2017 Gaspar et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Covalent dye attachment influences the dynamics and conformational properties of flexible peptides

PubMed Central

Crevenna, Alvaro H.; Bomblies, Rainer; Lamb, Don C.

2017-01-01

Fluorescence spectroscopy techniques like Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) have become important tools for the in vitro and in vivo investigation of conformational dynamics in biomolecules. These methods rely on the distance-dependent quenching of the fluorescence signal of a donor fluorophore either by a fluorescent acceptor fluorophore (FRET) or a non-fluorescent quencher, as used in FCS with photoinduced electron transfer (PET). The attachment of fluorophores to the molecule of interest can potentially alter the molecular properties and may affect the relevant conformational states and dynamics especially of flexible biomolecules like intrinsically disordered proteins (IDP). Using the intrinsically disordered S-peptide as a model system, we investigate the impact of terminal fluorescence labeling on the molecular properties. We perform extensive molecular dynamics simulations on the labeled and unlabeled peptide and compare the results with in vitro PET-FCS measurements. Experimental and simulated timescales of end-to-end fluctuations were found in excellent agreement. Comparison between simulations with and without labels reveal that the π-stacking interaction between the fluorophore labels traps the conformation of S-peptide in a single dominant state, while the unlabeled peptide undergoes continuous conformational rearrangements. Furthermore, we find that the open to closed transition rate of S-peptide is decreased by at least one order of magnitude by the fluorophore attachment. Our approach combining experimental and in silico methods provides a benchmark for the simulations and reveals the significant effect that fluorescence labeling can have on the conformational dynamics of small biomolecules, at least for inherently flexible short peptides. The presented protocol is not only useful for comparing PET-FCS experiments with simulation results but provides a strategy to minimize the influence on molecular properties when chosing labeling positions for fluorescence experiments. PMID:28542243

Fluorescent TEM-1 β-lactamase with wild-type activity as a rapid drug sensor for in vitro drug screening

PubMed Central

Cheong, Wing-Lam; Tsang, Ming-San; So, Pui-Kin; Chung, Wai-Hong; Leung, Yun-Chung; Chan, Pak-Ho

2014-01-01

We report the development of a novel fluorescent drug sensor from the bacterial drug target TEM-1 β-lactamase through the combined strategy of Val216→Cys216 mutation and fluorophore labelling for in vitro drug screening. The Val216 residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216 residue in the V216C mutant for sensing drug binding at the active site. The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when β-lactam antibiotics bind to the active site. The labelled V216C mutant can differentiate between potent and impotent β-lactam antibiotics and can distinguish active-site binders from non-binders (including aggregates formed by small molecules in aqueous solution) by giving characteristic time-course fluorescence profiles. Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state. Given the ancestor's role of TEM-1 in the TEM family, the fluorescent TEM-1 drug sensor represents a good model to demonstrate the general combined strategy of Val216→Cys216 mutation and fluorophore labelling for fabricating tailor-made fluorescent drug sensors from other clinically significant TEM-type β-lactamase variants for in vitro drug screening. PMID:25074398

Fluorescent labeling of proteins with amine-specific 1,3,2-(2H)-dioxaborine polymethine dye.

PubMed

Gerasov, Andriy; Shandura, Mykola; Kovtun, Yuriy; Losytskyy, Mykhaylo; Negrutska, Valentyna; Dubey, Igor

2012-01-15

A novel water-soluble amine-reactive dioxaborine trimethine dye was synthesized in a good yield and characterized. The potential of the dye as a specific reagent for protein labeling was demonstrated with bovine serum albumin and lysozyme. Its interaction with proteins was studied by fluorescence spectroscopy and gel electrophoresis. The covalent binding of this almost nonfluorescent dye to proteins results in a 75- to 78-fold increase of its emission intensity accompanied by a red shift of the fluorescence emission maximum by 27 to 45 nm, with fluorescence wavelengths of labeled biomolecules being more than 600 nm. The dye does not require activation for the labeling reaction and can be used in a variety of bioassay applications. Copyright © 2011 Elsevier Inc. All rights reserved.

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection

PubMed Central

Brown, Koshonna; Thurn, Ted; Xin, Lun; Liu, William; Bazak, Remon; Chen, Si; Lai, Barry; Vogt, Stefan; Jacobsen, Chris; Paunesku, Tatjana; Woloschak, Gayle E.

2018-01-01

Titanium dioxide (TiO2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here. PMID:29541425

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection.

PubMed

Brown, Koshonna; Thurn, Ted; Xin, Lun; Liu, William; Bazak, Remon; Chen, Si; Lai, Barry; Vogt, Stefan; Jacobsen, Chris; Paunesku, Tatjana; Woloschak, Gayle E

2018-01-01

Titanium dioxide (TiO 2 ) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.

Trace fluorescent labeling for protein crystallization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pusey, Marc, E-mail: marc.pusey@ixpressgenes.com; Barcena, Jorge; Morris, Michelle

2015-06-27

The presence of a covalently bound fluorescent probe at a concentration of <0.5% does not affect the outcome of macromolecule crystallization screening experiments. Additionally, the fluorescence can be used to determine new, not immediately apparent, lead crystallization conditions. Fluorescence can be a powerful tool to aid in the crystallization of proteins. In the trace-labeling approach, the protein is covalently derivatized with a high-quantum-yield visible-wavelength fluorescent probe. The final probe concentration typically labels ≤0.20% of the protein molecules, which has been shown to not affect the crystal nucleation or diffraction quality. The labeled protein is then used in a plate-screening experimentmore » in the usual manner. As the most densely packed state of the protein is the crystalline form, then crystals show as the brightest objects in the well under fluorescent illumination. A study has been carried out on the effects of trace fluorescent labeling on the screening results obtained compared with nonlabeled protein, and it was found that considering the stochastic nature of the crystal nucleation process the presence of the probe did not affect the outcomes obtained. Other effects are realised when using fluorescence. Crystals are clearly seen even when buried in precipitate. This approach also finds ‘hidden’ leads, in the form of bright spots, with ∼30% of the leads found being optimized to crystals in a single-pass optimization trial. The use of visible fluorescence also enables the selection of colors that bypass interfering substances, and the screening materials do not have to be UV-transparent.« less

Comparison of 68Ga- and fluorescence-labeled microspheres for measurement of relative pulmonary perfusion in anesthetized pigs.

PubMed

Braune, Anja; Scharffenberg, Martin; Naumann, Anne; Bluth, Thomas; de Abreu, Marcelo Gama; Kotzerke, Jörg

2018-06-01

We compared 68 Gallium ( 68 Ga)- and fluorescence-labeled microspheres for measurement of pulmonary perfusion distribution in anesthetized pigs without lung injury. In two mechanically ventilated pigs, the distribution of pulmonary perfusion was marked in vivo with 68 Ga- and fluorescence-labeled microspheres in supine and prone position. After each injection, the distribution of 68 Ga-labeled microspheres was measured in vivo with positron emission tomography/ computed tomography (PET/CT) in the position in which microspheres were injected and vice versa. The distribution of fluorescence-labeled microspheres was measured ex vivo . Perfusion distributions were compared between methods and postures within four lung regions and along the ventro-dorsal gradient. After each injection of 68 Ga-labeled microspheres, changes in ventro-dorsal perfusion gradients induced by repositioning were compared for volume- and mass-normalized PET/CT measurements. Regional and gradient analyses of in vivo and ex vivo measurements, respectively, consistently revealed higher pulmonary perfusion in dorsal than ventral regions in supine positioned animals. Both methods showed more pronounced perfusion gradients in supine compared to prone position. Changes in animal position were associated with alterations in the ventro-dorsal perfusion gradient when volume-, but not mass-normalization was conducted for PET/CT data. Ex vivo fluorescence- and in vivo 68 Ga-labeled microspheres measurements revealed similar perfusion distributions. Mass-normalized perfusion measurements by 68 Ga-labeled microspheres and PET/CT were not affected by positioning artifacts. Schattauer GmbH.

Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.

2010-07-23

Research highlights: {yields} Successful fusion of GFP to M.EcoKI DNA methyltransferase. {yields} GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. {yields} FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerstermore » resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.« less

Kinetics of T-cell receptor-dependent antigen recognition determined in vivo by multi-spectral normalized epifluorescence laser scanning

NASA Astrophysics Data System (ADS)

Favicchio, Rosy; Zacharakis, Giannis; Oikonomaki, Katerina; Zacharopoulos, Athanasios; Mamalaki, Clio; Ripoll, Jorge

2012-07-01

Detection of multiple fluorophores in conditions of low signal represents a limiting factor for the application of in vivo optical imaging techniques in immunology where fluorescent labels report for different functional characteristics. A noninvasive in vivo Multi-Spectral Normalized Epifluorescence Laser scanning (M-SNELS) method was developed for the simultaneous and quantitative detection of multiple fluorophores in low signal to noise ratios and used to follow T-cell activation and clonal expansion. Colocalized DsRed- and GFP-labeled T cells were followed in tandem during the mounting of an immune response. Spectral unmixing was used to distinguish the overlapping fluorescent emissions representative of the two distinct cell populations and longitudinal data reported the discrete pattern of antigen-driven proliferation. Retrieved values were validated both in vitro and in vivo with flow cytometry and significant correlation between all methodologies was achieved. Noninvasive M-SNELS successfully quantified two colocalized fluorescent populations and provides a valid alternative imaging approach to traditional invasive methods for detecting T cell dynamics.

Synthesis, characterization and bioimaging of fluorescent labeled polyoxometalates.

PubMed

Geisberger, Georg; Gyenge, Emina Besic; Hinger, Doris; Bösiger, Peter; Maake, Caroline; Patzke, Greta R

2013-07-21

A fluorescent labeled Wells-Dawson type POM ({P2W17O61Fluo}) was newly synthesized and characterized by a wide range of analytical methods. {P2W17O61Fluo} was functionalized with fluorescein amine through a stable amide bond, and its long time stability was verified by UV/vis spectroscopic techniques at physiologically relevant pH values. No significant impact on the cell viability or morphology of HeLa cells was observed for POM concentrations up to 100 μg mL(-1). Cellular uptake of fluorescent {P2W17O61Fluo} was monitored by confocal laser scanning microscopy. POM uptake occurs mainly after prolonged incubation times of 24 h resulting in different intracellular patterns, i.e. randomly distributed over the entire cytoplasm, or aggregated in larger clusters. This direct monitoring strategy for the interaction of POMs with cells opens up new pathways for elucidating their unknown mode of action on the way to POM-based drug development.

Rapid, Sensitive Detection of Botulinum Toxin on a Flexible Microfluidics Platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warner, Marvin G.; Dockendorff, Brian P.; Feldhaus, Michael J.

2004-10-30

In this paper we will describe how high affinity reagents and a sensor configuration enabling rapid mass transport can be combined for rapid, sensitive biodetection. The system that we have developed includes a renewable surface immunoassay, which involves on-column detection of a fluorescently labeled secondary antibody in a sandwich immunoassay. Yeast display and directed molecular evolution were used to create high affinity antibodies to the botulinum toxin heavy chain receptor binding domain, AR1 and 3D12. A rotating rod renewable surface microcolumn was used to form a microliter-sized column containing beads functionalized with the capture antibody (AR1). The column was perfusedmore » with sample, wash solutions, and a fluorescently labeled secondary antibody (3D12) while the on-column fluorescence was monitored. Detection was accomplished in less than 5 minutes, with a total processing time of about 10 minutes. On-column detection of botulinum toxin was more sensitive and much faster than flow cytometry analysis on microbeads using the same reagents.« less

Intracellular bimodal nanoparticles based on quantum dots for high-field MRI at 21.1 T.

PubMed

Rosenberg, Jens T; Kogot, Joshua M; Lovingood, Derek D; Strouse, Geoffrey F; Grant, Samuel C

2010-09-01

Multimodal, biocompatible contrast agents for high magnetic field applications represent a new class of nanomaterials with significant potential for tracking of fluorescence and MR in vitro and vivo. Optimized for high-field MR applications-including biomedical imaging at 21.1 T, the highest magnetic field available for MRI-these nanoparticles capitalize on the improved performance of chelated Dy(3+) with increasing magnetic field coupled to a noncytotoxic Indium Phosphide/Zinc Sulfide (InP/ZnS) quantum dot that provides fluorescence detection, MR responsiveness, and payload delivery. By surface modifying the quantum dot with a cell-penetrating peptide sequence coupled to an MR contrast agent, the bimodal nanomaterial functions as a self-transfecting high-field MR/optical contrast agent for nonspecific intracellular labeling. Fluorescent images confirm sequestration in perinuclear vesicles of labeled cells, with no apparent cytotoxicity. These techniques can be extended to impart cell selectivity or act as a delivery vehicle for genetic or pharmaceutical interventions. 2010 Wiley-Liss, Inc.

Development of a fluorescent label tool based on lanthanide nanophosphors for viral biomedical application

NASA Astrophysics Data System (ADS)

Le, Quoc Minh; Huong Tran, Thu; Huong Nguyen, Thanh; Khuyen Hoang, Thi; Binh Nguyen, Thanh; Do, Khanh Tung; Tran, Kim Anh; Hien Nguyen, Dang; Luan Le, Thi; Quy Nguyen, Thi; Dung Dang, Mai; Thu Nguyen, Nu Anh; Nguyen, Van Man

2012-09-01

We report for the first time the preparation of luminescent lanthanide nanomaterial (LLN) linked bioconjugates and their application as a label tool for recognizing virus in the processing line of vaccine industrial fabrication. Several LLNs with the nanostructure forms of particles or rods/wires with europium (III) and terbium (III) ions in lattices of vanadate, phosphate and metal organic complex were prepared to develop novel fluorescent conjugates able to be applied as labels in fluorescence immunoassay analysis of virus/vaccine. With regard to the LLNs, we have successfully synthesized nanoparticles around 10 nm of YVO4:Eu(III), with high emission in the red spectral region, nanorod and nanowire of TbPO4·H2O and Eu1-xTbxPO4·H2O, width 5-7 nm and length 300 nm, showing very bright luminescence in green, and core/shell nanosized Eu(III) and Tb(III)/Eu(III) complexes with naphthoyl trifluoroacetone and tri-n-octylphosphineoxide (Eu.NTA.TOPO@PVP, EuXTb1-X.NTA.TOPO). The appropriated core/shell structures can play a double role, one for enhancing luminescence efficiency and another for providing nanophosphors with better stability in water media for facilitating the penetration of nanophosphor core into a biomedical environment. The organic functionalizations of the obtained LLNs were done through their surface encapsulation with a functional polysiloxane including active groups such as amine (NH2), thiocyanate (SCN) or mecarpto (SH). The properties of functional sol-gel matrix have great influence on the luminescence properties, especially luminescence intensity of YVO4:Eu(III), Eu.NTA.TOPO@PVP, TbPO4·H2O and EuxTb1-xPO4·H2O. Bioconjugation processes of the functionalized LLNs have been studied with some bioactive molecules such as biotin, protein immunoglobulin G (IgG) or bovine serum albumin (BSA). The results of LLN-bioconjugate linking with IgG for recognizing virus (vaccine) will be presented in brief. It is consistent to state that the LLN bioconjugates prepared from YVO4:Eu(III)-nanoparticles, TbPO4·H2O nanorod or wire and EuNTA.TOPO@PVP nanosized core/shell complex could be used as labels for recognizing virus in diagnosis or in vaccine production by use of the fluorescence immunoassay (FIA) method. The fluorescence images of the incubated specimens consisting of LLN bioconjugate and vaccine fabricate could be obtained well in terms of sharpness, reproductivity and stability. However, much work still needs to be done to develop an ordinary LLN-conjugate using the FIA method for analysis of virus and, moreover, to extend the study of biomedical cell processes at nano/microscale in practical application.

Design, synthesis, and application of the trimethoprim-based chemical tag for live-cell imaging.

PubMed

Jing, Chaoran; Cornish, Virginia W

2013-01-01

Over the past decade, chemical tags have been developed to complement the use of fluorescent proteins in live-cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E. coli dihydrofolate reductase and the antibiotic trimethoprim and was subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live-cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live-cell imaging. Alternate protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. © 2013 by John Wiley & Sons, Inc.

Development of background-free tame fluorescent probes for intracellular live cell imaging

PubMed Central

Alamudi, Samira Husen; Satapathy, Rudrakanta; Kim, Jihyo; Su, Dongdong; Ren, Haiyan; Das, Rajkumar; Hu, Lingna; Alvarado-Martínez, Enrique; Lee, Jung Yeol; Hoppmann, Christian; Peña-Cabrera, Eduardo; Ha, Hyung-Ho; Park, Hee-Sung; Wang, Lei; Chang, Young-Tae

2016-01-01

Fluorescence labelling of an intracellular biomolecule in native living cells is a powerful strategy to achieve in-depth understanding of the biomolecule's roles and functions. Besides being nontoxic and specific, desirable labelling probes should be highly cell permeable without nonspecific interactions with other cellular components to warrant high signal-to-noise ratio. While it is critical, rational design for such probes is tricky. Here we report the first predictive model for cell permeable background-free probe development through optimized lipophilicity, water solubility and charged van der Waals surface area. The model was developed by utilizing high-throughput screening in combination with cheminformatics. We demonstrate its reliability by developing CO-1 and AzG-1, a cyclooctyne- and azide-containing BODIPY probe, respectively, which specifically label intracellular target organelles and engineered proteins with minimum background. The results provide an efficient strategy for development of background-free probes, referred to as ‘tame' probes, and novel tools for live cell intracellular imaging. PMID:27321135

Optimized Design and Synthesis of Cell Permeable Biarsenical Cyanine Probe for Imaging Tagged Cytosolic Bacterial Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Na; Xiong, Yijia; Squier, Thomas C.

2013-01-21

To optimize cellular delivery and specific labeling of tagged cytosolic proteins by biarsenical fluorescent probes build around a cyanine dye scaffold, we have systematically varied the polarity of the hydrophobic tails (i.e., 4-5 methylene groups appended by a sulfonate or methoxy ester moiety) and arsenic capping reagent (ethanedithiol versus benzenedithiol). Targeted labeling of the cytosolic proteins SlyD and the alpha subunit of RNA polymerase engineered with a tetracysteine tagging sequences demonstrate the utility of the newly synthesized probes for live-cell visualization, albeit with varying efficiencies and background intensities. Optimal routine labeling and visualization is apparent using the ethanedithiol capping reagentmore » with the uncharged methoxy ester functionalized acyl chains. These measurements demonstrate the general utility of this class of photostable and highly fluorescent biarsenical reagents based on the cyanine scaffold for in vivo targeting of tagged cellular proteins for live cell measurements of protein dynamics.« less

L-Cysteine capped CdTe-CdS core-shell quantum dots: preparation, characterization and immuno-labeling of HeLa cells.

PubMed

Zhang, Hongyan; Sun, Pan; Liu, Chang; Gao, Huanyu; Xu, Linru; Fang, Jin; Wang, Meng; Liu, Jinling; Xu, Shukun

2011-01-01

Functionalized CdTe-CdS core-shell quantum dots (QDs) were synthesized in aqueous solution via water-bathing combined hydrothermal method using L-cysteine (L-Cys) as a stabilizer. This method possesses both the advantages of water-bathing and hydrothermal methods for preparing high-quality QDs with markedly reduced synthesis time, and better stability than a lone hydrothermal method. The QDs were characterized by transmission electronic microscopy and powder X-ray diffraction and X-ray photoelectron spectroscopy. The CdTe-CdS QDs with core-shell structure showed both enhanced fluorescence and better photo stability than nude CdTe QDs. After conjugating with antibody rabbit anti-CEACAM8 (CD67), the as-prepared l-Cys capped CdTe-CdS QDs were successfully used as fluorescent probes for the direct immuno-labeling and imaging of HeLa cells. It was indicated that this kind of QD would have application potential in bio-labeling and cell imaging. Copyright © 2009 John Wiley & Sons, Ltd.

Luminescent Quantum Dots as Ultrasensitive Biological Labels

NASA Astrophysics Data System (ADS)

Nie, Shuming

2000-03-01

Highly luminescent semiconductor quantum dots have been covalently coupled to biological molecules for use in ultrasensitive biological detection. This new class of luminescent labels is considerably brighter and more resistant againt photobleaching in comparison with organic dyes. Quantum dots labeled with the protein transferrin undergo receptor-mediated endocytosis (RME) in cultured HeLa cells, and those dots that were conjugated to immunomolecules recognize specific antibodies or antigens. In addition, we show that DNA functionalized quantum dots can be used to target specific genes by hybridization. We expect that quantum dot bioconjugates will have a broad range of biological applications, such as ligand-receptor interactions, real-time monitoring of molecular trafficking inside living cells, multicolor fluorescence in-situ hybridization (FISH), high-sensitivity detection in miniaturized devices (e.g., DNA chips), and fluorescent tagging of combinatorial chemical libraries. A potential clinical application is the use of quantum dots for ultrasensitive viral RNA detection, in which as low as 100 copies of hepatitis C and HIV viruses per ml blood should be detected.

Synthesis and characterization of photoswitchable fluorescent silica nanoparticles.

PubMed

Fölling, Jonas; Polyakova, Svetlana; Belov, Vladimir; van Blaaderen, Alfons; Bossi, Mariano L; Hell, Stefan W

2008-01-01

We have designed and synthesized a new functional (amino reactive) highly efficient fluorescent molecular switch (FMS) with a photochromic diarylethene and a rhodamine fluorescent dye. The reactive group in this FMS -N-hydroxysuccinimide ester- allows selective labeling of amino containing molecules or other materials. In ethanolic solutions, the compound displays a large fluorescent quantum yield of 52 % and a large fluorescence modulation ratio (94 %) between two states that may be interconverted with red and near-UV light. Silica nanoparticles incorporating the new FMS were prepared and characterized, and their spectroscopic and switching properties were also studied. The dye retained its properties after the incorporation into the silica, thereby allowing light-induced reversible high modulation of the fluorescence signal of a single particle for up to 60 cycles, before undergoing irreversible photobleaching. Some applications of these particles in fluorescence microscopy are also demonstrated. In particular, subdiffraction images of nanoparticles were obtained, in the focal plane of a confocal microscope.

Development of PCR internal controls for DNA profiling with the AmpFℓSTR® SGM Plus® amplification kit.

PubMed

Nathalie, Zahra; Hadi, Sibte; Goodwin, William

2012-09-01

Forensic DNA profiling uses a series of commercial kits that co-amplify several loci in one reaction; the products of the PCR are fluorescently labelled and analysed using CE. Before CE, an aliquot of the PCR is mixed with formamide and an internal lane size standard. Using the SGM Plus amplification kit, we have developed two internal non-amplified controls of 80 bp and 380 bp that are labelled with ROX fluorescent dye and added to the PCR. Combined with two internal amplification controls of 90 bp and 410 bp, they provide additional controls for the PCR, electrokinetic injection, and CE and also function as an internal size standard. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mitochondrial dynamics and optical conformation changes in DsRed as studied by Fourier imaging correlation spectroscopy

NASA Astrophysics Data System (ADS)

Senning, Eric Nicolas

Novel experiments that probe the dynamics of intracellular species, including the center-of-mass displacements and internal conformational transitions of biological macromolecules, have the potential to reveal the complex biochemical mechanisms operating within the cell. This work presents the implementation and development of Fourier imaging correlation spectroscopy (FICS), a phase-selective approach to fluorescence spectroscopy that measures the collective coordinate fluctuations of fluorescently labeled microscopic particles. In FICS experiments, a spatially modulated optical grating excites a fluorescently labeled sample. Phase-synchronous detection of the fluorescence, with respect to the phase of the exciting optical grating, can be used to monitor the fluctuations of partially averaged spatial coordinates. These data are then analyzed by two-point and four-point time correlation functions to provide a statistically meaningful understanding of the dynamics under observation. FICS represents a unique route to elevate signal levels, while acquiring detailed information about molecular coordinate trajectories. Mitochondria of mammalian cells are known to associate with cytoskeletal proteins, and their motions are affected by the stability of microtubules and microfilaments. Within the cell it is possible to fluorescently label the mitochondria and study its dynamic behavior with FICS. The dynamics of S. cerevisiae yeast mitochondria are characterized at four discrete length scales (ranging from 0.6--1.19 mum) and provide detailed information about the influence of specific cytoskeletal elements. Using the microtubule and microfilament destabilizing agents, Nocodazole and Latrunculin A, it is determined that microfilaments are required for normal yeast mitochondrial motion while microtubules have no effect. Experiments with specific actin mutants revealed that actin is responsible for enhanced mobility on length scales greater than 0.6 mum. The versatility of FICS expands when individual molecules are labeled with fluorescent chromophores. In recent experiments on the tetrameric fluorescent protein DsRed, polarization-modulated FICS (PM-FICS) is demonstrated to separate conformational dynamics from molecular translational dynamics. The optical switching pathways of DsRed, a tetrameric complex of fluorescent protein subunits, are examined. An analysis of PM-FICS coordinate trajectories, in terms of 2D spectra and joint probability distributions, provides detailed information about the transition pathways between distinct dipole-coupled DsRed conformations. This dissertation includes co-authored and previously published material.

Differentiation of artery wall lesions using porphyrins and fiberoptic sensor in rabbits

NASA Astrophysics Data System (ADS)

Vari, Sandor G.; van der Veen, Maurits J.; Papazoglou, Theodore G.; Fishbein, Michael C.; Stavridi, Marigo; Papaioannou, Thanassis; Grundfest, Warren S.

1994-02-01

We investigated the ability of fluorescence spectroscopy, and photosensitizers to differentiate normal, hyperplastic and atherosclerotic arterial wall lesions in vivo. Hyperplastic lesions were induced in the abdominal aorta (AB) of 24 rabbits by balloon injury (BI). Atherosclerotic arterial wall lesions were induced by BI and diet. Fluorescence signals from thoracic n equals 16 and AB n equals 15 sites were analyzed by computer. A ratio was used as an index of drug presence. Use of PPS or BPD and LIFS may be a feasible, in vivo method for the differentiation between normal, hyperplastic and atherosclerotic arterial wall lesions.

Site-Specific Antibody Labeling by Covalent Photoconjugation of Z Domains Functionalized for Alkyne-Azide Cycloaddition Reactions.

PubMed

Perols, Anna; Arcos Famme, Melina; Eriksson Karlström, Amelie

2015-11-01

Antibodies are extensively used in research, diagnostics, and therapy, and for many applications the antibodies need to be labeled. Labeling is typically performed by using amine-reactive probes that target surface-exposed lysine residues, resulting in heterogeneously labeled antibodies. An alternative labeling strategy is based on the immunoglobulin G (IgG)-binding protein domain Z, which binds to the Fc region of IgG. Introducing the photoactivable amino acid benzoylphenylalanine (BPA) into the Z domain makes it possible for a covalent bond to be be formed between the Z domain and the antibody on UV irradiation, to produce a site-specifically labeled product. Z32 BPA was synthesized by solid-phase peptide synthesis and further functionalized to give alkyne-Z32 BPA and azide-Z32 BPA for Cu(I) -catalyzed cycloaddition, as well as DBCO-Z32 BPA for Cu-free strain-promoted cycloaddition. The Z32 BPA variants were conjugated to the human IgG1 antibody trastuzumab and site-specifically labeled with biotin or fluorescein. The fluorescently labeled trastuzumab showed specific staining of the membranes of HER2-expressing cells in immunofluorescence microscopy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Method for rapid base sequencing in DNA and RNA with two base labeling

DOEpatents

Jett, J.H.; Keller, R.A.; Martin, J.C.; Posner, R.G.; Marrone, B.L.; Hammond, M.L.; Simpson, D.J.

1995-04-11

A method is described for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand. 4 figures.

Fluorescent labels and their use in separations

DOEpatents

Mathies, Richard A.; Glazer, Alexander; Ju, Jingyue

1997-01-01

Compositions are provided comprising sets of fluorescent labels carrying pairs of donor and acceptor dye molecules, designed for efficient excitation of the donors at a single wavelength and emission from the acceptor in each of the pairs at different wavelengths. The different molecules having different donor-acceptor pairs can be modified to have substantially the same mobility under separation conditions, by varying the distance between the donor and acceptor in a given pair. Particularly, the fluorescent compositions find use as labels in sequencing nucleic acids.

Method for rapid base sequencing in DNA and RNA with two base labeling

DOEpatents

Jett, James H.; Keller, Richard A.; Martin, John C.; Posner, Richard G.; Marrone, Babetta L.; Hammond, Mark L.; Simpson, Daniel J.

1995-01-01

Method for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand.

Synthesis, structure and photoluminescence properties of amine-templated open-framework bismuth sulfates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marri, Subba R.; Behera, J.N., E-mail: jnbehera@niser.ac.in

2014-02-15

Two organically-templated bismuth sulfates of the compositions, [C{sub 6}N{sub 2}H{sub 14}] [Bi(SO{sub 4}){sub 2}(NO{sub 3})], (1) and [C{sub 4}N{sub 2}H{sub 12}]{sub 4}[Bi{sub 4}(SO{sub 4}){sub 10}(H{sub 2}O){sub 4}], (2), with open architecture have been synthesized and their structures determined by single crystal X-ray diffraction. 1 has a corrugated layered structure with 8-membered aperture wherein the SO{sub 4} tetrahedra and the BiO{sub 8} polyhedra join together to form (4, 4) net sheets of the metal centers while 2 has a three-dimensional structure possessing 8- and 12-membered channels. Both the compounds show good fluorescence properties exhibiting blue luminescence. Time-resolved fluorescence behavior of 1more » and 2 shows mean fluorescence life time of 0.9 and 1.0 ns, respectively. - Graphical abstract: Two open-framework bismuth sulfates with the layered and three-dimensional structures have been synthesized and characterized. Both the compounds show good fluorescence properties exhibiting blue luminescence. Display Omitted - Highlights: • Two organically-templated bismuth sulfates with open architecture have been synthesized and characterized. • One has a corrugated layered structure while the other one has a three-dimensional structure possessing channels. • They are novel in that open-framework three-dimensional main group metal sulfates are first to be reported. • They show good fluorescence properties exhibiting blue luminescence.« less

Label-Free Biosensors Based on Bimodal Waveguide (BiMW) Interferometers.

PubMed

Herranz, Sonia; Gavela, Adrián Fernández; Lechuga, Laura M

2017-01-01

The bimodal waveguide (BiMW) sensor is a novel common path interferometric transducer based on the evanescent field detection principle, which in combination with a bio-recognition element allows the direct detection of biomolecular interactions in a label-free scheme. Due to its inherent high sensitivity it has great potential to become a powerful analytical tool for monitoring substances of interest in areas such as environmental control, medical diagnostics and food safety, among others. The BiMW sensor is fabricated using standard silicon-based technology allowing cost-effective production, and meeting the requirements of portability and disposability necessary for implementation in a point-of-care (POC) setting.In this chapter we describe the design and fabrication of the BiMW transducer, as well as its application for bio-sensing purposes. We show as an example the biosensor capabilities two different applications: (1) the immunodetection of Irgarol 1051 biocide useful in the environmental field, and (2) the detection of human growth hormone as used in clinical diagnostics. The detection is performed in real time by monitoring changes in the intensity pattern of light exiting the BiMW transducer resulting from antigen-antibody interactions on the surface of the sensor.

3D multiplexed immunoplasmonics microscopy

NASA Astrophysics Data System (ADS)

Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

2016-07-01

Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed technology is simple and compatible with standard epi-fluorescence microscopes used in biological and clinical laboratories. Thus, 3D multiplexed immunoplasmonics microscopy is ready for clinical applications as a cost-efficient alternative to immunofluorescence.Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed technology is simple and compatible with standard epi-fluorescence microscopes used in biological and clinical laboratories. Thus, 3D multiplexed immunoplasmonics microscopy is ready for clinical applications as a cost-efficient alternative to immunofluorescence. Electronic supplementary information (ESI) available: Characterization of functionalized nanoparticles by UV-visible-NIR spectroscopy, standard dark field microscopy and reflected light microscopy. Immunofluorescence of cells. See DOI: 10.1039/c6nr01257d

Near-infrared fluorescent protein iRFP713 as a reporter protein for optogenetic vectors, a transgenic Cre-reporter rat, and other neuronal studies

PubMed Central

Richie, Christopher T.; Whitaker, Leslie R.; Whitaker, Keith W.; Necarsulmer, Julie; Baldwin, Heather A.; Zhang, Yajun; Fortuno, Lowella; Hinkle, Josh J.; Koivula, Pyry; Henderson, Mark J.; Sun, Wenzhi; Wang, Kai; Smith, Jeffrey C.; Pickel, Jim; Ji, Na; Hope, Bruce T.; Harvey, Brandon K.

2017-01-01

Background The use of genetically-encoded fluorescent reporters is essential for the identification and observation of cells that express transgenic modulatory proteins. Near-infrared (NIR) fluorescent proteins have superior light penetration through biological tissue, but are not yet widely adopted. New Method Using the near-infrared fluorescent protein, iRFP713, improves the imaging resolution in thick tissue sections or the intact brain due to the reduced light-scattering at the longer, NIR wavelengths used to image the protein. Additionally, iRFP713 can be used to identify transgenic cells without photobleaching other fluorescent reporters or affecting opsin function. We have generated a set of adeno-associated vectors in which iRFP713 has been fused to optogenetic channels, and can be expressed constitutively or Cre-dependently. Results iRFP713 is detectable when expressed in neurons both in vitro and in vivo without exogenously supplied chromophore biliverdin. Neuronally-expressed iRFP713 has similar properties to GFP-like fluorescent proteins, including the ability to be translationally fused to channelrhodopsin or halorhodopsin, however, it shows superior photostability compared to EYFP. Furthermore, electrophysiological recordings from iRFP713-labeled cells compared to cells labeled with mCherry suggest that iRFP713 cells are healthier and therefore more stable and reliable in an ex vivo preparation. Lastly, we have generated a transgenic rat that expresses iRFP713 in a Cre-dependent manner. Conclusions Overall, we have demonstrated that iRFP713 can be used as a reporter in neurons without the use of exogenous biliverdin, with minimal impact on viability and function thereby making it feasible to extend the capabilities for imaging genetically-tagged neurons in slices and in vivo. PMID:28380331

Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding.

PubMed

Branigan, Emma; Pliotas, Christos; Hagelueken, Gregor; Naismith, James H

2013-11-01

Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins.

In vivo labeling of cortical astrocytes with sulforhodamine 101 (SR101).

PubMed

Nimmerjahn, Axel; Helmchen, Fritjof

2012-03-01

Fluorescent markers that stain particular cell types in the intact brain are essential tools for fluorescence microscopy because they enable studies of structure and function of cells identified in this way. Although cell type-specific fluorescence staining can be achieved through promoter-driven expression of fluorescent proteins, this genetic approach is generally labor- and cost-intensive. Alternative viral approaches for targeted fluorophore expression are relatively invasive. For astrocytes, there is a simple alternative. This protocol describes an easy and robust method for rapid (within minutes) and high-contrast staining of astrocytes in defined regions of the intact rodent cortex using the synthetic, water-soluble but non-fixable red fluorescent dye sulforhodamine 101 (SR101). Selective staining is achieved through local uptake and gap junction-mediated spread of SR101 following its topical application or injection into tissue. Applications, technical pitfalls, and limitations of the SR101-staining technique are discussed. Given its simplicity and reliability, SR101 staining is a valuable tool for the study of astrocyte function in the intact brain and for in vivo fluorescence microscopy in general.

Inhibition of beta-amyloid aggregation by fluorescent dye labels

NASA Astrophysics Data System (ADS)

Amaro, Mariana; Wellbrock, Thorben; Birch, David J. S.; Rolinski, Olaf J.

2014-02-01

The fluorescence decay of beta-amyloid's (Aβ) intrinsic fluorophore tyrosine has been used for sensing the oligomer formation of dye-labelled Aβ monomers and the results compared with previously studied oligomerization of the non-labelled Aβ peptides. It has been demonstrated that two different sized, covalently bound probes 7-diethylaminocoumarin-3-carbonyl and Hilyte Fluor 488 (HLF), alter the rate and character of oligomerization to different extents. The ability of HLF to inhibit formation of highly ordered structures containing beta-sheets was also shown. The implications of our findings for using fluorescence methods in amyloidosis research are discussed and the advantages of this auto-fluorescence approach highlighted.

Time-lapse monitoring of TLR2 ligand internalization with newly developed fluorescent probes.

PubMed

Arai, Yohei; Yokoyama, Kouhei; Kawahara, Yuki; Feng, Qi; Ohta, Ippei; Shimoyama, Atsushi; Inuki, Shinsuke; Fukase, Koichi; Kabayama, Kazuya; Fujimoto, Yukari

2018-05-23

As a mammalian toll-like receptor family member protein, TLR2 recognizes lipoproteins from bacteria and modulates the immune response by inducing the expression of various cytokines. We have developed fluorescence-labeled TLR2 ligands with either hydrophilic or hydrophobic fluorescence groups. The labeled ligands maintained the inflammatory IL-6 induction activity and enabled us to observe the internalization and colocalization of the TLR2 ligands using live-cell imaging. The time-lapse monitoring in the live-cell imaging of the fluorescence-labeled TLR2 ligand showed that TLR2/CD14 expression in the host cells enhanced the internalization of TLR2 ligand molecules.

Investigation of structural, optical, catalytic, fluorescence studies of eco-friendly synthesized Bi2S3 nanostructures

NASA Astrophysics Data System (ADS)

Ayodhya, Dasari; Veerabhadram, Guttena

2017-02-01

A simple solution phase method has been developed for the synthesis of ribonucleosides capped Bi2S3 nanostructures (NSs) with an average diameter of 15 nm and length of below 100 nm. Transmission electron microscopy (TEM), selected-area electron diffraction (SAED), and X-ray diffraction (XRD) studies revealed that these NSs were grown from a colloidal dispersion of amorphous Bi2S3 particles, which was first formed through a thermal reaction at a temperature of 60 °C. The phase and structure of the Bi2S3 NSs have been identified by using X-ray powder diffraction. The crystal structure had orthorhombic structure. The surface properties and morphology have been investigated using scanning electron microscope (SEM) technique. The N2 sorption-desorption experiments showed that the surface area of the NSs was 6.35 m2 g-1 by Brunauer-Emmett-Teller (BET). The experiments showed that the Bi2S3 NSs prepared in the present work could be used as catalyst for the reduction of SO dye using a reducing agent. It was found that the as-obtained Bi2S3 NSs contributed to the best catalytic activity. Photoluminescence experiments showed a quenching of the Bi2S3 fluorescence with increasing L-cysteine (Cys) content in the solution. Furthermore, the proposed NSs as sensor were employed for the determination of metal ions with satisfactory results.

Generic epitaxial graphene biosensors for ultrasensitive detection of cancer risk biomarker

NASA Astrophysics Data System (ADS)

Tehrani, Z.; Burwell, G.; Mohd Azmi, M. A.; Castaing, A.; Rickman, R.; Almarashi, J.; Dunstan, P.; Miran Beigi, A.; Doak, S. H.; Guy, O. J.

2014-09-01

A generic electrochemical method of ‘bioreceptor’ antibody attachment to phenyl amine functionalized graphitic surfaces is demonstrated. Micro-channels of chemically modified multi-layer epitaxial graphene (MLEG) have been used to provide a repeatable and reliable response to nano-molar (nM) concentrations of the cancer risk (oxidative stress) biomarker 8-hydroxydeoxyguanosine (8-OHdG). X-ray photoelectron spectroscopy, Raman spectroscopy are used to characterize the functionalized MLEG. Confocal fluorescence microscopy using fluorescent-labelled antibodies indicates that the anti-8-OHdG antibody selectively binds to the phenyl amine-functionalized MLEG’s channel. Current-voltage measurements on functionalized channels showed repeatable current responses from antibody-biomarker binding events. This technique is scalable, reliable, and capable of providing a rapid, quantitative, label-free assessment of biomarkers at nano-molar (<20 nM) concentrations in analyte solutions. The sensitivity of the sensor device was investigated using varying concentrations of 8-OHdG, with changes in the sensor’s channel resistance observed upon exposure to 8-OHdG. Detection of 8-OHdG concentrations as low as 0.1 ng ml-1 (0.35 nM) has been demonstrated. This is five times more sensitive than reported enzyme linked immunosorbent assay tests (0.5 ng ml-1).

Wide-field imaging and flow cytometric analysis of cancer cells in blood by fluorescent nanodiamond labeling and time gating

NASA Astrophysics Data System (ADS)

Hui, Yuen Yung; Su, Long-Jyun; Chen, Oliver Yenjyh; Chen, Yit-Tsong; Liu, Tzu-Ming; Chang, Huan-Cheng

2014-07-01

Nanodiamonds containing high density ensembles of negatively charged nitrogen-vacancy (NV-) centers are promising fluorescent biomarkers due to their excellent photostability and biocompatibility. The NV- centers in the particles have a fluorescence lifetime of up to 20 ns, which distinctly differs from those (<10 ns) of cell and tissue autofluorescence, making it possible to achieve background-free detection in vivo by time gating. Here, we demonstrate the feasibility of using fluorescent nanodiamonds (FNDs) as optical labels for wide-field time-gated fluorescence imaging and flow cytometric analysis of cancer cells with a nanosecond intensified charge-coupled device (ICCD) as the detector. The combined technique has allowed us to acquire fluorescence images of FND-labeled HeLa cells in whole blood covered with a chicken breast of ~0.1-mm thickness at the single cell level, and to detect individual FND-labeled HeLa cells in blood flowing through a microfluidic device at a frame rate of 23 Hz, as well as to locate and trace FND-labeled lung cancer cells in the blood vessels of a mouse ear. It opens a new window for real-time imaging and tracking of transplanted cells (such as stem cells) in vivo.

Ultrasound Induced Fluorescence of Nanoscale Liposome Contrast Agents

PubMed Central

Zhang, Qimei; Morgan, Stephen P.; O’Shea, Paul; Mather, Melissa L.

2016-01-01

A new imaging contrast agent is reported that provides an increased fluorescent signal upon application of ultrasound (US). Liposomes containing lipids labelled with pyrene were optically excited and the excimer fluorescence emission intensity was detected in the absence and presence of an ultrasound field using an acousto-fluorescence setup. The acousto-fluorescence dynamics of liposomes containing lipids with pyrene labelled on the fatty acid tail group (PyPC) and the head group (PyPE) were compared. An increase in excimer emission intensity following exposure to US was observed for both cases studied. The increased intensity and time constants were found to be different for the PyPC and PyPE systems, and dependent on the applied US pressure and exposure time. The greatest change in fluorescence intensity (130%) and smallest rise time constant (0.33 s) are achieved through the use of PyPC labelled liposomes. The mechanism underlying the observed increase of the excimer emission intensity in PyPC labelled liposomes is proposed to arise from the “wagging” of acyl chains which involves fast response and requires lower US pressure. This is accompanied by increased lipid lateral diffusivity at higher ultrasound pressures, a mechanism that is also active in the PyPE labelled liposomes. PMID:27467748

Live Imaging of Cellular Internalization of Single Colloidal Particle by Combined Label-Free and Fluorescence Total Internal Reflection Microscopy.

PubMed

Byrne, Gerard D; Vllasaliu, Driton; Falcone, Franco H; Somekh, Michael G; Stolnik, Snjezana

2015-11-02

In this work we utilize the combination of label-free total internal reflection microscopy and total internal reflectance fluorescence (TIRM/TIRF) microscopy to achieve a simultaneous, live imaging of single, label-free colloidal particle endocytosis by individual cells. The TIRM arm of the microscope enables label free imaging of the colloid and cell membrane features, while the TIRF arm images the dynamics of fluorescent-labeled clathrin (protein involved in endocytosis via clathrin pathway), expressed in transfected 3T3 fibroblasts cells. Using a model polymeric colloid and cells with a fluorescently tagged clathrin endocytosis pathway, we demonstrate that wide field TIRM/TIRF coimaging enables live visualization of the process of colloidal particle interaction with the labeled cell structure, which is valuable for discerning the membrane events and route of colloid internalization by the cell. We further show that 500 nm in diameter model polystyrene colloid associates with clathrin, prior to and during its cellular internalization. This association is not apparent with larger, 1 μm in diameter colloids, indicating an upper particle size limit for clathrin-mediated endocytosis.

Biodetection using fluorescent quantum dots

NASA Astrophysics Data System (ADS)

Speckman, Donna M.; Jennings, Travis L.; LaLumondiere, Steven D.; Klimcak, Charles M.; Moss, Steven C.; Loper, Gary L.; Beck, Steven M.

2002-07-01

Multi-pathogen biosensors that take advantage of sandwich immunoassay detection schemes and utilize conventional fluorescent dye reporter molecules are difficult to make into extremely compact and autonomous packages. The development of a multi-pathogen, immunoassay-based, fiber optic detector that utilizes varying sized fluorescent semiconductor quantum dots (QDs) as the reporter labels has the potential to overcome these problems. In order to develop such a quantum dot-based biosensor, it is essential to demonstrate that QDs can be attached to antibody proteins, such that the specificity of the antibody is maintained. We have been involved in efforts to develop a reproducible method for attaching QDs to antibodies for use in biodetection applications. We have synthesized CdSe/ZnS core-shell QDs of differing size, functionalized their surfaces with several types of organic groups for water solubility, and covalently attached these functionalized QDs to rabbit anti-ovalbumin antibody protein. We also demonstrated that these labeled antibodies exhibit selective binding to ovalbumin antigen. We characterized the QDs at each step in the overall synthesis by UV-VIS absorption spectroscopy and by picosecond (psec) transient photoluminescence (TPL) spectroscopy. TPL spectroscopy measurements indicate that QD lifetime depends on the size of the QD, the intensity of the optical excitation source, and whether or not they are functionalized and conjugated to antibodies. We describe details of these experiments and discuss the impact of our results on our biosensor development program.

Integrated SRS and fluorescence imaging for study of thermogenesis and lipid metabolism in vivo

NASA Astrophysics Data System (ADS)

He, Sicong; An, Yitai; Li, Xuesong; Wu, Zhenguo; Qu, Jianan Y.

2018-02-01

In this work, we developed a label-free imaging and spectroscopy method to assess the metabolism and thermogenesis of mouse adipose tissues in vivo. An optical redox ratio based on the endogenous fluorescence of mitochondrial coenzymes was used as a biomarker to determine the metabolic state of adipocytes during thermogenesis. The morphological and functional characteristics of different types of adipocytes were assessed in vivo and their thermogenic activities were monitored in real time with a robust spectroscope system.

Method for detecting point mutations in DNA utilizing fluorescence energy transfer

DOEpatents

Parkhurst, Lawrence J.; Parkhurst, Kay M.; Middendorf, Lyle

2001-01-01

A method for detecting point mutations in DNA using a fluorescently labeled oligomeric probe and Forster resonance energy transfer (FRET) is disclosed. The selected probe is initially labeled at each end with a fluorescence dye, which act together as a donor/acceptor pair for FRET. The fluorescence emission from the dyes changes dramatically from the duplex stage, wherein the probe is hybridized to the complementary strand of DNA, to the single strand stage, when the probe is melted to become detached from the DNA. The change in fluorescence is caused by the dyes coming into closer proximity after melting occurs and the probe becomes detached from the DNA strand. The change in fluorescence emission as a function of temperature is used to calculate the melting temperature of the complex or T.sub.m. In the case where there is a base mismatch between the probe and the DNA strand, indicating a point mutation, the T.sub.m has been found to be significantly lower than the T.sub.m for a perfectly match probelstand duplex. The present invention allows for the detection of the existence and magnitude of T.sub.m, which allows for the quick and accurate detection of a point mutation in the DNA strand and, in some applications, the determination of the approximate location of the mutation within the sequence.

Upconversion Nanoparticles-Encoded Hydrogel Microbeads-Based Multiplexed Protein Detection

NASA Astrophysics Data System (ADS)

Shikha, Swati; Zheng, Xiang; Zhang, Yong

2018-06-01

Fluorescently encoded microbeads are in demand for multiplexed applications in different fields. Compared to organic dye-based commercially available Luminex's xMAP technology, upconversion nanoparticles (UCNPs) are better alternatives due to their large anti-Stokes shift, photostability, nil background, and single wavelength excitation. Here, we developed a new multiplexed detection system using UCNPs for encoding poly(ethylene glycol) diacrylate (PEGDA) microbeads as well as for labeling reporter antibody. However, to prepare UCNPs-encoded microbeads, currently used swelling-based encapsulation leads to non-uniformity, which is undesirable for fluorescence-based multiplexing. Hence, we utilized droplet microfluidics to obtain encoded microbeads of uniform size, shape, and UCNPs distribution inside. Additionally, PEGDA microbeads lack functionality for probe antibodies conjugation on their surface. Methods to functionalize the surface of PEGDA microbeads (acrylic acid incorporation, polydopamine coating) reported thus far quench the fluorescence of UCNPs. Here, PEGDA microbeads surface was coated with silica followed by carboxyl modification without compromising the fluorescence intensity of UCNPs. In this study, droplet microfluidics-assisted UCNPs-encoded microbeads of uniform shape, size, and fluorescence were prepared. Multiple color codes were generated by mixing UCNPs emitting red and green colors at different ratios prior to encapsulation. UCNPs emitting blue color were used to label the reporter antibody. Probe antibodies were covalently immobilized on red UCNPs-encoded microbeads for specific capture of human serum albumin (HSA) as a model protein. The system was also demonstrated for multiplexed detection of both human C-reactive protein (hCRP) and HSA protein by immobilizing anti-hCRP antibodies on green UCNPs.

Novel fabrication of fluorescent silk utilized in biotechnological and medical applications.

PubMed

Kim, Dong Wook; Lee, Ok Joo; Kim, Seong-Wan; Ki, Chang Seok; Chao, Janet Ren; Yoo, Hyojong; Yoon, Sung-Il; Lee, Jeong Eun; Park, Ye Ri; Kweon, HaeYong; Lee, Kwang Gill; Kaplan, David L; Park, Chan Hum

2015-11-01

Silk fibroin (SF) is a natural polymer widely used and studied for diverse applications in the biomedical field. Recently, genetically modified silks, particularly fluorescent SF fibers, were reported to have been produced from transgenic silkworms. However, they are currently limited to textile manufacturing. To expand the use of transgenic silkworms for biomedical applications, a solution form of fluorescent SF needed to be developed. Here, we describe a novel method of preparing a fluorescent SF solution and demonstrate long-term fluorescent function up to one year after subcutaneous insertion. We also show that fluorescent SF labeled p53 antibodies clearly identify HeLa cells, indicating the applicability of fluorescent SF to cancer detection and bio-imaging. Furthermore, we demonstrate the intraoperative use of fluorescent SF in an animal model to detect a small esophageal perforation (0.5 mm). This study suggests how fluorescent SF biomaterials can be applied in biotechnology and clinical medicine. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fluorometric graphene oxide-based detection of Salmonella enteritis using a truncated DNA aptamer.

PubMed

Chinnappan, Raja; AlAmer, Saleh; Eissa, Shimaa; Rahamn, Anas Abdel; Abu Salah, Khalid M; Zourob, Mohammed

2017-12-18

The work describes a fluorescence-based study for mapping the highest affinity truncated aptamer from the full length sequence and its integration in a graphene oxide platform for the detection of Salmonella enteriditis. To identify the best truncated sequence, molecular beacons and a displacement assay design are applied. In the fluorescence displacement assay, the truncated aptamer was hybridized with fluorescein and quencher-labeled complementary sequences to form a fluorescence/quencher pair. In the presence of S. enteritidis, the aptamer dissociates from the complementary labeled oligonucleotides and thus, the fluorescein/quencher pair becomes physically separated. This leads to an increase in fluorescence intensity. One of the truncated aptamers identified has a 2-fold lower dissociation constant (3.2 nM) compared to its full length aptamer (6.3 nM). The truncated aptamer selected in this process was used to develop a fluorometric graphene oxide (GO) based assay. If fluorescein-labeled aptamer is adsorbed on GO via π stacking interaction, fluorescence is quenched. However, in the presence of target (S. enteriditis), the labeled aptamers is released from surface to form a stable complex with the bacteria and fluorescence is restored, depending on the quantity of bacteria being present. The resulting assay has an unsurpassed detection limit of 25 cfu·mL -1 in the best case. The cross reactivity to Salmonella typhimurium, Staphylococcus aureus and Escherichia coli is negligible. The assay was applied to analyze doped milk samples for and gave good recovery. Thus, we believe that the truncated aptamer/graphene oxide platform is a potential tool for the detection of S. Enteritidis. Graphical abstract Fluorescently labelled aptamer against Salmonella enteritidis was adsorbed on the surface of graphene oxide by π-stacking interaction. This results in quenching of the fluorescence of the label. Addition of Salmonella enteritidis restores fluorescence, and this effect is used for quantification of this food-borne pathogen.

Fluorescent Applications to Crystallization

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Forsythe, Elizabeth; Achari, Aniruddha

2006-01-01

By covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and tests with model proteins have shown that labeling u to 5 percent of the protein molecules does not affect the X-ray data quality obtained . The presence of the trace fluorescent label gives a number of advantages. Since the label is covalently attached to the protein molecules, it "tracks" the protein s response to the crystallization conditions. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a darker background. Non-protein structures, such as salt crystals, do not show up under fluorescent illumination. Crystals have the highest protein concentration and are readily observed against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. Preliminary tests, using model proteins, indicates that we can use high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that more rapid amorphous precipitation kinetics may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Experiments are now being carried out to test this approach using a wider range, of proteins. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons.

Optimization of Antibody-Conjugated Magnetic Nanoparticles for Target Preconcentration and Immunoassays

DTIC Science & Technology

2010-01-01

protein, AlexaFluor647– chicken IgG (Alexa647–chick IgG). Antibody-labeled MNPs (Alexa647– chick–MNPs) were used to preconcentrate the target via magnetic...separation and as the tracer to dem- onstrate binding to slides modified with anti- chicken IgG as a capture agent. A full optimization study of the...magnetically assisted transport evanescent field fluoroimmunoassay; Alexa647–chick–MNPs, MNPs functionalized with fluorescently labeled target chicken IgG

Human induced pluripotent stem cells labeled with fluorescent magnetic nanoparticles for targeted imaging and hyperthermia therapy for gastric cancer.

PubMed

Li, Chao; Ruan, Jing; Yang, Meng; Pan, Fei; Gao, Guo; Qu, Su; Shen, You-Lan; Dang, Yong-Jun; Wang, Kan; Jin, Wei-Lin; Cui, Da-Xiang

2015-09-01

Human induced pluripotent stem (iPS) cells exhibit great potential for generating functional human cells for medical therapies. In this paper, we report for use of human iPS cells labeled with fluorescent magnetic nanoparticles (FMNPs) for targeted imaging and synergistic therapy of gastric cancer cells in vivo. Human iPS cells were prepared and cultured for 72 h. The culture medium was collected, and then was co-incubated with MGC803 cells. Cell viability was analyzed by the MTT method. FMNP-labeled human iPS cells were prepared and injected into gastric cancer-bearing nude mice. The mouse model was observed using a small-animal imaging system. The nude mice were irradiated under an external alternating magnetic field and evaluated using an infrared thermal mapping instrument. Tumor sizes were measured weekly. iPS cells and the collected culture medium inhibited the growth of MGC803 cells. FMNP-labeled human iPS cells targeted and imaged gastric cancer cells in vivo, as well as inhibited cancer growth in vivo through the external magnetic field. FMNP-labeled human iPS cells exhibit considerable potential in applications such as targeted dual-mode imaging and synergistic therapy for early gastric cancer.

Developing a novel fiber optic fluorescence device for multiplexed high-throughput cytotoxic screening.

PubMed

Lee, Dennis; Barnes, Stephen

2010-01-01

The need for new pharmacological agents is unending. Yet the drug discovery process has changed substantially over the past decade and continues to evolve in response to new technologies. There is presently a high demand to reduce discovery time by improving specific lab disciplines and developing new technology platforms in the area of cell-based assay screening. Here we present the developmental concept and early stage testing of the Ab-Sniffer, a novel fiber optic fluorescence device for high-throughput cytotoxicity screening using an immobilized whole cell approach. The fused silica fibers are chemically functionalized with biotin to provide interaction with fluorescently labeled, streptavidin functionalized alginate-chitosan microspheres. The microspheres are also functionalized with Concanavalin A to facilitate binding to living cells. By using lymphoma cells and rituximab in an adaptation of a well-known cytotoxicity protocol we demonstrate the utility of the Ab-Sniffer for functional screening of potential drug compounds rather than indirect, non-functional screening via binding assay. The platform can be extended to any assay capable of being tied to a fluorescence response including multiple target cells in each well of a multi-well plate for high-throughput screening.

Cryo-imaging of fluorescently labeled single cells in a mouse

NASA Astrophysics Data System (ADS)

Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

2009-02-01

We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron-scale, fluorescence, and bright field image data. Here we describe our image preprocessing, analysis, and visualization techniques. Processing improves axial resolution, reduces subsurface fluorescence by 97%, and enables single cell detection and counting. High quality 3D volume renderings enable us to evaluate cell distribution patterns. Applications include the myriad of biomedical experiments using fluorescent reporter gene and exogenous fluorophore labeling of cells in applications such as stem cell regenerative medicine, cancer, tissue engineering, etc.

A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags.

PubMed

Müller, Andreas; Neukam, Martin; Ivanova, Anna; Sönmez, Anke; Münster, Carla; Kretschmar, Susanne; Kalaidzidis, Yannis; Kurth, Thomas; Verbavatz, Jean-Marc; Solimena, Michele

2017-02-02

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

Rational design of a monomeric and photostable far-red fluorescent protein for fluorescence imaging in vivo.

PubMed

Yu, Dan; Dong, Zhiqiang; Gustafson, William Clay; Ruiz-González, Rubén; Signor, Luca; Marzocca, Fanny; Borel, Franck; Klassen, Matthew P; Makhijani, Kalpana; Royant, Antoine; Jan, Yuh-Nung; Weiss, William A; Guo, Su; Shu, Xiaokun

2016-02-01

Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue-shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP-labeled nucleus and tdTomato-labeled plasma membrane, time-lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two-color protein labeling in living cells and in two-color tumor labeling in mice. © 2015 The Protein Society.

Effect of Dye and Conjugation Chemistry on the Biodistribution Profile of Near-Infrared-Labeled Nanobodies as Tracers for Image-Guided Surgery.

PubMed

Debie, Pieterjan; Van Quathem, Jannah; Hansen, Inge; Bala, Gezim; Massa, Sam; Devoogdt, Nick; Xavier, Catarina; Hernot, Sophie

2017-04-03

Advances in optical imaging technologies have stimulated the development of near-infrared (NIR) fluorescently labeled targeted probes for use in image-guided surgery. As nanobodies have already proven to be excellent candidates for molecular imaging, we aimed in this project to design NIR-conjugated nanobodies targeting the tumor biomarker HER2 for future applications in this field and to evaluate the effect of dye and dye conjugation chemistry on their pharmacokinetics during development. IRDye800CW or IRdye680RD were conjugated either randomly (via lysines) or site-specifically (via C-terminal cysteine) to the anti-HER2 nanobody 2Rs15d. After verification of purity and functionality, the biodistribution and tumor targeting of the NIR-nanobodies were assessed in HER2-positive and -negative xenografted mice. Site-specifically IRDye800CW- and IRdye680RD-labeled 2Rs15d as well as randomly labeled 2Rs15d-IRDye680RD showed rapid tumor accumulation and low nonspecific uptake, resulting in high tumor-to-muscle ratios at early time points (respectively 6.6 ± 1.0, 3.4 ± 1.6, and 3.5 ± 0.9 for HER2-postive tumors at 3 h p.i., while <1.0 for HER2-negative tumors at 3 h p.i., p < 0.05). Contrarily, using the randomly labeled 2Rs15d-IRDye800CW, HER2-positive and -negative tumors could only be distinguished after 24 h due to high nonspecific signals. Moreover, both randomly labeled 2Rs15d nanobodies were not only cleared via the kidneys but also partially via the hepatobiliary route. In conclusion, near-infrared fluorescent labeling of nanobodies allows rapid, specific, and high contrast in vivo tumor imaging. Nevertheless, the fluorescent dye as well as the chosen conjugation strategy can affect the nanobodies' properties and consequently have a major impact on their pharmacokinetics.

Detection of Sessile Serrated Adenomas in the Proximal Colon Using Wide-Field Fluorescence Endoscopy.

PubMed

Joshi, Bishnu P; Dai, Zhenzhen; Gao, Zhenghong; Lee, Jeong Hoon; Ghimire, Navin; Chen, Jing; Prabhu, Anoop; Wamsteker, Erik J; Kwon, Richard S; Elta, Grace H; Stoffel, Elena M; Pant, Asha; Kaltenbach, Tonya; Soetikno, Roy M; Appelman, Henry D; Kuick, Rork; Turgeon, D Kim; Wang, Thomas D

2017-04-01

Many cancers in the proximal colon develop via from sessile serrated adenomas (SSAs), which have flat, subtle features that are difficult to detect with conventional white-light colonoscopy. Many SSA cells have the V600E mutation in BRAF. We investigated whether this feature could be used with imaging methods to detect SSAs in patients. We used phage display to identify a peptide that binds specifically to SSAs, using subtractive hybridization with HT29 colorectal cancer cells containing the V600E mutation in BRAF and Hs738.St/Int cells as a control. Binding of fluorescently labeled peptide to colorectal cancer cells was evaluated with confocal fluorescence microscopy. Rats received intra-colonic 0.0086 mg/kg, 0.026 mg/kg, or 0.86 mg/kg peptide or vehicle and morbidity, mortality, and injury were monitored twice daily to assess toxicity. In the clinical safety study, fluorescently labeled peptide was topically administered, using a spray catheter, to the proximal colon of 25 subjects undergoing routine outpatient colonoscopies (3 subjects were given 2.25 μmol/L and 22 patients were given 76.4 μmol/L). We performed blood cell count, chemistry, liver function, and urine analyses approximately 24 hours after peptide administration. In the clinical imaging study, 38 subjects undergoing routine outpatient colonoscopies, at high risk for colorectal cancer, or with a suspected unresected proximal colonic polyp, were first evaluated by white-light endoscopy to identify suspicious regions. The fluorescently labeled peptide (76.4 μmol/L) was administered topically to proximal colon, unbound peptide was washed away, and white-light, reflectance, and fluorescence videos were recorded digitally. Fluorescence intensities of SSAs were compared with those of normal colonic mucosa. Endoscopists resected identified lesions, which were analyzed histologically by gastrointestinal pathologists (reference standard). We also analyzed the ability of the peptide to identify SSAs vs adenomas, hyperplastic polyps, and normal colonic mucosa in specimens obtained from the tissue bank at the University of Michigan. We identified the peptide sequence KCCFPAQ and measured an apparent dissociation constant of K d  = 72 nM and an apparent association time constant of K = 0.174 min -1 (5.76 minutes). During fluorescence imaging of patients during endoscopy, regions of SSA had 2.43-fold higher mean fluorescence intensity than that for normal colonic mucosa. Fluorescence labeling distinguished SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity. The peptide had no observed toxic effects in animals or patients. In the analysis of ex vivo specimens, peptide bound to SSAs had significantly higher mean fluorescence intensity than to hyperplastic polyps. We have identified a fluorescently labeled peptide that has no observed toxic effects in animals or humans and can be used for wide-field imaging of lesions in the proximal colon. It distinguishes SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity. This targeted imaging method might be used in early detection of premalignant serrated lesions during routine colonoscopies. ClinicalTrials.gov ID: NCT02156557. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Detection of Sessile Serrated Adenomas in Proximal Colon Using Wide-field Fluorescence Endoscopy

PubMed Central

Joshi, Bishnu P.; Dai, Zhenzhen; Gao, Zhenghong; Lee, Jeong Hoon; Ghimire, Navin; Chen, Jing; Prabhu, Anoop; Wamsteker, Erik J.; Kwon, Richard S.; Elta, Grace H.; Stoffel, Elena M.; Pant, Asha; Kaltenbach, Tonya; Soetikno, Roy M.; Appelman, Henry D.; Kuick, Rork; Turgeon, D. Kim; Wang, Thomas D.

2018-01-01

Background & Aims Many cancers in the proximal colon develop via from sessile serrated adenomas (SSAs), which have flat, subtle features that are difficult to detect with conventional white-light colonoscopy. Many SSA cells have the V600E mutation in BRAF. We investigated whether this feature could be used with imaging methods to detect SSAs in patients. Methods We used phage display to identify a peptide that binds specifically to SSAs, using subtractive hybridization with HT29 colorectal cancer cells containing the V600E mutation in BRAF and Hs738.St/Int cells as a control. Binding of fluorescently labeled peptide to colorectal cancer cells was evaluated with confocal fluorescence microscopy. Rats received intra-colonic 0.0086 mg/kg, 0.026 mg/kg, or 0.86 mg/kg peptide or vehicle and morbidity, mortality, and injury were monitored twice daily to assess toxicity. In the clinical safety study, fluorescently labeled peptide was topically administered, using a spray catheter, to the proximal colon of 25 subjects undergoing routine outpatient colonoscopies (3 subjects were given 2.25 µmol/L and 22 patients were given 76.4 µmol/L). We performed blood cell count, chemistry, liver function, and urine analyses approximately 24 hrs after peptide administration. In the clinical imaging study, 38 subjects undergoing routine outpatient colonoscopies, at high risk for colorectal cancer, or with a suspected unresected proximal colonic polyp, were first evaluated by white-light endoscopy, to identify suspicious regions. The fluorescently labeled peptide (76.4 µmol/L) was administered topically to proximal colon, unbound peptide was washed away, and white-light, reflectance, and fluorescence videos were recorded digitally. Fluorescence intensities of SSAs were compared with those of normal colonic mucosa. Endoscopists resected identified lesions, which were analyzed histologically by gastrointestinal pathologists (reference standard). We also analyzed the ability of the peptide to identify SSAs vs adenomas, hyperplastic polyps, and normal colonic mucosa in specimens obtained from the tissue bank at the University of Michigan. Results We identified the peptide sequence KCCFPAQ, and measured an apparent dissociation constant of kd = 72 nM and an apparent association time constant of k = 0.174 min−1 (5.76 min). During fluorescence imaging of patients during endoscopy, regions of SSA had 2.43-fold higher mean fluorescence intensity than that for normal colonic mucosa. Fluorescence labeling distinguished SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity. The peptide had no observed toxic effects in animals or patients. In the analysis of ex vivo specimens, peptide bound to SSAs had significantly higher mean fluorescence intensity than to hyperplastic polyps. Conclusions We have identified a fluorescently labeled peptide that has no observed toxic effects in animals or humans and can be used for wide-field imaging of lesions in the proximal colon. It distinguishes SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity. This targeted imaging method might be used in early detection of pre-malignant serrated lesions during routine colonoscopies. ClinicalTrials.gov no: NCT02156557 PMID:28012848

Novel functionalized fluorescent polymeric nanoparticles for immobilization of biomolecules

NASA Astrophysics Data System (ADS)

Jain, Swati; Chattopadhyay, Sruti; Jackeray, Richa; Abid, C. K. V. Zainul; Singh, Harpal

2013-07-01

Novel, size controlled fluorescent polymeric nanoparticles (FPNP) were synthesized having acetoacetoxy functionality on the surface for immobilization of biomolecules which can be utilized as biomarkers and labels in fluoroimmunoassays. Core-shell nanoparticles of poly(styrene, St-methyl methacrylate, MMA-acetoacetoxy ethyl methacrylate, AAEM), stabilized by various concentrations of surfactant, sodium lauryl sulphate (SLS), were obtained by facile miniemulsion co-polymerization encapsulated with pyrene molecules in their hydrophobic core. Analytical, spectroscopic and imaging characterization techniques revealed the formation of stable, monodisperse, spherical nano sized particles exhibiting high luminescence properties. Particles with 1% SLS (S1) showed good dispersion stability and fluorescence intensity and were chosen as ideal candidates for further immobilization studies. Steady state fluorescence studies showed 10 times higher fluorescence intensity of S1 nanoparticles than that of pyrene solution in solvent-toluene at the same concentration. Environmental factors such as pH, ionic strength and time were found to have no effect on fluorescence intensity of FPNPs. Surface β-di-ketone groups were utilized for the covalent immobilization of enzyme conjugated antibodies without any activation or pre-treatment of nanoparticles.Novel, size controlled fluorescent polymeric nanoparticles (FPNP) were synthesized having acetoacetoxy functionality on the surface for immobilization of biomolecules which can be utilized as biomarkers and labels in fluoroimmunoassays. Core-shell nanoparticles of poly(styrene, St-methyl methacrylate, MMA-acetoacetoxy ethyl methacrylate, AAEM), stabilized by various concentrations of surfactant, sodium lauryl sulphate (SLS), were obtained by facile miniemulsion co-polymerization encapsulated with pyrene molecules in their hydrophobic core. Analytical, spectroscopic and imaging characterization techniques revealed the formation of stable, monodisperse, spherical nano sized particles exhibiting high luminescence properties. Particles with 1% SLS (S1) showed good dispersion stability and fluorescence intensity and were chosen as ideal candidates for further immobilization studies. Steady state fluorescence studies showed 10 times higher fluorescence intensity of S1 nanoparticles than that of pyrene solution in solvent-toluene at the same concentration. Environmental factors such as pH, ionic strength and time were found to have no effect on fluorescence intensity of FPNPs. Surface β-di-ketone groups were utilized for the covalent immobilization of enzyme conjugated antibodies without any activation or pre-treatment of nanoparticles. Electronic supplementary information (ESI) available: Resulting ATR-FTIR spectrum and procedure to study fluorescence of nanoparticles, effect of particle size, concentration, pH, ionic strength and time on Fl intensity of FPNP. See DOI: 10.1039/c3nr34100c

Aptamer-fluorescent silica nanoparticles bioconjugates based dual-color flow cytometry for specific detection of Staphylococcus aureus.

PubMed

He, Xiaoxiao; Li, Yuhong; He, Dinggen; Wang, Kemin; Shangguan, Jingfang; Shi, Hui

2014-07-01

This paper describes a sensitive and specific determination strategy for Staphylococcus aureus (S. aureus) detection using aptamer recognition and fluorescent silica nanoparticles (FSiNPs) label based dual-color flow cytometry assay (Aptamer/FSiNPs-DCFCM). In the protocol, an aptamer, having high affinity to S. aureus, was first covalently immobilized onto chloropropyl functionalized FSiNPs through a click chemistry approach to generate aptamer-nanoparticles bioconjugates (Aptamer/FSiNPs). Next, S. aureus was incubated with Aptamer/FSiNPs, and then stained with SYBR Green I (a special staining material for the duplex DNA). Upon target binding and nucleic acid staining with SYBR Green I, the S. aureus was determined using two-color flow cytometry. The method took advantage of the specificity of aptamer, signal amplification of FSiNPs label and decreased false positives of two-color flow cytometry assay. It was demonstrated that these Aptamer/FSiNPs could efficiently recognize and fluorescently label target S. aureus. Through multiparameter determination with flow cytometry, this assay allowed for detection of as low as 1.5 x 10(2) and 7.6 x 10(2) cells mL(-1) S. aureus in buffer and spiked milk, respectively, with higher sensitivity than the Aptamer/FITC based flow cytometry.

In vitro and in vivo documentation of quantum dots labeled Trypanosoma cruzi--Rhodnius prolixus interaction using confocal microscopy.

PubMed

Feder, Denise; Gomes, Suzete A O; de Thomaz, André A; Almeida, Diogo B; Faustino, Wagner M; Fontes, Adriana; Stahl, Cecília V; Santos-Mallet, Jacenir R; Cesar, Carlos L

2009-12-01

Semiconductor quantum dots (QDs) are highly fluorescent nanocrystals markers that allow long photobleaching and do not destroy the parasites. In this paper, we used fluorescent core shell quantum dots to perform studies of live parasite-vector interaction processes without any observable effect on the vitality of parasites. These nanocrystals were synthesized in aqueous medium and physiological pH, which is very important for monitoring live cells activities, and conjugated with molecules such as lectins to label specific carbohydrates involved on the parasite-vector interaction. These QDs were successfully used for the study of in vitro and in vivo interaction of Trypanosoma cruzi and the triatomine Rhodnius prolixus. These QDs allowed us to acquire real time confocal images sequences of live T. cruzi-R. prolixus interactions for an extended period, causing no damage to the cells. By zooming to the region of interest, we have been able to acquire confocal images at the three to four frames per second rate. Our results show that QDs are physiological fluorescent markers capable to label living parasites and insect vector cells. QDs can be functionalized with lectins to specifically mark surface carbohydrates on perimicrovillar membrane of R. prolixus to follow, visualize, and understand interaction between vectors and its parasites in real-time.

Probing effects of pressure release on virus capture during virus filtration using confocal microscopy.

PubMed

Dishari, Shudipto K; Venkiteshwaran, Adith; Zydney, Andrew L

2015-10-01

Virus filtration is used to ensure drug safety in the production of biotherapeutics. Several recent studies have shown a dramatic decrease in virus retention as a result of a process disruption, e.g., a transient pressure release. In this work, a novel two-label fluorescence technique was developed to probe virus capture within virus filtration membranes using confocal microscopy. Experiments were performed with Ultipor® DV20, Viresolve® Pro, and Viresolve® NFP membranes using bacteriophage φx174 as a model virus. The filters were challenged with two batches of fluorescently labeled phage: one labeled with red dye (Cy5) and one with green dye (SYBR Gold) to visualize captured phage from before and after the pressure release. The capture patterns seen in the confocal images were a strong function of the underlying membrane morphology and pore structure. The DV20 and Viresolve® NFP showed migration of previously captured phage further into the filter, consistent with the observed loss of virus retention after the pressure release. In contrast, there was no migration of captured virus in the Viresolve® Pro membranes, and these filters were also the only ones to show stable virus retention after a pressure release. The direct visualization of virus capture using the two-label fluorescence technique provides unique insights into the factors controlling the retention characteristics of virus filters with different pore structure. © 2015 Wiley Periodicals, Inc.

Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy

PubMed Central

Shimura, Mari; Shindou, Hideo; Szyrwiel, Lukasz; Tokuoka, Suzumi M.; Hamano, Fumie; Matsuyama, Satoshi; Okamoto, Mayumi; Matsunaga, Akihiro; Kita, Yoshihiro; Ishizaka, Yukihito; Yamauchi, Kazuto; Kohmura, Yoshiki; Lobinski, Ryszard; Shimizu, Isao; Shimizu, Takao

2016-01-01

Fatty acids are taken up by cells and incorporated into complex lipids such as neutral lipids and glycerophospholipids. Glycerophospholipids are major constituents of cellular membranes. More than 1000 molecular species of glycerophospholipids differ in their polar head groups and fatty acid compositions. They are related to cellular functions and diseases and have been well analyzed by mass spectrometry. However, intracellular imaging of fatty acids and glycerophospholipids has not been successful due to insufficient resolution using conventional methods. Here, we developed a method for labeling fatty acids with bromine (Br) and applied scanning X-ray fluorescence microscopy (SXFM) to obtain intracellular Br mapping data with submicrometer resolution. Mass spectrometry showed that cells took up Br-labeled fatty acids and metabolized them mainly into glycerophospholipids in CHO cells. Most Br signals observed by SXFM were in the perinuclear region. Higher resolution revealed a spot-like distribution of Br in the cytoplasm. The current method enabled successful visualization of intracellular Br-labeled fatty acids. Single-element labeling combined with SXFM technology facilitates the intracellular imaging of fatty acids, which provides a new tool to determine dynamic changes in fatty acids and their derivatives at the single-cell level.—Shimura, M., Shindou, H., Szyrwiel, L., Tokuoka, S. M., Hamano, F., Matsuyama, S., Okamoto, M., Matsunaga, A., Kita, Y., Ishizaka, Y., Yamauchi, K., Kohmura, Y., Lobinski, R., Shimizu, I., Shimizu, T. Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy. PMID:27601443

Confocal fluorescence microscopy in a murine model of microdissection testicular sperm extraction to improve sperm retrieval.

PubMed

Smith, Ryan P; Lowe, Greg J; Kavoussi, Parviz K; Steers, William D; Costabile, Raymond A; Herr, John C; Shetty, Jagathpala; Lysiak, Jeffrey J

2012-05-01

Microdissection testicular sperm extraction markedly improves the sperm retrieval rates in men with nonobstructive azoospermia. However, localizing sperm foci can be time-consuming and it is not always successful. Fiberoptic confocal fluorescent microscopy offers the advantage of rapid in vivo detection of fluorescently labeled sperm in the seminiferous tubules. After establishing the feasibility of fiberoptic confocal fluorescent microscopy to identify antibody labeled sperm in vivo C57/B6 mice underwent intraperitoneal injection of busulfan to induce azoospermia. During spermatogenesis reestablishment at approximately 16 weeks the mice were anesthetized and the testes were delivered through a low midline incision. Fluorescein isothiocyanate labeled antibody to intra-acrosomal protein Hs-14 was injected retrograde into a single murine rete testis. The testes were imaged in vivo with fiberoptic confocal fluorescent microscopy and sperm foci were detected. The respective seminiferous tubules were excised and squash prepared for immunofluorescence microscopy. Sperm foci were identified in the testis injected with fluorescently tagged antibody by in vivo fiberoptic confocal fluorescence microscopy. The contralateral control testis of each mouse showed no specific signal. Immunofluorescence microscopy of the excised tubules provided morphological confirmation of the presence of labeled sperm with an absence in controls. Findings were consistent in the feasibility portion of the study and in the busulfan model of nonobstructive azoospermia. Fiberoptic confocal fluorescent microscopy was feasible during microdissection testicular sperm extraction in an azoospermic mouse model to identify fluorescently labeled sperm in vivo. Translation to the clinical setting could decrease operative time and improve the sperm harvest rate. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Aptamer-Functionalized Fluorescent Silica Nanoparticles for Highly Sensitive Detection of Leukemia Cells

NASA Astrophysics Data System (ADS)

Tan, Juntao; Yang, Nuo; Hu, Zixi; Su, Jing; Zhong, Jianhong; Yang, Yang; Yu, Yating; Zhu, Jianmeng; Xue, Dabin; Huang, Yingying; Lai, Zongqiang; Huang, Yong; Lu, Xiaoling; Zhao, Yongxiang

2016-06-01

A simple, highly sensitive method to detect leukemia cells has been developed based on aptamer-modified fluorescent silica nanoparticles (FSNPs). In this strategy, the amine-labeled Sgc8 aptamer was conjugated to carboxyl-modified FSNPs via amide coupling between amino and carboxyl groups. Sensitivity and specificity of Sgc8-FSNPs were assessed using flow cytometry and fluorescence microscopy. These results showed that Sgc8-FSNPs detected leukemia cells with high sensitivity and specificity. Aptamer-modified FSNPs hold promise for sensitive and specific detection of leukemia cells. Changing the aptamer may allow the FSNPs to detect other types of cancer cells.

Intracellular in situ labeling of TiO 2 nanoparticles for fluorescence microscopy detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Koshonna; Thurn, Ted; Xin, Lun

Titanium dioxide (TiO 2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. In this paper, we describe two in situ posttreatmentmore » labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyneconjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Finally and therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.« less

Intracellular in situ labeling of TiO 2 nanoparticles for fluorescence microscopy detection

DOE PAGES

Brown, Koshonna; Thurn, Ted; Xin, Lun; ...

2017-07-19

Titanium dioxide (TiO 2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. In this paper, we describe two in situ posttreatmentmore » labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyneconjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Finally and therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.« less

MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection.

PubMed

Presley, Andrew D; Fuller, Kathryn M; Arriaga, Edgar A

2003-08-05

MitoTracker Green (MTG) is a mitochondrial-selective fluorescent label commonly used in confocal microscopy and flow cytometry. It is expected that this dye selectively accumulates in the mitochondrial matrix where it covalently binds to mitochondrial proteins by reacting with free thiol groups of cysteine residues. Here we demonstrate that MTG can be used as a protein labeling reagent that is compatible with a subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Although the MTG-labeled proteins and MTG do not seem to electrophoretically separate, an enhancement in fluorescence intensity of the product indicates that only proteins with free thiol groups are capable of reacting with MTG. In addition we propose that MTG is a partially selective label towards some mitochondrial proteins. This selectivity stems from the high MTG concentration in the mitochondrial matrix that favors alkylation of the available thiol groups in this subcellular compartment. To that effect we treated mitochondria-enriched fractions that had been prepared by differential centrifugation of an NS-1 cell lysate. This fraction was solubilized with an SDS-containing buffer and analyzed by CE-LIF. The presence of a band with fluorescence stronger than MTG alone also indicated the presence of an MTG-protein product. Confirming that MTG is labeling mitochondrial proteins was done by treating the solubilized mitochondrial fraction with 5-furoylquinoline-3-carboxaldehyde (FQ), a fluorogenic reagent that reacts with primary amino groups, and analysis by CE-LIF using two separate detection channels: 520 nm for MTG-labeled species and 635 nm for FQ-labeled species. In addition, these results indicate that MTG labels only a subset of proteins in the mitochondria-enriched fraction.

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform.

PubMed

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-12-14

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.

Aminoquinolines as fluorescent labels for hydrophilic interaction liquid chromatography of oligosaccharides.

PubMed

Struwe, Weston B; Rudd, Pauline M

2012-08-01

In this study, we investigated the potential of four different aminoquinoline (AQ) compounds as fluorescent labels for glycan analysis using hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection (FLD). We confirmed the optimal excitation and emission wavelengths of 3-AQ and 6-AQ conjugated to glycan standards using three-dimensional fluorescent spectral scanning. The optimal excitation and emission wavelengths for 6-AQ were confirmed at λ(ex)=355 nm and λ(em)=440 nm. We concluded that the optimal wavelengths for 3-AQ were λ(ex)=355 nm and λ(em)=420 nm, which differed considerably from the wavelengths applied in previous reports. HILIC-FLD chromatograms using experimentally determined wavelengths were similar to 2-aminobenzamide controls, but the peak capacity and resolution differed significantly when published 3-AQ λ(ex/em) values were applied. Furthermore, we found that 5-AQ and 8-AQ labeled maltohexaose did not display any fluorescent properties when used as a carbohydrate tag for HPLC analysis. Finally, we applied experimentally determined wavelengths to 3-AQ labeled N-glycans released from human IgG to illustrate changes in retention time as well as to demonstrate that AQ labeling is applicable to complex sample analysis via exoglycosidase sequencing.

Novel water-soluble near-infrared cyanine dyes: synthesis, spectral properties, and use in the preparation of internally quenched fluorescent probes.

PubMed

Bouteiller, Cédric; Clavé, Guillaume; Bernardin, Aude; Chipon, Bertrand; Massonneau, Marc; Renard, Pierre-Yves; Romieu, Anthony

2007-01-01

In this paper, we describe the synthesis and the photophysical properties of two novel near-infrared (NIR) cyanine dyes (NIR5.5-2 and NIR7.0-2) which are water soluble potential substitutes of the commercially available Cy 5.5 and Cy 7.0 fluorescent labels respectively. For each one of these cyanine dyes, the synthetic strategy relies on the postsynthetic derivatization of a cyanine precursor in order to introduce the key functionalities required for bioconjugation of these NIR fluorophores. For NIR5.5-2, a reactive amino group was acylated with an original trisulfonated linker for water solubility. For NIR7.0-2, a vinylic chlorine atom was derivatized through a SRN1 reaction for the introduction of a monoreactive carboxyl group for labeling purposes. Unexpectedly, when these two fluorophores were closely associated within a peptidic architecture, mutual fluorescence quenching between NIR5.5-2 and NIR7.0-2 was observed both at 705 (NIR5.5-2) and 798 nm (NIR7.0-2). On the basis of this property, a novel internally quenched caspase-3-sensitive NIR fluorescent probe was prepared.

Solvothermal modification of BiOCl nanosheets with Bi nanoparticles using ascorbic acid as reductant and the superoxide radicals dominated photocatalytic performance

NASA Astrophysics Data System (ADS)

Cui, Zhankui; Gao, Keke; Ge, Suxiang; Fa, Wenjun

2017-11-01

BiOCl nanosheets were solvothermally modified with Bi nanoparticles (NPs) using ascorbic acid as the reductant. The structures of Bi/BiOCl composites were characterized by XRD, Raman spectroscopy, FTIR spectroscopy and SEM. The light absorption properties were measured by UV-vis-NIR spectroscopy. The photocatalytic performances were evaluated by photodegrading methyl orange (MO) and the photocatalytic mechanism was investigated using trapping experiments and a fluorescent probe method. The results show that Bi NPs are uniformly distributed on the surfaces of BiOCl nanosheets and the modification amount of Bi NPs could be well controlled because of the mild property of ascorbic acid as reducing agent. The photocatalytic activities for the composites are improved obviously and the best photocatalytic performance is obtained when the weight ratio of Bi and BiOCl is1:10 and the photochemical reaction rate is 3.5 times that of pure BiOCl nanosheets and 19.7 times of Bi powders. The enhanced photocatalytic efficiency is ascribed to the favorable formation of dominant \\cdot O2- radicals caused by the increased photoinduced electrons from both Bi NPs and BiOCl nanosheets.

Label-free imaging to study phenotypic behavioural traits of cells in complex co-cultures

NASA Astrophysics Data System (ADS)

Suman, Rakesh; Smith, Gabrielle; Hazel, Kathryn E. A.; Kasprowicz, Richard; Coles, Mark; O'Toole, Peter; Chawla, Sangeeta

2016-02-01

Time-lapse imaging is a fundamental tool for studying cellular behaviours, however studies of primary cells in complex co-culture environments often requires fluorescent labelling and significant light exposure that can perturb their natural function over time. Here, we describe ptychographic phase imaging that permits prolonged label-free time-lapse imaging of microglia in the presence of neurons and astrocytes, which better resembles in vivo microenvironments. We demonstrate the use of ptychography as an assay to study the phenotypic behaviour of microglial cells in primary neuronal co-cultures through the addition of cyclosporine A, a potent immune-modulator.

Multifunctional recombinant phycobiliprotein-based fluorescent constructs and phycobilisome display

DOEpatents

Glazer, Alexander N.; Cai, Yuping

2007-01-30

The invention provides multifunctional fusion constructs which are rapidly incorporated into a macromolecular structure such as a phycobilisome such that the fusion proteins are separated from one another and unable to self-associate. The invention provides methods and compositions for displaying a functional polypeptide domain on an oligomeric phycobiliprotein, including fusion proteins comprising a functional displayed domain and a functional phycobiliprotein domain incorporated in a functional oligomeric phycobiliprotein. The fusion proteins provide novel specific labeling reagents.

Multifunctional recombinant phycobiliprotein-based fluorescent constructs and phycobilisome display

DOEpatents

Glazer, Alexander N.; Cai, Yuping

2007-02-13

The invention provides multifunctional fusion constructs which are rapidly incorporated into a macromolecular structure such as a phycobilisome such that the fusion proteins are separated from one another and unable to self-associate. The invention provides methods and compositions for displaying a functional polypeptide domain on an oligomeric phycobiliprotein. including fusion proteins comprising a functional displayed domain and a functional phycobiliprotein domain incorporated in a functional oligomeric phycobiliprotein. The fusion proteins provide novel specific labeling reagents.

Multifunctional recombinant phycobiliprotein-based fluorescent constructs and phycobilisome display

DOEpatents

Glazer, Alexander N.; Cai, Yuping

2003-11-18

The invention provides multifunctional fusion constructs which are rapidly incorporated into a macromolecular structure such as a phycobilisome such that the fusion proteins are separated from one another and unable to self-associate. The invention provides methods and compositions for displaying a functional polypeptide domain on an oligomeric phycobiliprotein, including fusion proteins comprising a functional displayed domain and a functional phycobiliprotein domain incorporated in a functional oligomeric phycobiliprotein. The fusion proteins provide novel specific labeling reagents.

Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

PubMed

Murgha, Yusuf; Beliveau, Brian; Semrau, Kassandra; Schwartz, Donald; Wu, Chao-Ting; Gulari, Erdogan; Rouillard, Jean-Marie

2015-06-01

Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

Organosilane-functionalized graphene quantum dots and their encapsulation into bi-layer hollow silica spheres for bioimaging applications.

PubMed

Wen, Ting; Yang, Baocheng; Guo, Yanzhen; Sun, Jing; Zhao, Chunmei; Zhang, Shouren; Zhang, Miao; Wang, Yonggang

2014-11-14

Graphene quantum dots (GQDs) represent an important class of luminescent quantum dots owing to their low toxicity and superior biocompatibility. Chemical functionalization of GQDs and subsequent combination with other materials further provide attractive techniques for advanced bioapplications. Herein, we report the facile fabrication of fluorescent organosilane-functionalized graphene quantum dots (Si-GQDs) and their embedding into mesoporous hollow silica spheres as a biolabel for the first time. Well-proportioned Si-GQDs with bright and excitation dependent tunable emissions in the visible region were obtained via a simple and economical solvothermal route adopting graphite oxide as a carbon source and 3-(2-aminoethylamino)-propyltrimethoxysilane as a surface modifier. The as-synthesized Si-GQDs can be well dispersed and stored in organic solvents, easily manufactured into transparent film and bulk form, and particularly provide great potential to be combined with other materials. As a proof-of-principle experiment, we demonstrate the successful incorporation of Si-GQDs into hollow mesoporous silica spheres and conduct preliminary cellular imaging experiments. Interestingly, the Si-GQDs not only serve as fluorescent chromophores in the composite material, but also play a crucial role in the formation of mesoporous hollow silica spheres with a distinctive bi-layer architecture. The layer thickness and optical properties can be precisely controlled by simply adjusting the silane coupling agent addition procedure in the preparation process. Our demonstration of low-cost Si-GQDs and their encapsulation into multifunctional composites may expand the applications of carbon-based nanomaterials for future biomedical imaging and other optoelectronic applications.

Silver nanoparticles-enhanced time-resolved fluorescence sensor for VEGF(165) based on Mn-doped ZnS quantum dots.

PubMed

Zhu, Dong; Li, Wei; Wen, Hong-Mei; Yu, Sheng; Miao, Zhao-Yi; Kang, An; Zhang, Aihua

2015-12-15

A silver nanoparticles (AgNPs)-enhanced time-resolved fluorescence (TR-FL) sensor based on long-lived fluorescent Mn-doped ZnS quantum dots (QDs) is developed for the sensitive detection of vascular endothelial growth factor-165 (VEGF165), a predominant cancer biomarker in cancer angiogenesis. The aptamers bond with the Mn-doped ZnS QDs and the BHQ-2 quencher-labelling strands hybridized in duplex are coupled with streptavidin (SA)-functionalized AgNPs to form the AgNPs-enhanced TR-FL sensor, showing lower fluorescence intensity in the duplex state due to the fluorescence resonance energy transfer (FRET) between the Mn-doped ZnS QDs and quenchers. Upon the addition of VEGF165, the BHQ-2 quencher-labelling strands of the duplex are displaced, leading to the disruption of the FRET. As a result, the fluorescence of the Mn-doped QDs within the proximity of the AgNPs is recovered. The FL signal can be measured free of the interference of short-lived background by setting appropriate delay time and gate time, which offers a signal with high signal-to-noise ratio in photoluminescent biodetection. Compared with the bare TR-FL sensor, the AgNPs-based TR-FL sensor showed a huge improvement in fluorescence based on metal-enhanced fluorescence (MEF) effect, and the sensitivity increased 11-fold with the detection limit of 0.08 nM. In addition, the sensor provided a wide range of linear detection from 0.1 nM to 16 nM. Copyright © 2015 Elsevier B.V. All rights reserved.

Frequency-domain photoacoustic and fluorescence microscopy: application on labeled and unlabeled cells

NASA Astrophysics Data System (ADS)

Langer, Gregor; Buchegger, Bianca; Jacak, Jaroslaw; Pfeffer, Karoline; Wohlfarth, Sven; Hannesschläger, Günther; Klar, Thomas A.; Berer, Thomas

2018-02-01

In this paper, multimodal optical-resolution frequency-domain photoacoustic and fluorescence scanning microscopy is presented on labeled and unlabeled cells. In many molecules, excited electrons relax radiatively and non-radiatively, leading to fluorescence and photoacoustic signals, respectively. Both signals can then be detected simultaneously. There also exist molecules, e.g. hemoglobin, which do not exhibit fluorescence, but provide photoacoustic signals solely. Other molecules, especially fluorescent dyes, preferentially exhibit fluorescence. The fluorescence quantum yield of a molecule and with it the strength of photoacoustic and fluorescence signals depends on the local environment, e.g. on the pH. Therefore, the local distribution of the simultaneously recorded photoacoustic and fluorescence signals may be used in order to obtain information about the local chemistry.

Reconstruction of thin fluorophore-filled capillaries in thick scattering medium using fluorescence diffuse optical tomography within the diffusion approximation

NASA Astrophysics Data System (ADS)

Desrochers, Johanne; Vermette, Patrick; Fontaine, Réjean; Bérubé-Lauzière, Yves

2009-02-01

Current efforts in tissue engineering target the growth of 3D volumes of tissue cultures in bioreactor conditions. Fluorescence optical tomography has the potential to monitor cells viability and tissue growth non-destructively directly within the bioreactor via bio-molecular fluorescent labelling strategies. We currently work on developing the imaging instrumentation for tissue cultures in bioreactor conditions. Previously, we localized in 3D thin fluorescent-labelled capillaries in a cylindrically shaped bioreactor phantom containing a diffusive medium with our time-of-flight localization technique. Here, we present our first reconstruction results of the spatial distribution of fluorophore concentrations for labelled capillaries embedded in a bioreactor phantom.

Achieving second order advantage with multi-way partial least squares and residual bi-linearization with total synchronous fluorescence data of monohydroxy-polycyclic aromatic hydrocarbons in urine samples.

PubMed

Calimag-Williams, Korina; Knobel, Gaston; Goicoechea, H C; Campiglia, A D

2014-02-06

An attractive approach to handle matrix interference in samples of unknown composition is to generate second- or higher-order data formats and process them with appropriate chemometric algorithms. Several strategies exist to generate high-order data in fluorescence spectroscopy, including wavelength time matrices, excitation-emission matrices and time-resolved excitation-emission matrices. This article tackles a different aspect of generating high-order fluorescence data as it focuses on total synchronous fluorescence spectroscopy. This approach refers to recording synchronous fluorescence spectra at various wavelength offsets. Analogous to the concept of an excitation-emission data format, total synchronous data arrays fit into the category of second-order data. The main difference between them is the non-bilinear behavior of synchronous fluorescence data. Synchronous spectral profiles change with the wavelength offset used for sample excitation. The work presented here reports the first application of total synchronous fluorescence spectroscopy to the analysis of monohydroxy-polycyclic aromatic hydrocarbons in urine samples of unknown composition. Matrix interference is appropriately handled by processing the data either with unfolded-partial least squares and multi-way partial least squares, both followed by residual bi-linearization. Copyright © 2013 Elsevier B.V. All rights reserved.

Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

PubMed Central

Kuzma-Kuzniarska, Maria; Yapp, Clarence; Pearson-Jones, Thomas W.; Jones, Andrew K.; Hulley, Philippa A.

2014-01-01

Abstract. Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures. PMID:24390370

Direct labeling of serum proteins by fluorescent dye for antibody microarray.

PubMed

Klimushina, M V; Gumanova, N G; Metelskaya, V A

2017-05-06

Analysis of serum proteome by antibody microarray is used to identify novel biomarkers and to study signaling pathways including protein phosphorylation and protein-protein interactions. Labeling of serum proteins is important for optimal performance of the antibody microarray. Proper choice of fluorescent label and optimal concentration of protein loaded on the microarray ensure good quality of imaging that can be reliably scanned and processed by the software. We have optimized direct serum protein labeling using fluorescent dye Arrayit Green 540 (Arrayit Corporation, USA) for antibody microarray. Optimized procedure produces high quality images that can be readily scanned and used for statistical analysis of protein composition of the serum. Copyright © 2017 Elsevier Inc. All rights reserved.

A Label-Free Fluorescent Array Sensor Utilizing Liposome Encapsulating Calcein for Discriminating Target Proteins by Principal Component Analysis

PubMed Central

Imamura, Ryota; Murata, Naoki; Shimanouchi, Toshinori; Yamashita, Kaoru; Fukuzawa, Masayuki; Noda, Minoru

2017-01-01

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots. PMID:28714873

A Label-Free Fluorescent Array Sensor Utilizing Liposome Encapsulating Calcein for Discriminating Target Proteins by Principal Component Analysis.

PubMed

Imamura, Ryota; Murata, Naoki; Shimanouchi, Toshinori; Yamashita, Kaoru; Fukuzawa, Masayuki; Noda, Minoru

2017-07-15

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots.

Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes.

PubMed

Segers-Nolten, G M J; Wyman, C; Wijgers, N; Vermeulen, W; Lenferink, A T M; Hoeijmakers, J H J; Greve, J; Otto, C

2002-11-01

We used scanning confocal fluorescence microscopy to observe and analyze individual DNA- protein complexes formed between human nucleotide excision repair (NER) proteins and model DNA substrates. For this purpose human XPA protein was fused to EGFP, purified and shown to be functional. Binding of EGFP-labeled XPA protein to a Cy3.5-labeled DNA substrate, in the presence and absence of RPA, was assessed quantitatively by simultaneous excitation and emission detection of both fluorophores. Co-localization of Cy3.5 and EGFP signals within one diffraction limited spot indicated complexes of XPA with DNA. Measurements were performed on samples in a 1% agarose matrix in conditions that are compatible with protein activity and where reactions can be studied under equilibrium conditions. In these samples DNA alone was freely diffusing and protein-bound DNA was immobile, whereby they could be discriminated resulting in quantitative data on DNA binding. On the single molecule level approximately 10% of XPA co-localized with DNA; this increased to 32% in the presence of RPA. These results, especially the enhanced binding of XPA in the presence of RPA, are similar to those obtained in bulk experiments, validating the utility of scanning confocal fluorescence microscopy for investigating functional interactions at the single molecule level.

Methods and kits for nucleic acid analysis using fluorescence resonance energy transfer

DOEpatents

Kwok, Pui-Yan; Chen, Xiangning

1999-01-01

A method for detecting the presence of a target nucleotide or sequence of nucleotides in a nucleic acid is disclosed. The method is comprised of forming an oligonucleotide labeled with two fluorophores on the nucleic acid target site. The doubly labeled oligonucleotide is formed by addition of a singly labeled dideoxynucleoside triphosphate to a singly labeled polynucleotide or by ligation of two singly labeled polynucleotides. Detection of fluorescence resonance energy transfer upon denaturation indicates the presence of the target. Kits are also provided. The method is particularly applicable to genotyping.

Deconstructing autofluorescence: non-invasive detection and monitoring of biochemistry in cells and tissues (Conference Presentation)

NASA Astrophysics Data System (ADS)

Goldys, Ewa M.; Gosnell, Martin E.; Anwer, Ayad G.; Cassano, Juan C.; Sue, Carolyn M.; Mahbub, Saabah B.; Pernichery, Sandeep M.; Inglis, David W.; Adhikary, Partho P.; Jazayeri, Jalal A.; Cahill, Michael A.; Saad, Sonia; Pollock, Carol; Sutton-Mcdowall, Melanie L.; Thompson, Jeremy G.

2016-03-01

Automated and unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biology and medicine. Label-free cell imaging provides information about endogenous fluorescent metabolites, enzymes and cofactors in cells. However extracting high content information from imaging of native fluorescence has been hitherto impossible. Here, we quantitatively characterise cell populations in different tissue types, live or fixed, by using novel image processing and a simple multispectral upgrade of a wide-field fluorescence microscope. Multispectral intrinsic fluorescence imaging was applied to patient olfactory neurosphere-derived cells, cell model of a human metabolic disease MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like syndrome). By using an endogenous source of contrast, subtle metabolic variations have been detected between living cells in their full morphological context which made it possible to distinguish healthy from diseased cells before and after therapy. Cellular maps of native fluorophores, flavins, bound and free NADH and retinoids unveiled subtle metabolic signatures and helped uncover significant cell subpopulations, in particular a subpopulation with compromised mitochondrial function. The versatility of our method is further illustrated by detecting genetic mutations in cancer, non-invasive monitoring of CD90 expression, label-free tracking of stem cell differentiation, identifying stem cell subpopulations with varying functional characteristics, tissue diagnostics in diabetes, and assessing the condition of preimplantation embryos. Our optimal discrimination approach enables statistical hypothesis testing and intuitive visualisations where previously undetectable differences become clearly apparent.

PVP-coated gold nanoparticles for the selective determination of ochratoxin A via quenching fluorescence of the free aptamer.

PubMed

Lv, Lei; Jin, Yongdong; Kang, Xiaojiao; Zhao, Yangyang; Cui, Chengbi; Guo, Zhijun

2018-05-30

This paper describes an aptamer/gold nanoparticle-based assay for ochratoxin A (OTA) detection. This assay is based on the use of an aptamer labeled with carboxyfluorescein (FAM) at its 5'-end and gold nanoparticles (AuNPs) that act as quenchers of fluorescence. When OTA is absent in the system, the fluorescently labeled aptamers are adsorbed on the surface of AuNPs. The fluorescence signal of the fluorescein-labeled OTA aptamer generated is quenched by the fluorescence resonance energy transfer effect of AuNPs. When OTA is present in the system, the fluorescently labeled aptamer binds to OTA and forms a folded structure, which can resist the adsorption of AuNPs. Thus, the fluorescent signal can be retained. The detection limit of this sensing platform is 5 nM, and the linear detection range is 10-1000 nM (R 2  = 0.994). The procedure was validated by the quantitation of OTA in spiked ginger powder samples and were found to be free of interference by the sample matrix. The recoveries and the relative standard deviation varied from 89.0% to 117.8% and from 1.9% to 6.3%, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Modulation of ICT probability in bi(polyarene)-based O-BODIPYs: towards the development of low-cost bright arene-BODIPY dyads.

PubMed

Gartzia-Rivero, Leire; Sánchez-Carnerero, Esther M; Jiménez, Josue; Bañuelos, Jorge; Moreno, Florencio; Maroto, Beatriz L; López-Arbeloa, Iñigo; de la Moya, Santiago

2017-09-12

We report the synthesis, and spectroscopic and electrochemical properties of a selected library of novel spiranic O-BODIPYs bearing a phenol-based bi(polyarene) unit tethered to the boron center through oxygen atoms. These dyes constitute an interesting family of arene-BODIPY dyads useful for the development of photonic applications due to their synthetic accessibility and tunable photonic properties. It is demonstrated that the electron-donor capability of the involved arene moiety switches on a non-emissive intramolecular charge transfer (ICT) state, which restricts the fluorescence efficiency of the dyad. Interestingly, the influence of this non-radiative deactivation channel can be efficiently modulated by the substitution pattern, either at the dipyrrin ligand or at the polyarene moiety. Thus, dyads featuring electron-rich dipyrrin and electron-poor polyarene show lower or almost negligible ICT probability, and hence display bright fluorescence upon dual excitation at far-away spectral regions. This synthetic approach has allowed the easy development of low-cost efficient ultraviolet-absorbing visible-emitting cassettes by selecting properly the substitution pattern of the involved key units, dipyrrin and bi(polyarene), to modulate not only absorption and emission wavelengths, but also fluorescence efficiencies.

Quantum dot-doped silica nanoparticles as probes for targeting of T-lymphocytes.

PubMed

Bottini, Massimo; D'Annibale, Federica; Magrini, Andrea; Cerignoli, Fabio; Arimura, Yutaka; Dawson, Marcia I; Bergamaschi, Enrico; Rosato, Nicola; Bergamaschi, Antonio; Mustelin, Tomas

2007-01-01

To enhance diagnostic or therapeutic efficacy, novel nanomaterials must be engineered to function in biologically relevant environments, be visible by conventional fluorescent microscopy, and have multivalent loading capacity for easy detection or effective drug delivery. Here we report the fabrication of silica nanoparticles doped with quantum dots and superficially functionalized with amino and phosphonate groups. The amino groups were acylated with a water-soluble biotin-labeling reagent. The biotinylated nanoparticles were subsequently decorated with neutravidin by exploiting the strong affinity between neutravidin and biotin. The resultant neutravidin-decorated fluorescent silica nanoparticles stably dispersed under physiological conditions, were visible by conventional optical and confocal fluorescent microscopy, and could be further functionalized with macromolecules, nucleic acids, and polymers. We also coated the surface of the nanoparticles with biotinylated mouse anti-human CD3 (alphaCD3). The resultant fluorescent nanoassembly was taken up by Jurkat T cells through receptor-mediated endocytosis and was partially released to lysosomes. Thus, quantum dot-doped silica nanoparticles decorated with neutravidin represent a potentially excellent scaffold for constructing specific intracellular nanoprobes and transporters.

Validation of fluorescent-labeled microspheres for measurement of relative blood flow in severely injured lungs

NASA Technical Reports Server (NTRS)

Hubler, M.; Souders, J. E.; Shade, E. D.; Hlastala, M. P.; Polissar, N. L.; Glenny, R. W.

1999-01-01

The aim of the study was to validate a nonradioactive method for relative blood flow measurements in severely injured lungs that avoids labor-intensive tissue processing. The use of fluorescent-labeled microspheres was compared with the standard radiolabeled-microsphere method. In seven sheep, lung injury was established by using oleic acid. Five pairs of radio- and fluorescent-labeled microspheres were injected before and after established lung injury. Across all animals, 175 pieces were selected randomly. The radioactivity of each piece was determined by using a scintillation counter. The fluorescent dye was extracted from each piece with a solvent without digestion or filtering. The fluorescence was determined with an automated fluorescent spectrophotometer. Perfusion was calculated for each piece from both the radioactivity and fluorescence and volume normalized. Correlations between flow determined by the two methods were in the range from 0.987 +/- 0.007 (SD) to 0.991 +/- 0.002 (SD) after 9 days of soaking. Thus the fluorescent microsphere technique is a valuable tool for investigating regional perfusion in severely injured lungs and can replace radioactivity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mukundan, Harshini; Xei, Hongshi; Anderson, Aaron S

We have developed a waveguide-based optical biosensor for the sensitive and specific detection of biomarkers associated with disease. Our technology combines the superior optical properties of single-mode planar waveguides, the robust nature of functionalized self-assembled monolayer sensing films and the specificity of fluorescence sandwich immunoassays to detect biomarkers in complex biological samples such as serum, urine and sputum. We have previously reported the adaptation of our technology to the detection of biomarkers associated with breast cancer and anthrax. However, these approaches primarily used phospholipid bilayers as the functional film and organic dyes (ex: AlexaFluors) as the fluorescence reporter. Organic dyesmore » are easily photodegraded and are not amenable to multiplexing because of their narrow Stokes' shift. Here we have developed strategies for conjugation of the detector antibodies with quantum dots for use in a multiplex detection platform. We have previously evaluated dihydroxylipoic acid quantum dots for the detection of a breast cancer biomarker. In this manuscript, we investigate the detection of the Bacillus anthracis protective antigen using antibodies conjugated with polymer-coated quantum dots. Kinetics of binding on the waveguide-based biosensor is reported. We compare the sensitivity of quantum dot labeled antibodies to those labeled with AlexaFluor and demonstrate the photostability of the former in our assay platform. In addition, we compare sulfydryl labeling of the antibody in the hinge region to that of nonspecific amine labeling. This is but the first step in developing a multiplex assay for such biomarkers on our waveguide platform.« less

Toward photostable multiplex analyte detection on a single mode planar optical waveguide

NASA Astrophysics Data System (ADS)

Mukundan, Harshini; Xie, Hongzhi; Anderson, Aaron; Grace, W. Kevin; Martinez, Jennifer S.; Swanson, Basil

2009-02-01

We have developed a waveguide-based optical biosensor for the sensitive and specific detection of biomarkers associated with disease. Our technology combines the superior optical properties of single-mode planar waveguides, the robust nature of functionalized self-assembled monolayer sensing films and the specificity of fluorescence sandwich immunoassays to detect biomarkers in complex biological samples such as serum, urine and sputum. We have previously reported the adaptation of our technology to the detection of biomarkers associated with breast cancer and anthrax. However, these approaches primarily used phospholipid bilayers as the functional film and organic dyes (ex: AlexaFluors) as the fluorescence reporter. Organic dyes are easily photodegraded and are not amenable to multiplexing because of their narrow Stokes' shift. Here we have developed strategies for conjugation of the detector antibodies with quantum dots for use in a multiplex detection platform. We have previously evaluated dihydroxylipoic acid quantum dots for the detection of a breast cancer biomarker. In this manuscript, we investigate the detection of the Bacillus anthracis protective antigen using antibodies conjugated with polymer-coated quantum dots. Kinetics of binding on the waveguide-based biosensor is reported. We compare the sensitivity of quantum dot labeled antibodies to those labeled with AlexaFluor and demonstrate the photostability of the former in our assay platform. In addition, we compare sulfydryl labeling of the antibody in the hinge region to that of nonspecific amine labeling. This is but the first step in developing a multiplex assay for such biomarkers on our waveguide platform.

Direct fluorescence anisotropy assay for cocaine using tetramethylrhodamine-labeled aptamer.

PubMed

Liu, Yingxiong; Zhao, Qiang

2017-06-01

Development of simple, sensitive, and rapid method for cocaine detection is important in medicine and drug abuse monitoring. Taking advantage of fluorescence anisotropy and aptamer, this study reports a direct fluorescence anisotropy (FA) assay for cocaine by employing an aptamer probe with tetramethylrhodamine (TMR) labeled on a specific position. The binding of cocaine and the aptamer causes a structure change of the TMR-labeled aptamer, leading to changes of the interaction between labeled TMR and adjacent G bases in aptamer sequence, so FA of TMR varies with increasing of cocaine. After screening different labeling positions of the aptamer, including thymine (T) bases and terminals of the aptamer, we obtained a favorable aptamer probe with TMR labeled on the 25th base T in the sequence, which exhibited sensitive and significant FA-decreasing responses upon cocaine. Under optimized assay conditions, this TMR-labeled aptamer allowed for direct FA detection of cocaine as low as 5 μM. The maximum FA change reached about 0.086. This FA method also enabled the detection of cocaine spiked in diluted serum and urine samples, showing potential for applications. Graphical Abstract The binding of cocaine to the TMR-labeled aptamer causes conformation change and alteration of the intramolecular interaction between TMR and bases of aptamer, leading to variance of fluorescence anisotropy (FA) of TMR, so direct FA analyis of cocaine is achieved.

Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging

PubMed Central

Schvartz, Tomer; Aloush, Noa; Goliand, Inna; Segal, Inbar; Nachmias, Dikla; Arbely, Eyal; Elia, Natalie

2017-01-01

Genetic code expansion and bioorthogonal labeling provide for the first time a way for direct, site-specific labeling of proteins with fluorescent-dyes in live cells. Although the small size and superb photophysical parameters of fluorescent-dyes offer unique advantages for high-resolution microscopy, this approach has yet to be embraced as a tool in live cell imaging. Here we evaluated the feasibility of this approach by applying it for α-tubulin labeling. After a series of calibrations, we site-specifically labeled α-tubulin with silicon rhodamine (SiR) in live mammalian cells in an efficient and robust manner. SiR-labeled tubulin successfully incorporated into endogenous microtubules at high density, enabling video recording of microtubule dynamics in interphase and mitotic cells. Applying this labeling approach to structured illumination microscopy resulted in an increase in resolution, highlighting the advantages in using a smaller, brighter tag. Therefore, using our optimized assay, genetic code expansion provides an attractive tool for labeling proteins with a minimal, bright tag in quantitative high-resolution imaging. PMID:28835375

High-level fluorescence labeling of gram-positive pathogens.

PubMed

Aymanns, Simone; Mauerer, Stefanie; van Zandbergen, Ger; Wolz, Christiane; Spellerberg, Barbara

2011-01-01

Fluorescence labeling of bacterial pathogens has a broad range of interesting applications including the observation of living bacteria within host cells. We constructed a novel vector based on the E. coli streptococcal shuttle plasmid pAT28 that can propagate in numerous bacterial species from different genera. The plasmid harbors a promoterless copy of the green fluorescent variant gene egfp under the control of the CAMP-factor gene (cfb) promoter of Streptococcus agalactiae and was designated pBSU101. Upon transfer of the plasmid into streptococci, the bacteria show a distinct and easily detectable fluorescence using a standard fluorescence microscope and quantification by FACS-analysis demonstrated values that were 10-50 times increased over the respective controls. To assess the suitability of the construct for high efficiency fluorescence labeling in different gram-positive pathogens, numerous species were transformed. We successfully labeled Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, Streptococcus anginosus and Staphylococcus aureus strains utilizing the EGFP reporter plasmid pBSU101. In all of these species the presence of the cfb promoter construct resulted in high-level EGFP expression that could be further increased by growing the streptococcal and enterococcal cultures under high oxygen conditions through continuous aeration.

Multiple tag labeling method for DNA sequencing

DOEpatents

Mathies, Richard A.; Huang, Xiaohua C.; Quesada, Mark A.

1995-01-01

A DNA sequencing method described which uses single lane or channel electrophoresis. Sequencing fragments are separated in said lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radio-isotope labels.

Vestibular efferent neurons project to the flocculus

NASA Technical Reports Server (NTRS)

Shinder, M. E.; Purcell, I. M.; Kaufman, G. D.; Perachio, A. A.

2001-01-01

A bilateral projection from the vestibular efferent neurons, located dorsal to the genu of the facial nerve, to the cerebellar flocculus and ventral paraflocculus was demonstrated. Efferent neurons were double-labeled by the unilateral injections of separate retrograde tracers into the labyrinth and into the floccular and ventral parafloccular lobules. Efferent neurons were found with double retrograde tracer labeling both ipsilateral and contralateral to the sites of injection. No double labeling was found when using a fluorescent tracer with non-fluorescent tracers such as horseradish peroxidase (HRP) or biotinylated dextran amine (BDA), but large percentages of efferent neurons were found to be double labeled when using two fluorescent substances including: fluorogold, microruby dextran amine, or rhodamine labeled latex beads. These data suggest a potential role for vestibular efferent neurons in modulating the dynamics of the vestibulo-ocular reflex (VOR) during normal and adaptive conditions.

A new approach of light microscopic immunohistochemical triple-staining: combination of Fos labeling with diaminobenzidine-nickel and neuropeptides labeled with Alexa488 and Alexa555 fluorescent dyes.

PubMed

Majercikova, Z; Weering, H van; Scsukova, S; Mikkelsen, J D; Kiss, A

2012-10-01

The aim of the present study was to introduce a new approach of the light microscopic immunohistochemical triple-staining enabling to study the differences in the activity of at least two different phenotypes of neurons on the same histological section. For this purpose combination of Fos (a product of the immediate early gene) labeling with nickel intensified diaminobenzidine (DAB-Ni) and two neuropeptides labeled with Alexa488 and Alexa555 fluorescent dyes on cryo-processed 35-40 µm thick free-floating brain sections was selected. The parallel occurrence of three antibodies studied, i.e. Fos, hypocretin (HCRT), and melanin-concentrating hormone (MCH), was studied by a new methodic approach utilizing combination of Fos immunolabeled with DAB-Ni and HCRT and MCH labeled with Alexa488 and Alexa555 fluorescent dyes, respectively. Fos stimulation was induced by a single immobilization (IM0) for 120 min. Then, the rats were sacrificed, the brains removed, soaked with 30% sucrose in 0.1 M phosphate buffer (PB), cryo-sectioned throughout the hypothalamus into 35-40 μm thick coronal sections, collected, and washed in the same buffer for 10-15 min. Fos was revealed by avidin-biotin-peroxidase (ABC) complex and visualized by diaminobenzidine chromogen containing nickel chloride salt. HCRT and MCH neurons were visualized by the above mentioned fluorescent dyes. Evaluation of the Fos and fluorescent staining was performed in the computerized Axo Imager Carl Zeiss microscope using light and fluorescent illuminations. All the antibodies used showed clear immunoreactive staining. Fos staining occurred in the form of black color located in the cell nuclei. HCRH and MCH neuropeptides showed clear green and red fluorescence in the cell perikarya, respectively. The final merged picture showed Fos protein in the activated green HCRT or red MCH neurons in the form of white nuclei. The present study clearly demonstrate that the combination of Fos labeling with DAB-Ni and neuropeptides labeled with Alexa488 and Alexa555 on cryo-processed 35-40 µm thick free-floating brain sections is an excellent approach providing further advantages for quick and reproducible triple immuno-staining enabling to compare the activity of at least two phenotypes of neurons on the same section. Alexa488 and Alexa555 fluorescent dyes, Fos, hypocretin, melanin-concentrating hormone, cryostat sections, triple labeling immunohistochemistry, rat.

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform

NASA Astrophysics Data System (ADS)

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-11-01

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05839b

The Effect of a Fluorophore Photo-Physics on the Lipid Vesicle Diffusion Coefficient Studied by Fluorescence Correlation Spectroscopy.

PubMed

Drabik, Dominik; Przybyło, Magda; Sikorski, Aleksander; Langner, Marek

2016-03-01

Fluorescence Correlation Spectroscopy (FCS) is a technique, which allows determination of the diffusion coefficient and concentration of fluorescent objects suspended in the solution. The measured parameter is the fluctuation of the fluorescence signal emitted by diffusing molecules. When 100 nm DOPC vesicles labeled with various fluorescent dyes (Fluorescein-PE, NBD-PE, Atto488 DOPE or βBodipy FL) were measured, different values of diffusion coefficients have been obtained. These diffusion coefficients were different from the expected values measured using the dynamic light scattering method (DLS). The FCS was initially developed for solutions containing small fluorescent molecules therefore the observed inconsistency may result from the nature of vesicle suspension itself. The duration of the fluorescence signal may depend on the following factors: the exposure time of the labeled object to the excitation beam, the photo-physical properties (e.g., stability) of a fluorophore, the theoretical model used for the calculations of the diffusion coefficient and optical properties of the vesicle suspension. The diffusion coefficients determined for differently labeled liposomes show that its dependence on vesicle size and quantity of fluorescent probed used for labeling was significant demonstrating that the fluorescence properties of the fluorophore itself (bleaching and/or blinking) were critical factors for a correct outcome of FCS experiment. The new, based on combined FCS and DLS measurements, method for the determination of the focal volume prove itself to be useful for the evaluation of a fluorescence dye with respect to its applicability for FCS experiment.

Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx.

PubMed

Löschberger, Anna; Niehörster, Thomas; Sauer, Markus

2014-05-01

Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide-alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid.

PubMed

Dukes, Madeline J; Peckys, Diana B; de Jonge, Niels

2010-07-27

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7x12 nm were visible in a 5 microm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.

Correlative Fluorescence Microscopy and Scanning Transmission Electron Microscopy of Quantum Dot Labeled Proteins in Whole Cells in Liquid

PubMed Central

Dukes, Madeline J.; Peckys, Diana B.; de Jonge, Niels

2010-01-01

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7 × 12 nm were visible in a 5 μm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs. PMID:20550177

Fluorescence intensity- and lifetime-based glucose sensing using glucose/galactose-binding protein.

PubMed

Pickup, John C; Khan, Faaizah; Zhi, Zheng-Liang; Coulter, Jonathan; Birch, David J S

2013-01-01

We review progress in our laboratories toward developing in vivo glucose sensors for diabetes that are based on fluorescence labeling of glucose/galactose-binding protein. Measurement strategies have included both monitoring glucose-induced changes in fluorescence resonance energy transfer and labeling with the environmentally sensitive fluorophore, badan. Measuring fluorescence lifetime rather than intensity has particular potential advantages for in vivo sensing. A prototype fiber-optic-based glucose sensor using this technology is being tested. © 2013 Diabetes Technology Society.

Application of magnetic carriers to two examples of quantitative cell analysis

NASA Astrophysics Data System (ADS)

Zhou, Chen; Qian, Zhixi; Choi, Young Suk; David, Allan E.; Todd, Paul; Hanley, Thomas R.

2017-04-01

The use of magnetophoretic mobility as a surrogate for fluorescence intensity in quantitative cell analysis was investigated. The objectives of quantitative fluorescence flow cytometry include establishing a level of labeling for the setting of parameters in fluorescence activated cell sorters (FACS) and the determination of levels of uptake of fluorescently labeled substrates by living cells. Likewise, the objectives of quantitative magnetic cytometry include establishing a level of labeling for the setting of parameters in flowing magnetic cell sorters and the determination of levels of uptake of magnetically labeled substrates by living cells. The magnetic counterpart to fluorescence intensity is magnetophoretic mobility, defined as the velocity imparted to a suspended cell per unit of magnetic ponderomotive force. A commercial velocimeter available for making this measurement was used to demonstrate both applications. Cultured Gallus lymphoma cells were immunolabeled with commercial magnetic beads and shown to have adequate magnetophoretic mobility to be separated by a novel flowing magnetic separator. Phagocytosis of starch nanoparticles having magnetic cores by cultured Chinese hamster ovary cells, a CHO line, was quantified on the basis of magnetophoretic mobility.

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

PubMed Central

Schwab, Rebekka A.V.; Niedzwiedz, Wojciech

2011-01-01

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development. Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope. PMID:22064662

Novel online security system based on rare-earth-doped glass microbeads

NASA Astrophysics Data System (ADS)

Officer, Simon; Prabhu, G. R.; Pollard, Pat; Hunter, Catherine; Ross, Gary A.

2004-06-01

A novel fluorescent security label has been produced that could replace numerous conventional fluorescent dyes in document security. This label utilizes rare earth ions doped in a borosilicate glass matrix to produce sharp spectral fluorescence peaks with characteristic long lifetimes due to the rare earth ions. These are subsequently detected by an online detection system based on fluorescence and the long lifetimes to avoid any interference from other fluorophores present in the background. Security is further enhanced by the interaction of the rare earth ions with each other and the effect of the host on the emission spectra and therefore the number of permutations that could be produced. This creates a very secure label with various applications for the security market.

Fluorescence Immunofiltration Assay of Brucella Melitensis.

DTIC Science & Technology

1995-01-01

second urease -labelled antibody directed against fluorescein. The assay system is useful for measuring protein, virus and bacteria in aqueous...binding site for the signal-generating urease -labelled antibody, it is a highly fluorescent molecule and has signal-generating capacity of its own

Development of a facile and sensitive HPLC-FLD method via fluorescence labeling for triterpenic acid bioavailability investigation.

PubMed

You, Jinmao; Wu, Di; Zhao, Mei; Li, Guoliang; Gong, Peiwei; Wu, Yueyue; Guo, Yu; Chen, Guang; Zhao, Xianen; Sun, Zhiwei; Xia, Lian; Wu, Yongning

2017-06-01

Triterpenic acids are widely distributed in many fruits and are known for their medicinal benefits. The study of bioavailability has been an important task for a better understanding of the triterpenic acids. Although many methods based on fluorescence labeling for triterpenic acid determination have been established, these reported methods needed anhydrous conditions, which are not suitable for the convenient study of triterpenic acid bioavailability. Inspired by that, a versatile method, which overcomes the difficulty of the reported methods, has been first developed in this study. The novel method using 2-[12-benzo[b]acridin-5- (12H)-yl]-acetohydrazide (BAAH) as the fluorescence labeling reagent coupled with high-performance liquid chromatography with fluorescence detection was first developed for the study of triterpenic acid bioavailability. Furthermore, the labeling conditions have been optimized in order to achieve the best fluorescence labeling yield. Under the optimal conditions, the quantitative linear range of analytes was 2-1000 ng mL -1 , and the correlation coefficients were >0.9998. The detection limits for all triterpenic acid derivatives were achieved within the range of 0.28-0.29 ng mL -1 . The proposed method was successfully applied to the study of triterpenic acid bioavailability with excellent applicability and good reproducibility. Copyright © 2016 John Wiley & Sons, Ltd.

Enhancing the Photocatalytic Activity of Sr4 Al14 O25 : Eu2+ , Dy3+ Persistent Phosphors by Codoping with Bi3+ Ions.

PubMed

García, Carlos R; Oliva, Jorge; Romero, Maria Teresa; Diaz-Torres, Luis A

2016-03-01

The photocatalytic activity of Bismuth-codoped Sr 4 Al 14 O 25 : Eu 2+ , Dy 3+ persistent phosphors is studied by monitoring the degradation of the blue methylene dye UV light irradiation. Powder phosphors are obtained by a combustion synthesis method and a postannealing process in reductive atmosphere. The XRD patterns show a single orthorhombic phase Sr 4 Al 14 O 25 : Eu 2+ , Dy 3+ , Bi 3+ phosphors even at high Bismuth dopant concentrations of 12 mol%, suggesting that Bi ions are well incorporated into the host lattice. SEM micrographs show irregular micrograins with sizes in the range of 0.5-20 μm. The samples present an intense greenish-blue fluorescence and persistent emissions at 495 nm, attributed to the 5d-4f allowed transitions of Eu 2+ . The fluorescence decreases as Bi concentration increases; that suggest bismuth-induced traps formation that in turn quench the luminescence. The photocatalytic evaluation of the powders was studied under both 365 nm UV and solar irradiations. Sample with 12 mol% of Bi presented the best MB degradation activity; 310 min of solar irradiation allow 100% MB degradation, whereas only 62.49% MB degradation is achieved under UV irradiation. Our results suggest that codoping the persistent phosphors with Bi 3+ can be an alternative to enhance their photocatalytic activity. © 2016 The American Society of Photobiology.

Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer.

PubMed Central

Tsuji, A; Sato, Y; Hirano, M; Suga, T; Koshimoto, H; Taguchi, T; Ohsuka, S

2001-01-01

We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells. PMID:11423432

Variability of Performance: A "Signature" Characteristic of Learning Disabled Children?

ERIC Educational Resources Information Center

Fuchs, Douglas; And Others

Two studies were conducted to compare the performance instability of children (grades 3-9) labeled learning disabled/brain injured (LD/BI) to the performance instability of emotionally handicapped (EH) children. In the first study, 50 LD/BI and 37 EH students were measured on three third grade reading passages twice, once within one sitting and…

Goat anti-rabbit IgG conjugated fluorescent dye-doped silica nanoparticles for human breast carcinoma cell recognition.

PubMed

Chen, Min-Yan; Chen, Ze-Zhong; Wu, Ling-Ling; Tang, Hong-Wu; Pang, Dai-Wen

2013-11-12

We report an indirect method for cancer cell recognition using photostable fluorescent silica nanoprobes as biological labels. The dye-doped fluorescent silica nanoparticles were synthesized using the water-in-oil (W/O) reverse microemulsion method. The silica matrix was produced by the controlled hydrolysis of tetraethylorthosilicate (TEOS) in water nanodroplets with the initiation of ammonia (NH3·H2O). Fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate conjugated with dextran (RBITC-Dextran) was doped in silica nanoparticles (NPs) with a size of 60 ± 5 nm as a fluorescent signal element by covalent bonding and steric hindrance, respectively. The secondary antibody, goat anti-rabbit IgG, was conjugated on the surface of the PEG-terminated modified FITC-doped or RBITC-Dextran-doped silica nanoparticles (PFSiNPs or PBSiNPs) by covalent binding to the PEG linkers using the cyanogen bromide method. The concentrations of goat anti-rabbit IgG covering the nanoprobes were quantified via the Bradford method. In the proof-of-concept experiment, an epithelial cell adhesion molecule (EpCAM) on the human breast cancer SK-Br-3 cell surface was used as the tumor marker, and the nanoparticle functionalized with rabbit anti-EpCAM antibody was employed as the nanoprobe for cancer cell recognition. Compared with fluorescent dye labeled IgG (FITC-IgG and RBITC-IgG), the designed nanoprobes display dramatically increased stability of fluorescence as well as photostability under continuous irradiation.

Multiple tag labeling method for DNA sequencing

DOEpatents

Mathies, R.A.; Huang, X.C.; Quesada, M.A.

1995-07-25

A DNA sequencing method is described which uses single lane or channel electrophoresis. Sequencing fragments are separated in the lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radioisotope labels. 5 figs.

Hybrid inorganic/organic photonic crystal biochips for cancer biomarkers detection

NASA Astrophysics Data System (ADS)

Sinibaldi, Alberto; Danz, Norbert; Munzert, Peter; Michelotti, Francesco

2018-06-01

We report on hybrid inorganic/organic one-dimensional photonic crystal biochips sustaining Bloch surface waves. The biochips were used, together with an optical platform operating in a label-free and fluorescence configuration simultaneously, to detect the cancer biomarker Angiopoietin 2 in a protein base buffer. The hybrid photonic crystals embed in their geometry a thin functionalization poly-acrylic acid layer deposited by plasma polymerization, which is used to immobilize a monoclonal antibody for highly specific biological recognition. The fluorescence operation mode is described in detail, putting into evidence the role of field enhancement and localization at the photonic crystal surface in the shaping and intensification of the angular fluorescence pattern. In the fluorescence operation mode, the hybrid biochips can attain the limit of detection 6 ng/ml.

Fluorescent Quantum Dots for Biological Labeling

NASA Technical Reports Server (NTRS)

McDonald, Gene; Nadeau, Jay; Nealson, Kenneth; Storrie-Lomardi, Michael; Bhartia, Rohit

2003-01-01

Fluorescent semiconductor quantum dots that can serve as "on/off" labels for bacteria and other living cells are undergoing development. The "on/off" characterization of these quantum dots refers to the fact that, when properly designed and manufactured, they do not fluoresce until and unless they come into contact with viable cells of biological species that one seeks to detect. In comparison with prior fluorescence-based means of detecting biological species, fluorescent quantum dots show promise for greater speed, less complexity, greater sensitivity, and greater selectivity for species of interest. There are numerous potential applications in medicine, environmental monitoring, and detection of bioterrorism.

Myosin conformational states determined by single fluorophore polarization

PubMed Central

Warshaw, David M.; Hayes, Eric; Gaffney, Donald; Lauzon, Anne-Marie; Wu, Junru; Kennedy, Guy; Trybus, Kathleen; Lowey, Susan; Berger, Christopher

1998-01-01

Muscle contraction is powered by the interaction of the molecular motor myosin with actin. With new techniques for single molecule manipulation and fluorescence detection, it is now possible to correlate, within the same molecule and in real time, conformational states and mechanical function of myosin. A spot-confocal microscope, capable of detecting single fluorophore polarization, was developed to measure orientational states in the smooth muscle myosin light chain domain during the process of motion generation. Fluorescently labeled turkey gizzard smooth muscle myosin was prepared by removal of endogenous regulatory light chain and re-addition of the light chain labeled at cysteine-108 with the 6-isomer of iodoacetamidotetramethylrhodamine (6-IATR). Single myosin molecule fluorescence polarization data, obtained in a motility assay, provide direct evidence that the myosin light chain domain adopts at least two orientational states during the cyclic interaction of myosin with actin, a randomly disordered state, most likely associated with myosin whereas weakly bound to actin, and an ordered state in which the light chain domain adopts a finite angular orientation whereas strongly bound after the powerstroke. PMID:9653135

Biotin and fluorescent labeling of RNA using T4 RNA ligase.

PubMed Central

Richardson, R W; Gumport, R I

1983-01-01

Biotin, fluorescein, and tetramethylrhodamine derivatives of P1-(6-aminohex-1-yl)-P2-(5'-adenosine) pyrophosphate were synthesized and used as substrates with T4 RNA ligase. In the absence of ATP, the non-adenylyl portion of these substrates is transferred to the 3'-hydroxyl of an RNA acceptor to form a phosphodiester bond and the AMP portion is released. E. coli and D. melanogaster 5S RNA, yeast tRNAPhe, (Ap)3C, and (Ap)3A serve as acceptors with yields of products varying from 50 to 100%. Biotin-labeled oligonucleotides are bound selectively and quantitatively to avidin-agarose and may be eluted with 6 M guanidine hydrochloride, pH 2.5. Fluorescein and tetramethylrhodamine-labeled oligonucleotides are highly fluorescent and show no quenching due to attachment to the acceptor. The diverse structures of the appended groups and of the chain lengths and compositions of the acceptor RNAs show that T4 RNA ligase will be a useful modification reagent for the addition of various functional groups to the 3'-terminus of RNA molecules. Images PMID:6194506

The internalization of fluorescence-labeled PLA nanoparticles by macrophages.

PubMed

Li, Fengjuan; Zhu, Aiping; Song, Xiaoli; Ji, Lijun; Wang, Juan

2013-09-10

Rhodamine B (RhB)-labeled PLA nanoparticles were prepared through surface grafting copolymerization of glycidyl methacrylate (GMA) onto PLA nanoparticles during the emulsion/evaporation process. RhB firstly interacts with sodium dodecyl sulfate (SDS) through electrostatic interaction to form hydrophobic complex (SDS-RhB). Due to the high-affinity of SDS-RhB with GMA, hydrophilic RhB can be successfully combined into PLA nanoparticles. The internalization of RhB-labeled PLA nanoparticles by macrophages was investigated with fluorescence microscope technology. The effects of the PLA nanoparticle surface nature and size on the internalization were investigated. The results indicate that the PLA particles smaller than 200 nm can avoid the uptake of phagocytosis. The bigger PLA particles (300 nm) with polyethylene glycol (PEG) surface showed less internalization by macrophage compared with those with poly(ethylene oxide-propylene oxide) copolymer (F127) or poly(vinyl alcohol) (PVA) surface. The "stealth" function of PEG on the PLA nanoparticles from internalization of macrophages due to the low protein adsorption is revealed by electrochemical impedance technology. Copyright © 2013 Elsevier B.V. All rights reserved.

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy.

PubMed

Peckys, Diana B; de Jonge, Niels

2015-09-11

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e., one emitting at 655 nm and at 800 revealed similar characteristic results.

Protein labeling for live cell fluorescence microscopy with a highly photostable renewable signal† †Electronic supplementary information (ESI) available: Supplementary methods, figures, movies, and data. See DOI: 10.1039/c7sc01628j

PubMed Central

Bozhanova, Nina G.; Baranov, Mikhail S.; Klementieva, Natalia V.; Sarkisyan, Karen S.; Gavrikov, Alexey S.; Yampolsky, Ilia V.; Zagaynova, Elena V.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

2017-01-01

We present protein-PAINT – the implementation of the general principles of PAINT (Point Accumulation for Imaging in Nanoscale Topography) for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes. We demonstrate an order of magnitude higher photostability of the fluorescence signal in comparison with spectrally similar fluorescent proteins. Protein-PAINT ensures prolonged super-resolution fluorescence microscopy of living cells in both single molecule detection and stimulated emission depletion regimes. PMID:29147545

Fluorescent vancomycin and terephthalate comodified europium-doped layered double hydroxides nanoparticles: synthesis and application for bacteria labelling

NASA Astrophysics Data System (ADS)

Sun, Jianchao; Fan, Hai; Wang, Nan; Ai, Shiyun

2014-09-01

Vancomycin (Van)- and terephthalate (TA)-comodified europium-doped layered double hydroxides (Van-TA-Eu-LDHs) nanoparticles were successfully prepared by a two-step method, in which, TA acted as a sensitizer to enhance the fluorescent property and Van was modified on the surface of LDH to act as an affinity reagent to bacteria. The obtained products were characterized by X-ray diffraction, transmission electron microscope and fluorescent spectroscopy. The results demonstrated that the prepared Van- and TA-comodified europium-doped layered double hydroxides (Van-TA-Eu-LDHs) nanoparticles with diameter of 50 nm in size showed highly efficient fluorescent property. Furthermore, due to the high affinity of Van to bacteria, the prepared Van-TA-Eu-LDHs nanoparticles showed efficient bacteria labelling by fluorescent property. The prepared nanoparticles may have wide applications in the biological fields, such as biomolecular labelling and cell imaging.

End-specific strategies of attachment of long double stranded DNA onto gold-coated nanofiber arrays

NASA Astrophysics Data System (ADS)

Peckys, Diana B.; de Jonge, Niels; Simpson, Michael L.; McKnight, Timothy E.

2008-10-01

We report the effective and site-specific binding of long double stranded (ds)DNA to high aspect ratio carbon nanofiber arrays. The carbon nanofibers were first coated with a thin gold layer to provide anchorage for two controllable binding methods. One method was based on the direct binding of thiol end-labeled dsDNA. The second and enhanced method used amine end-labeled dsDNA bound with crosslinkers to a carboxyl-terminated self-assembled monolayer. The bound dsDNA was first visualized with a fluorescent, dsDNA-intercalating dye. The specific binding onto the carbon nanofiber was verified by a high resolution detection method using scanning electron microscopy in combination with the binding of neutravidin-coated fluorescent microspheres to the immobilized and biotinylated dsDNA. Functional activity of thiol end-labeled dsDNA on gold-coated nanofiber arrays was verified with a transcriptional assay, whereby Chinese hamster lung cells (V79) were impaled upon the DNA-modified nanofibers and scored for transgene expression of the tethered template. Thiol end-labeled dsDNA demonstrated significantly higher expression levels than nanofibers prepared with control dsDNA that lacked a gold-binding end-label. Employing these site-specific and robust techniques of immobilization of dsDNA onto nanodevices can be of advantage for the study of DNA/protein interactions and for gene delivery applications.

Fluorescence lifetime as a new parameter in analytical cytology measurements

NASA Astrophysics Data System (ADS)

Steinkamp, John A.; Deka, Chiranjit; Lehnert, Bruce E.; Crissman, Harry A.

1996-05-01

A phase-sensitive flow cytometer has been developed to quantify fluorescence decay lifetimes on fluorochrome-labeled cells/particles. This instrument combines flow cytometry (FCM) and frequency-domain fluorescence spectroscopy measurement principles to provide unique capabilities for making phase-resolved lifetime measurements, while preserving conventional FCM capabilities. Cells are analyzed as they intersect a high-frequency, intensity-modulated (sine wave) laser excitation beam. Fluorescence signals are processed by conventional and phase-sensitive signal detection electronics and displayed as frequency distribution histograms. In this study we describe results of fluorescence intensity and lifetime measurements on fluorescently labeled particles, cells, and chromosomes. Examples of measurements on intrinsic cellular autofluorescence, cells labeled with immunofluorescence markers for cell- surface antigens, mitochondria stains, and on cellular DNA and protein binding fluorochromes will be presented to illustrate unique differences in measured lifetimes and changes caused by fluorescence quenching. This innovative technology will be used to probe fluorochrome/molecular interactions in the microenvironment of cells/chromosomes as a new parameter and thus expand the researchers' understanding of biochemical processes and structural features at the cellular and molecular level.

Label-free fluorescent aptasensor for potassium ion using structure-switching aptamers and berberine

NASA Astrophysics Data System (ADS)

Guo, Yanqing; Chen, Yanxia; Wei, Yanli; Li, Huanhuan; Dong, Chuan

2015-02-01

A simple, rapid and label-free fluorescent aptasensor was fabricated for the detection of potassium ion (K+ ion) in aqueous solution using K+ ion-stabilized single stranded DNA (ssDNA) with G-rich sequence as the recognition element and a fluorescent dye, berberine, as the fluorescence probe. In the presence of K+ ion, the G-rich ssDNA is promoted to form the aptamer-target complex with a G-quadruplex conformation, and berberine binding to the G-quadruplex structure results in the enhancement of its fluorescence. The fluorescence intensity of the sensing system displayed a calibration response for K+ ion in the range of 0-1600 μM with a detection limit of 31 nM (S/N = 3) and a relative standard deviation (RSD) of 0.45%. This label-free fluorescence aptasensor is conveniently and effectively applicable for analysis of K+ ion in blood serum samples with the recovery range of 81.7-105.3%. The assay for detection of potassium ion is easy, economical, robust, and stable in rough conditions.

Synthesis, photophysical properties and application of dye doped water soluble silica-based nanoparticles to label bacteria E. coli O157:H7

NASA Astrophysics Data System (ADS)

Tan Pham, Minh; Van Nguyen, Thi; Thi, Thuy Duong Vu; Nghiem Thi, Ha Lien; Thuan Tong, Kim; Thuy Tran, Thanh; Chu, Viet Ha; Brochon, Jean-Claude; Nhung Tran, Hong

2012-12-01

Organically modified silicate (ORMOSIL) nanoparticles (NPs) doped with rhodamine 6G and rhodamine B (RB) dyes were synthesized by Stöber method from methyltriethoxysilane CH3Si(OCH3)3 precursor (MTEOS). The NPs are surface functionalized by cationic amino groups. The optical characterization of dye-doped ORMOSIL NPs was studied in comparison with that of free dye in solution. The synthesized NPs were used for labeling bacteria E. coli O157:H7. The number of bacteria have been counted using the fluorescent spectra and microscope images of labeled bacteria. The results show the ability of NPs to work as biomarkers.

Phosphorylation-Induced Conformational Changes of Photoactivated Rhodopsin Probed by Fluorescent Labeling at Cys140 and Cys316.

PubMed

Rodríguez, Sheerly; Silva, May-Li; Benaím, Gustavo; Bubis, José

2018-05-03

In order to monitor conformational changes following photoactivation and phosphorylation of bovine rhodopsin, the two reactive sulfhydryl groups at Cys 140 and Cys 316 were specifically labeled with the monobromobimane (mBBr) fluorophore. Although alterations in conformation after light exposure of rhodopsin were not detected by fluorescence excitation scans (300-450 nm) of the mBBr-labeled protein, the fluorescence signal was reduced ∼ 90% in samples containing photoactivated phosphorhodopsin. Predominant labeling at either Cys 140 or Cys 316 in light-activated and phosphorylated rhodopsin merely generated a decrease of ∼ 38% and 28%, respectively, in the fluorescence excitation intensity. Thus, neither mBBr-modified Cys 140 nor mBBr-modified Cys 316 were involved single-handedly in the remarkable fall seen on the signal following phosphorylation of the protein; rather, the incorporation of phosphate groups on the mBBr-labeled light-activated rhodopsin appeared to affect its fluorescence signal in a cooperative or synergistic manner. These findings demonstrated that the phosphorylation of specific hydroxyl groups at the carboxyl terminal tail of rhodopsin causes definite conformational changes in the three-dimensional fold of the protein. Apparently, amino acid residues that are buried in the interior of the inactive protein become accessible following bleaching and phosphorylation of rhodopsin, quenching in turn the fluorescence excitation signal of mBBr-modified rhodopsin. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Protistan Grazing Analysis by Flow Cytometry Using Prey Labeled by In Vivo Expression of Fluorescent Proteins

PubMed Central

Fu, Yutao; O'Kelly, Charles; Sieracki, Michael; Distel, Daniel L.

2003-01-01

Selective grazing by protists can profoundly influence bacterial community structure, and yet direct, quantitative observation of grazing selectivity has been difficult to achieve. In this investigation, flow cytometry was used to study grazing by the marine heterotrophic flagellate Paraphysomonas imperforata on live bacterial cells genetically modified to express the fluorescent protein markers green fluorescent protein (GFP) and red fluorescent protein (RFP). Broad-host-range plasmids were constructed that express fluorescent proteins in three bacterial prey species, Escherichia coli, Enterobacter aerogenes, and Pseudomonas putida. Micromonas pusilla, an alga with red autofluorescence, was also used as prey. Predator-prey interactions were quantified by using a FACScan flow cytometer and analyzed by using a Perl program described here. Grazing preference of P. imperforata was influenced by prey type, size, and condition. In competitive feeding trials, P. imperforata consumed algal prey at significantly lower rates than FP (fluorescent protein)-labeled bacteria of similar or different size. Within-species size selection was also observed, but only for P. putida, the largest prey species examined; smaller cells of P. putida were grazed preferentially. No significant difference in clearance rate was observed between GFP- and RFP-labeled strains of the same prey species or between wild-type and GFP-labeled strains. In contrast, the common chemical staining method, 5-(4,6-dichloro-triazin-2-yl)-amino fluorescein hydrochloride, depressed clearance rates for bacterial prey compared to unlabeled or RFP-labeled cells. PMID:14602649

In vivo administration of fluorescent dextrans for the specific and sensitive localization of brain vascular pericytes and their characterization in normal and neurotoxin exposed brains.

PubMed

Sarkar, Sumit; Schmued, Larry

2012-06-01

We have aimed to develop novel histochemical markers for the labeling of brain pericytes and characterize their morphology in the normal and the excitotoxin-exposed brain, as this class of cells has received little attention until recently. Pericyte labeling was accomplished by the intracerebroventricular injection of certain fluorescent dextran conjugates, such as Fluoro-Gold-dextran, FR-dextran, FITC-dextran and Fluoro-Turquoise (FT)-dextran. 1-7 days after the tracer injection, extensive labeling of vascular pericytes was seen throughout the entire brain. These cells were found distal to the endothelial cells and exhibited large dye containing vacuoles. The morphology of the pericytes was somewhat variable, exhibiting round or amoeboid shapes within larger intracellular vesicles, while those wrapping around capillaries exhibited a more elongated appearance with finger-like projections. The use of FG-dextran resulted in bluish yellow fluorescently labeled pericytes, while FR-dextran resulted in red fluorescent labeled pericytes, FITC-dextran exhibited green fluorescent pericytes and FT-dextran showed fluorescent blue pericytes in the brain. We have used these tracers to study possible changes in morphology and pericyte number following kainic acid insult, observing that the number of pericytes in the injured or lesioned areas of the brain is dramatically reduced compared to the non-injured areas. These novel fluorochromes should be of use for studies involving the detection and localization of pericytes in both normal and pathological brain tissues. Published by Elsevier B.V.

Parallel-multiplexed excitation light-sheet microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Xu, Dongli; Zhou, Weibin; Peng, Leilei

2017-02-01

Laser scanning light-sheet imaging allows fast 3D image of live samples with minimal bleach and photo-toxicity. Existing light-sheet techniques have very limited capability in multi-label imaging. Hyper-spectral imaging is needed to unmix commonly used fluorescent proteins with large spectral overlaps. However, the challenge is how to perform hyper-spectral imaging without sacrificing the image speed, so that dynamic and complex events can be captured live. We report wavelength-encoded structured illumination light sheet imaging (λ-SIM light-sheet), a novel light-sheet technique that is capable of parallel multiplexing in multiple excitation-emission spectral channels. λ-SIM light-sheet captures images of all possible excitation-emission channels in true parallel. It does not require compromising the imaging speed and is capable of distinguish labels by both excitation and emission spectral properties, which facilitates unmixing fluorescent labels with overlapping spectral peaks and will allow more labels being used together. We build a hyper-spectral light-sheet microscope that combined λ-SIM with an extended field of view through Bessel beam illumination. The system has a 250-micron-wide field of view and confocal level resolution. The microscope, equipped with multiple laser lines and an unlimited number of spectral channels, can potentially image up to 6 commonly used fluorescent proteins from blue to red. Results from in vivo imaging of live zebrafish embryos expressing various genetic markers and sensors will be shown. Hyper-spectral images from λ-SIM light-sheet will allow multiplexed and dynamic functional imaging in live tissue and animals.

Single-label kinase and phosphatase assays for tyrosine phosphorylation using nanosecond time-resolved fluorescence detection.

PubMed

Sahoo, Harekrushna; Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

2007-12-26

The collision-induced fluorescence quenching of a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) by hydrogen atom abstraction from the tyrosine residue in peptide substrates was introduced as a single-labeling strategy to assay the activity of tyrosine kinases and phosphatases. The assays were tested for 12 different combinations of Dbo-labeled substrates and with the enzymes p60c-Src Src kinase, EGFR kinase, YOP protein tyrosine phosphatase, as well as acid and alkaline phosphatases, thereby demonstrating a broad application potential. The steady-state fluorescence changed by a factor of up to 7 in the course of the enzymatic reaction, which allowed for a sufficient sensitivity of continuous monitoring in steady-state experiments. The fluorescence lifetimes (and intensities) were found to be rather constant for the phosphotyrosine peptides (ca. 300 ns in aerated water), while those of the unphosphorylated peptides were as short as 40 ns (at pH 7) and 7 ns (at pH 13) as a result of intramolecular quenching. Owing to the exceptionally long fluorescence lifetime of Dbo, the assays were alternatively performed by using nanosecond time-resolved fluorescence (Nano-TRF) detection, which leads to an improved discrimination of background fluorescence and an increased sensitivity. The potential for inhibitor screening was demonstrated through the inhibition of acid and alkaline phosphatases by molybdate.

Antibody-mediated fluorescence enhancement based on shifting the intramolecular dimer<-->monomer equilibrium of fluorescent dyes.

PubMed

Wei, A P; Blumenthal, D K; Herron, J N

1994-05-01

A novel concept is described for directly coupling fluorescence emission to protein-ligand binding. It is based on shifting the intramolecular monomer<-->dimer equilibrium of two fluorescent dyes linked by a short spacer. A 13-residue peptide, recognized by a monoclonal antibody against human chorionic gonadotrophin (hCG), was labeled with fluorescein (F) and tetramethylrhodamine (T) at its N- and C-terminus, respectively. Spectral evidence suggests that when the conjugate is free in solution, F and T exist as an intramolecular dimer. Fluorescence quenching of fluorescein and rhodamine is approximately 98% and approximately 90%, respectively, due to dimerization. When the double-labeled peptide is bound to anti-hCG, however, the rhodamine fluorescence increases by up to 7.8-fold, depending upon the excitation wavelength. This is attributed to the dissociation of intramolecular dimers brought about by conformational changes of the conjugate upon binding. Fluorescein fluorescence, on the other hand, was still quenched because of excited-state energy transfer and residual ground-state interactions. Antibody binding also resulted in a approximately 3.4-fold increase in fluorescence anisotropy of the peptide. These changes in intensity and anisotropy allow direct measurement of antigen-antibody binding with a fluorescence plate reader or a polarization analyzer, without the need for separation steps and labeling antibodies. Because recent advances in peptide technology have allowed rapid and economical identification of antigen-mimicking peptides, the double-labeled peptide approach offers many opportunities for developing new diagnostic assays and screening new therapeutic drugs. It also has many potential applications to techniques involving recombinant antibodies, biosensors, cell sorting, and DNA probes.

Two-site ionic labeling with pyranine: implications for structural dynamics studies of polymers and polypeptides by time-resolved fluorescence anisotropy.

PubMed

Sharma, Jai; Tleugabulova, Dina; Czardybon, Wojciech; Brennan, John D

2006-04-26

Time-resolved fluorescence anisotropy (TRFA) is widely used to study dynamic motions of biomolecules in a variety of environments. However, depolarization due to rapid side chain motions often complicates the interpretation of anisotropy decay data and interferes with the accurate observation of segmental motions. Here, we demonstrate a new method for two-point ionic labeling of polymers and biomolecules that have appropriately spaced amino groups using the fluorescent probe 8-hydroxyl-1,3,6-trisulfonated pyrene (pyranine). TRFA analysis shows that such labeling provides a more rigid attachment of the fluorophore to the macromolecule than the covalent or single-point ionic labeling of amino groups, leading to time-resolved anisotropy decays that better reflect the backbone motion of the labeled polymer segment. Optimal coupling of pyranine to biomolecule dynamics is shown to be obtained for appropriately spaced Arg groups, and in such cases the ionic binding is stable up to 150 mM ionic strength. TRFA was used to monitor the behavior of pyranine-labeled poly(allylamine) (PAM) and poly-d-lysine (PL) in sodium silicate derived sol-gel materials and revealed significant restriction of backbone motion upon entrapment for both polymers, an observation that was not readily apparent in a previous study with entrapped fluorescein-labeled PAM and PL. The implications of these findings for fluorescence studies of polymer and biomolecule dynamics are discussed.

Bio-Inspired Microsystem for Robust Genetic Assay Recognition

PubMed Central

Lue, Jaw-Chyng; Fang, Wai-Chi

2008-01-01

A compact integrated system-on-chip (SoC) architecture solution for robust, real-time, and on-site genetic analysis has been proposed. This microsystem solution is noise-tolerable and suitable for analyzing the weak fluorescence patterns from a PCR prepared dual-labeled DNA microchip assay. In the architecture, a preceding VLSI differential logarithm microchip is designed for effectively computing the logarithm of the normalized input fluorescence signals. A posterior VLSI artificial neural network (ANN) processor chip is used for analyzing the processed signals from the differential logarithm stage. A single-channel logarithmic circuit was fabricated and characterized. A prototype ANN chip with unsupervised winner-take-all (WTA) function was designed, fabricated, and tested. An ANN learning algorithm using a novel sigmoid-logarithmic transfer function based on the supervised backpropagation (BP) algorithm is proposed for robustly recognizing low-intensity patterns. Our results show that the trained new ANN can recognize low-fluorescence patterns better than an ANN using the conventional sigmoid function. PMID:18566679

Gold Functionalized Mesoporous Silica Nanoparticle Mediated Protein and DNA Codelivery to Plant Cells Via the Biolistic Method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin-Ortigosa, Susana; Valenstein, Justin S.; Lin, Victor S.-Y.

2012-09-11

The synthesis and characterization of a gold nanoparticle functionalized mesoporous silica nanoparticle (Au-MSN) platform for codelivery of proteins and plasmid DNA to plant tissues using a biolistic particle delivery system is reported. The in vitro uptake and release profiles of fluorescently labeled bovine serum albumin (BSA) and enhanced green fluorescent protein (eGFP) are investigated. As a proof-of-concept demonstration, Au-MSN with large average pore diameters (10 nm) are shown to deliver and subsequently release proteins and plasmid DNA to the same cell after passing through the plant cell wall upon bombardment. Release of fluorescent eGFP indicates the delivery of active, non-denaturedmore » proteins to plant cells. This advance represents the first example of biolistic-mediated codelivery of proteins and plasmid DNA to plant cells via gold-functionalized MSN and provides a powerful tool for both fundamental and applied research of plant sciences.« less

Location of the motoneurons of the mylohyoid muscle in the rat. A fluorescence and Nissl study.

PubMed

Badran, Darwish H; Al-Hadidi, Maher T; Ramadan, Hassan N; Abu-Ghaida, Jamal H

2005-01-01

To locate the neuronal motor cells of the mylohyoid muscle and discuss their topographical organization. The present study was conducted at the Department of Anatomy and Histology, Faculty of Medicine, University of Jordan, Amman, Jordan between 2002 and 2003. The mylohyoid muscle in 15 albino rats was injected with 15 mliter of a retrogradely transported fluorescent material DAPI-Pr. After a survival period of 48 hours, animals were sacrificed, fixed in situ and brains harvested. The caudorostral transverse sections of the hindbrains were examined under the fluorescence microscope to detect the fluorescing cells, which were immediately photographed. Sections containing the labeled cells were charted, stained with 1% thionine and photographs obtained through light and fluorescence microscopes at different magnifications. The place and shape of all labeled cells were singled out by asset of their charted referring photographs of hindbrain sections, which display the entire motor trigeminal nucleus. The results showed that the fluorescent cell increase was found to occupy the rostromedial part of the ipsilateral motor trigeminal nucleus. The nucleus was large at its caudal third; the labeled cells are mainly those of the medial "subgroup". These cells are rationally distinct and lie alongside the internal loop of the facial nerve. At the middle third, most of the medial "subgroup" was found labeled. At its middle, the nucleus found was well developed, attained an appreciable size and its medial "subgroup" was somewhat distinct. Whereas, at the rostral third, the nucleus was larger, the medial group was more distinct and all cells were labeled. The medial cellular mass of the nucleus showed reduced labeled cells at the rostral end. This study demonstrates that the rostromedial part of the motor trigeminal nucleus represents the absolute territorial domain of the mylohyoid muscle motoneurons.

Fluorescent Approaches to High Throughput Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Forsythe, Elizabeth

2005-01-01

X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, 51%, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear hits. Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons. This presentation will focus on the methodology for fluorescent labeling, the crystallization results, and the effects of the trace labeling on the crystal quality.

Fluorescent Approaches to High Throughput Crystallography

NASA Technical Reports Server (NTRS)

Minamitani, Elizabeth Forsythe; Pusey, Marc L.

2004-01-01

X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1%, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of a macromolecules purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals will show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "bits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons. This presentation will focus on the methodology for fluorescent labeling, the crystallization results, and the effects of the trace labeling on the crystal quality.

Fluorescent Approaches to High Throughput Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Forsythe, Elizabeth

2004-01-01

X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically can not reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1%, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "hits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons. This presentation will focus on the methodology for fluorescent labeling, the crystallization results, and the effects of the trace labeling on the crystal quality.

Flash lamp-excited time-resolved fluorescence microscope suppresses autofluorescence in water concentrates to deliver an 11-fold increase in signal-to-noise ratio.

PubMed

Connally, Russell; Veal, Duncan; Piper, James

2004-01-01

The ubiquity of naturally fluorescing components (autofluorophores) encountered in most biological samples hinders the detection and identification of labeled targets through fluorescence-based techniques. Time-resolved fluorescence (TRF) is a technique by which the effects of autofluorescence are reduced by using specific fluorescent labels with long fluorescence lifetimes (compared with autofluorophores) in conjunction with time-gated detection. A time-resolved fluorescence microscope (TRFM) is described that is based on a standard epifluorescence microscope modified by the addition of a pulsed excitation source and an image-intensified time-gateable CCD camera. The choice of pulsed excitation source for TRFM has a large impact on the price and performance of the instrument. A flash lamp with rapid discharge characteristics was selected for our instrument because of the high spectral energy in the UV region and short pulse length. However, the flash output decayed with an approximate lifetime of 18 micros and the TRFM required a long-lived lanthanide chelate label to ensure that probe fluorescence was visible after decay of the flash plasma. We synthesized a recently reported fluorescent chelate (BHHCT) and conjugated it to a monoclonal antibody directed against the waterborne parasite Giardia lamblia. For a 600-nm bandpass filter set and a gate delay of 60 micros, the TRFM provided an 11.3-fold improvement in the signal-to-noise ratio (S/N) of labeled Giardia over background. A smaller gain in an SNR of 9.69-fold was achieved with a 420-nm longpass filter set; however, the final contrast ratio between labeled cyst and background was higher (11.3 versus 8.5). Despite the decay characteristics of the light pulse, flash lamps have many practical advantages compared with optical chopper wheels and modulated lasers for applications in TRFM.

Fluorescence-based strategies to investigate the structure and dynamics of aptamer-ligand complexes

NASA Astrophysics Data System (ADS)

Perez-Gonzalez, Cibran; Lafontaine, Daniel; Penedo, J.

2016-08-01

In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labelling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labelled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these single-molecule FRET microscopy techniques.

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes

PubMed Central

Perez-Gonzalez, Cibran; Lafontaine, Daniel A.; Penedo, J. Carlos

2016-01-01

In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labeling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labeled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these single-molecule FRET microscopy techniques. PMID:27536656

High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

PubMed

Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo

2016-01-01

Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'. Copyright © 2015 Elsevier Ltd. All rights reserved.

Segmentation and classification of cell cycle phases in fluorescence imaging.

PubMed

Ersoy, Ilker; Bunyak, Filiz; Chagin, Vadim; Cardoso, M Christina; Palaniappan, Kannappan

2009-01-01

Current chemical biology methods for studying spatiotemporal correlation between biochemical networks and cell cycle phase progression in live-cells typically use fluorescence-based imaging of fusion proteins. Stable cell lines expressing fluorescently tagged protein GFP-PCNA produce rich, dynamically varying sub-cellular foci patterns characterizing the cell cycle phases, including the progress during the S-phase. Variable fluorescence patterns, drastic changes in SNR, shape and position changes and abundance of touching cells require sophisticated algorithms for reliable automatic segmentation and cell cycle classification. We extend the recently proposed graph partitioning active contours (GPAC) for fluorescence-based nucleus segmentation using regional density functions and dramatically improve its efficiency, making it scalable for high content microscopy imaging. We utilize surface shape properties of GFP-PCNA intensity field to obtain descriptors of foci patterns and perform automated cell cycle phase classification, and give quantitative performance by comparing our results to manually labeled data.

Multimodal quantitative phase and fluorescence imaging of cell apoptosis

NASA Astrophysics Data System (ADS)

Fu, Xinye; Zuo, Chao; Yan, Hao

2017-06-01

Fluorescence microscopy, utilizing fluorescence labeling, has the capability to observe intercellular changes which transmitted and reflected light microscopy techniques cannot resolve. However, the parts without fluorescence labeling are not imaged. Hence, the processes simultaneously happen in these parts cannot be revealed. Meanwhile, fluorescence imaging is 2D imaging where information in the depth is missing. Therefore the information in labeling parts is also not complete. On the other hand, quantitative phase imaging is capable to image cells in 3D in real time through phase calculation. However, its resolution is limited by the optical diffraction and cannot observe intercellular changes below 200 nanometers. In this work, fluorescence imaging and quantitative phase imaging are combined to build a multimodal imaging system. Such system has the capability to simultaneously observe the detailed intercellular phenomenon and 3D cell morphology. In this study the proposed multimodal imaging system is used to observe the cell behavior in the cell apoptosis. The aim is to highlight the limitations of fluorescence microscopy and to point out the advantages of multimodal quantitative phase and fluorescence imaging. The proposed multimodal quantitative phase imaging could be further applied in cell related biomedical research, such as tumor.

High-Level Fluorescence Labeling of Gram-Positive Pathogens

PubMed Central

Aymanns, Simone; Mauerer, Stefanie; van Zandbergen, Ger; Wolz, Christiane; Spellerberg, Barbara

2011-01-01

Fluorescence labeling of bacterial pathogens has a broad range of interesting applications including the observation of living bacteria within host cells. We constructed a novel vector based on the E. coli streptococcal shuttle plasmid pAT28 that can propagate in numerous bacterial species from different genera. The plasmid harbors a promoterless copy of the green fluorescent variant gene egfp under the control of the CAMP-factor gene (cfb) promoter of Streptococcus agalactiae and was designated pBSU101. Upon transfer of the plasmid into streptococci, the bacteria show a distinct and easily detectable fluorescence using a standard fluorescence microscope and quantification by FACS-analysis demonstrated values that were 10–50 times increased over the respective controls. To assess the suitability of the construct for high efficiency fluorescence labeling in different gram-positive pathogens, numerous species were transformed. We successfully labeled Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, Streptococcus anginosus and Staphylococcus aureus strains utilizing the EGFP reporter plasmid pBSU101. In all of these species the presence of the cfb promoter construct resulted in high-level EGFP expression that could be further increased by growing the streptococcal and enterococcal cultures under high oxygen conditions through continuous aeration. PMID:21731607

Fluorescent humanized anti-CEA antibody specifically labels metastatic pancreatic cancer in a patient-derived orthotopic xenograft (PDOX) mouse model

NASA Astrophysics Data System (ADS)

Lwin, Thinzar M.; Miyake, Kentaro; Murakami, Takashi; DeLong, Jonathan C.; Yazaki, Paul J.; Shivley, John E.; Clary, Bryan; Hoffman, Robert M.; Bouvet, Michael

2018-03-01

Specific tumor targeting can result in selective labeling of cancer in vivo for surgical navigation. In the present study, we show that the use of an anti-CEA antibody conjugated to the near-infrared (NIR) fluorescent dye, IRDye800CW, can selectively target and label pancreatic cancer and its metastases in a clinically relevant patient derived xenograft mouse model.

DiOlistic Labeling of Neurons in Tissue Slices: A Qualitative and Quantitative Analysis of Methodological Variations.

PubMed

Staffend, Nancy A; Meisel, Robert L

2011-01-01

Fine neuronal morphology, such as dendritic spines, classically has been studied using the Golgi technique; however, Golgi staining is difficult to combine with other histological techniques. With the increasing popularity of fluorescent imaging, a number of fluorescent dyes have been developed that enable the coupling of multiple fluorescent labels in a single preparation. These fluorescent dyes include the lipophilic dialkylcarbocyanine, DiI; traditionally used for anterograde and retrograde neuronal tracing. More recently, DiI labeling has been used in combination with the Gene Gun for "DiOlistic" labeling of neurons in slice preparations. DiI sequesters itself within and diffuses laterally along the neuronal membrane, however once the cell is permeabilized, the DiI begins to leak from the cell membrane. A DiI derivative, Cell Tracker™ CM-DiI, increases dye stability and labeling half-life in permeabilized tissue, however at much greater expense. Here, the DiI and CM-DiI DiOlistic labeling techniques were tested in side-by-side experiments evaluating dye stability within dendritic architecture in medium spiny neurons of the dorsal stratum in both non-permeabilized and permeabilized tissue sections. In tissue sections that were not permeabilized, spine density in DiI labeled sections was higher than in CM-DiI labeling. In contrast, tissue sections that were permeabilized had higher spine densities in CM-DiI labeled neurons. These results suggest that for experiments involving non-permeabilized tissue, traditional DiI will suffice, however for experiments involving permeabilized tissue CM-DiI provides more consistent data. These experiments provide the first quantitative analyses of the impact of methodological permutations on neuronal labeling with DiI.

Immune cells tracing using quantum dots

NASA Astrophysics Data System (ADS)

Hoshino, Akiyoshi; Fujioka, Kouki; Kawamura, Yuki I.; Toyama-Sorimachi, Noriko; Yasuhara, Masato; Dohi, Taeko; Yamamoto, Kenji

2006-02-01

Fluorescent nanoparticles, such as nanocrystal quantum dots (QDs), have potential to be applied to molecular biology and bioimaging, since some nanocrystals emit higher and longer lasting fluorescence than conventional organic probes do. Here we report an example of labeling immune cells by QDs. We collected splenic CD4 + T-lymphocyte and peritoneal macrophages from mice. Then cells were labeled with QDs. QDs are incorporated into the T-lymphocyte and macrophages immediately after addition and located in the cytoplasm via endocytosis pathway. The fluorescence of QDs held in the endosomes was easily detected for more than a week. In addition, T-lymphocytes labeled with QDs were stable and cell proliferation or cytokine production including IL-2 and IFN-γ was not affected. When QD-labeled T-lymphocytes were adoptively transferred intravenously to mice, they remained in the peripheral blood and spleen up to a week. Using QD-labeled peritoneal macrophages, we studied cell traffic during inflammation on viscera in peritoneum cavity. QD-labeled macrophages were transplanted into the peritoneum of the mouse, and colitis was induced by intracolonic injection of a hapten, trinitrobenzensulfonic acid. With the aid of stong signals of QDs, we found that macrophage accumuled on the inflammation site of the colon. These results suggested that fluorescent probes of QDs might be useful as bioimaging tools for tracing target cells in vivo.

Breast cancer cells synchronous labeling and separation based on aptamer and fluorescence-magnetic silica nanoparticles

NASA Astrophysics Data System (ADS)

Wang, Qiu-Yue; Huang, Wei; Jiang, Xing-Lin; Kang, Yan-Jun

2018-01-01

In this work, an efficient method based on biotin-labeled aptamer and streptavidin-conjugated fluorescence-magnetic silica nanoprobes (FITC@Fe3O4@SiNPs-SA) has been established for human breast carcinoma MCF-7 cells synchronous labeling and separation. Carboxyl-modified fluorescence-magnetic silica nanoparticles (FITC@Fe3O4@SiNPs-COOH) were first synthesized using the Stöber method. Streptavidin (SA) was then conjugated to the surface of FITC@Fe3O4@SiNPs-COOH. The MCF-7 cell suspension was incubated with biotin-labeled MUC-1 aptamer. After centrifugation and washing, the cells were then treated with FITC@Fe3O4@SiNPs-SA. Afterwards, the mixtures were separated by a magnet. The cell-probe conjugates were then imaged using fluorescent microscopy. The results show that the MUC-1 aptamer could recognize and bind to the targeted cells with high affinity and specificity, indicating the prepared FITC@Fe3O4@SiNPs-SA with great photostability and superparamagnetism could be applied effectively in labeling and separation for MCF-7 cell in suspension synchronously. In addition, the feasibility of MCF-7 cells detection in peripheral blood was assessed. The results indicate that the method above is also applicable for cancer cells synchronous labeling and separation in complex biological system.

Multicapillary SDS-gel electrophoresis for the analysis of fluorescently labeled mAb preparations: a high throughput quality control process for the production of QuantiPlasma and PlasmaScan mAb libraries.

PubMed

Székely, Andrea; Szekrényes, Akos; Kerékgyártó, Márta; Balogh, Attila; Kádas, János; Lázár, József; Guttman, András; Kurucz, István; Takács, László

2014-08-01

Molecular heterogeneity of mAb preparations is the result of various co- and post-translational modifications and to contaminants related to the production process. Changes in molecular composition results in alterations of functional performance, therefore quality control and validation of therapeutic or diagnostic protein products is essential. A special case is the consistent production of mAb libraries (QuantiPlasma™ and PlasmaScan™) for proteome profiling, quality control of which represents a challenge because of high number of mAbs (>1000). Here, we devise a generally applicable multicapillary SDS-gel electrophoresis process for the analysis of fluorescently labeled mAb preparations for the high throughput quality control of mAbs of the QuantiPlasma™ and PlasmaScan™ libraries. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemical Probes for Visualizing Intact Animal and Human Brain Tissue.

PubMed

Lai, Hei Ming; Ng, Wai-Lung; Gentleman, Steve M; Wu, Wutian

2017-06-22

Newly developed tissue clearing techniques can be used to render intact tissues transparent. When combined with fluorescent labeling technologies and optical sectioning microscopy, this allows visualization of fine structure in three dimensions. Gene-transfection techniques have proved very useful in visualizing cellular structures in animal models, but they are not applicable to human brain tissue. Here, we discuss the characteristics of an ideal chemical fluorescent probe for use in brain and other cleared tissues, and offer a comprehensive overview of currently available chemical probes. We describe their working principles and compare their performance with the goal of simplifying probe selection for neuropathologists and stimulating probe development by chemists. We propose several approaches for the development of innovative chemical labeling methods which, when combined with tissue clearing, have the potential to revolutionize how we study the structure and function of the human brain. Copyright © 2017 Elsevier Ltd. All rights reserved.

Visualization of the Nucleolus in Living Cells with Cell-Penetrating Fluorescent Peptides.

PubMed

Martin, Robert M; Herce, Henry D; Ludwig, Anne K; Cardoso, M Cristina

2016-01-01

The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of ribosomal RNA synthesis and assembly of ribosomes. The nucleolus plays also a major role in nuclear organization as the largest compartment within the nucleus. The prominent structure of the nucleolus can be detected using contrast light microscopy providing an approximate localization of the nucleolus, but this approach does not allow to determine accurately the three-dimensional structure of the nucleolus in cells and tissues. Immunofluorescence staining with antibodies specific to nucleolar proteins albeit very useful is time consuming, normally antibodies recognize their epitopes only within a small range of species and is applicable only in fixed cells. Here, we present a simple method to selectively and accurately label this ubiquitous subnuclear compartment in living cells of a large range of species using a fluorescently labeled cell-penetrating peptide.

Biotinylated dextran amine anterograde tracing of the canine corticospinal tract.

PubMed

Han, Xiao; Lv, Guangming; Wu, Huiqun; Ji, Dafeng; Sun, Zhou; Li, Yaofu; Tang, Lemin

2012-04-15

In this study, biotinylated dextran amine (BDA) was microinjected into the left cortical motor area of the canine brain. Fluorescence microscopy results showed that a large amount of BDA-labeled pyramidal cells were visible in the left cortical motor area after injection. In the left medulla oblongata, the BDA-labeled corticospinal tract was evenly distributed, with green fluorescence that had a clear boundary with the surrounding tissue. The BDA-positive corticospinal tract entered into the right lateral funiculus of the spinal cord and descended into the posterior part of the right lateral funiculus, close to the posterior horn, from cervical to sacral segments. There was a small amount of green fluorescence in the sacral segment. The distribution of BDA labeling in the canine central nervous system was consistent with the course of the corticospinal tract. Fluorescence labeling for BDA gradually diminished with time after injection. Our findings indicate that the BDA anterograde tracing technique can be used to visualize the localization and trajectory of the corticospinal tract in the canine central nervous system.

Fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis for quantitative determination of DNA methylation and demethylation status.

PubMed

Kageyama, Shinji; Shinmura, Kazuya; Yamamoto, Hiroko; Goto, Masanori; Suzuki, Koichi; Tanioka, Fumihiko; Tsuneyoshi, Toshihiro; Sugimura, Haruhiko

2008-04-01

The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

Superresolution microscopy with transient binding.

PubMed

Molle, Julia; Raab, Mario; Holzmeister, Susanne; Schmitt-Monreal, Daniel; Grohmann, Dina; He, Zhike; Tinnefeld, Philip

2016-06-01

For single-molecule localization based superresolution, the concentration of fluorescent labels has to be thinned out. This is commonly achieved by photophysically or photochemically deactivating subsets of molecules. Alternatively, apparent switching of molecules can be achieved by transient binding of fluorescent labels. Here, a diffusing dye yields bright fluorescent spots when binding to the structure of interest. As the binding interaction is weak, the labeling is reversible and the dye ligand construct diffuses back into solution. This approach of achieving superresolution by transient binding (STB) is reviewed in this manuscript. Different realizations of STB are discussed and compared to other localization-based superresolution modalities. We propose the development of labeling strategies that will make STB a highly versatile tool for superresolution microscopy at highest resolution. Copyright © 2015 Elsevier Ltd. All rights reserved.

Photoaffinity-labeling and fluorescence-distribution studies of gonadotropin-releasing hormone receptors in ovarian granulosa cells.

PubMed Central

Hazum, E; Nimrod, A

1982-01-01

Photoaffinity labeling of rat ovarian granulosa cells and membrane preparations with a bioactive photoaffinity derivative of gonadotropin-releasing hormone resulted in identification of two specific components with apparent molecular weights of 60,000 and 54,000. Fluorescent visualization of gonadotropin-releasing hormone receptors in these cells, by using a bioactive rhodamine derivative of the hormone, indicated that the fluorescently labeled receptors were initially distributed uniformly on the cell surface and then formed patches that subsequently internalized (at 37 degrees C) into endocytic vesicles. These processes were dependent on specific binding sites for the rhodamine-labeled peptide on the granulosa cells. These studies may provide an experimental basis for understanding the molecular events involved in the action of the hormone in the ovary. Images PMID:6281784

Tracking Image Correlation: Combining Single-Particle Tracking and Image Correlation

PubMed Central

Dupont, A.; Stirnnagel, K.; Lindemann, D.; Lamb, D.C.

2013-01-01

The interactions and coordination of biomolecules are crucial for most cellular functions. The observation of protein interactions in live cells may provide a better understanding of the underlying mechanisms. After fluorescent labeling of the interacting partners and live-cell microscopy, the colocalization is generally analyzed by quantitative global methods. Recent studies have addressed questions regarding the individual colocalization of moving biomolecules, usually by using single-particle tracking (SPT) and comparing the fluorescent intensities in both color channels. Here, we introduce a new method that combines SPT and correlation methods to obtain a dynamical 3D colocalization analysis along single trajectories of dual-colored particles. After 3D tracking, the colocalization is computed at each particle’s position via the local 3D image cross correlation of the two detection channels. For every particle analyzed, the output consists of the 3D trajectory, the time-resolved 3D colocalization information, and the fluorescence intensity in both channels. In addition, the cross-correlation analysis shows the 3D relative movement of the two fluorescent labels with an accuracy of 30 nm. We apply this method to the tracking of viral fusion events in live cells and demonstrate its capacity to obtain the time-resolved colocalization status of single particles in dense and noisy environments. PMID:23746509

Correlative cellular ptychography with functionalized nanoparticles at the Fe L-edge

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gallagher-Jones, Marcus; Dias, Carlos Sato Baraldi; Pryor, Alan

Precise localization of nanoparticles within a cell is crucial to the understanding of cell-particle interactions and has broad applications in nanomedicine. Here in this paper, we report a proof-of-principle experiment for imaging individual functionalized nanoparticles within a mammalian cell by correlative microscopy. Using a chemically-fixed HeLa cell labeled with fluorescent core-shell nanoparticles as a model system, we implemented a graphene-oxide layer as a substrate to significantly reduce background scattering. We identified cellular features of interest by fluorescence microscopy, followed by scanning transmission X-ray tomography to localize the particles in 3D, and ptychographic coherent diffractive imaging of the fine features inmore » the region at high resolution. By tuning the X-ray energy to the Fe L-edge, we demonstrated sensitive detection of nanoparticles composed of a 22 nm magnetic Fe 3O 4 core encased by a 25-nm-thick fluorescent silica (SiO 2) shell. These fluorescent core-shell nanoparticles act as landmarks and offer clarity in a cellular context. Our correlative microscopy results confirmed a subset of particles to be fully internalized, and high-contrast ptychographic images showed two oxidation states of individual nanoparticles with a resolution of ~16.5 nm. The ability to precisely localize individual fluorescent nanoparticles within mammalian cells will expand our understanding of the structure/function relationships for functionalized nanoparticles.« less

Correlative cellular ptychography with functionalized nanoparticles at the Fe L-edge

DOE PAGES

Gallagher-Jones, Marcus; Dias, Carlos Sato Baraldi; Pryor, Alan; ...

2017-07-06

Precise localization of nanoparticles within a cell is crucial to the understanding of cell-particle interactions and has broad applications in nanomedicine. Here in this paper, we report a proof-of-principle experiment for imaging individual functionalized nanoparticles within a mammalian cell by correlative microscopy. Using a chemically-fixed HeLa cell labeled with fluorescent core-shell nanoparticles as a model system, we implemented a graphene-oxide layer as a substrate to significantly reduce background scattering. We identified cellular features of interest by fluorescence microscopy, followed by scanning transmission X-ray tomography to localize the particles in 3D, and ptychographic coherent diffractive imaging of the fine features inmore » the region at high resolution. By tuning the X-ray energy to the Fe L-edge, we demonstrated sensitive detection of nanoparticles composed of a 22 nm magnetic Fe 3O 4 core encased by a 25-nm-thick fluorescent silica (SiO 2) shell. These fluorescent core-shell nanoparticles act as landmarks and offer clarity in a cellular context. Our correlative microscopy results confirmed a subset of particles to be fully internalized, and high-contrast ptychographic images showed two oxidation states of individual nanoparticles with a resolution of ~16.5 nm. The ability to precisely localize individual fluorescent nanoparticles within mammalian cells will expand our understanding of the structure/function relationships for functionalized nanoparticles.« less

Cell and Tissue Imaging with Molecularly Imprinted Polymers.

PubMed

Panagiotopoulou, Maria; Kunath, Stephanie; Haupt, Karsten; Tse Sum Bui, Bernadette

2017-01-01

Advanced tools for cell imaging are of particular interest as they can detect, localize and quantify molecular targets like abnormal glycosylation sites that are biomarkers of cancer and infection. Targeting these biomarkers is often challenging due to a lack of receptor materials. Molecularly imprinted polymers (MIPs) are promising artificial receptors; they can be tailored to bind targets specifically, be labeled easily, and are physically and chemically stable. Herein, we demonstrate the application of MIPs as artificial antibodies for selective labeling and imaging of cellular targets, on the example of hyaluronan and sialylation moieties on fixated human skin cells and tissues. Thus, fluorescently labeled MIP nanoparticles templated with glucuronic acid (MIPGlcA) and N-acetylneuraminic acid (MIPNANA) are respectively applied. Two different fluorescent probes are used: (1) MIPGlcA particles, ~400 nm in size are labeled with the dye rhodamine that target the extracellular hyaluronan on cells and tissue specimens and (2) MIP-coated InP/ZnS quantum dots (QDs) of two different colors, ~125 nm in size that target the extracellular and intracellular hyaluronan and sialylation sites. Green and red emitting QDs are functionalized with MIPGlcA and MIPNANA respectively, enabling multiplexed cell imaging. This is a general approach that can also be adapted to other target molecules on and in cells.

Hydrophobic pocket targeting probes for enteroviruses

NASA Astrophysics Data System (ADS)

Martikainen, Mari; Salorinne, Kirsi; Lahtinen, Tanja; Malola, Sami; Permi, Perttu; Häkkinen, Hannu; Marjomäki, Varpu

2015-10-01

Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron microscopes. The probe mildly stabilized the virus particle by increasing the melting temperature by 1-3 degrees, and caused a delay in the uncoating of the virus in the cellular endosomes, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding moiety of the probe was shown to bind to echovirus 1 particle by STD and tr-NOESY NMR methods. Furthermore, binding to echovirus 1 and Coxsackievirus A9, and to a lesser extent to Coxsackie virus B3 was verified by using a gold nanocluster labeled probe by TEM analysis. Molecular modelling suggested that the probe fits the hydrophobic pockets of EV1 and CVA9, but not of CVB3 as expected, correlating well with the variations in the infectivity and stability of the virus particles. EV1 conjugated to the fluorescent dye labeled probe was efficiently internalized into the cells. The virus-fluorescent probe conjugate accumulated in the cytoplasmic endosomes and caused infection starting from 6 hours onwards. Remarkably, before and during the time of replication, the fluorescent probe was seen to leak from the virus-positive endosomes and thus separate from the capsid proteins that were left in the endosomes. These results suggest that, like the physiological hydrophobic content, the probe may be released upon virus uncoating. Our results collectively thus show that the gold and fluorescently labeled probes may be used to track and visualize the studied enteroviruses during the early phases of infection opening new avenues to follow virus uncoating in cells.Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron microscopes. The probe mildly stabilized the virus particle by increasing the melting temperature by 1-3 degrees, and caused a delay in the uncoating of the virus in the cellular endosomes, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding moiety of the probe was shown to bind to echovirus 1 particle by STD and tr-NOESY NMR methods. Furthermore, binding to echovirus 1 and Coxsackievirus A9, and to a lesser extent to Coxsackie virus B3 was verified by using a gold nanocluster labeled probe by TEM analysis. Molecular modelling suggested that the probe fits the hydrophobic pockets of EV1 and CVA9, but not of CVB3 as expected, correlating well with the variations in the infectivity and stability of the virus particles. EV1 conjugated to the fluorescent dye labeled probe was efficiently internalized into the cells. The virus-fluorescent probe conjugate accumulated in the cytoplasmic endosomes and caused infection starting from 6 hours onwards. Remarkably, before and during the time of replication, the fluorescent probe was seen to leak from the virus-positive endosomes and thus separate from the capsid proteins that were left in the endosomes. These results suggest that, like the physiological hydrophobic content, the probe may be released upon virus uncoating. Our results collectively thus show that the gold and fluorescently labeled probes may be used to track and visualize the studied enteroviruses during the early phases of infection opening new avenues to follow virus uncoating in cells. Electronic supplementary information (ESI) available: Details of the synthesis of the probes, UV-Vis absorption spectra of the probe (2), PAGE separation and the absorption spectra of the gold labeled probe (3), details of the NMR experiments, determination of the cytotoxicity of the studied molecules, TEM micrographs of the gold labeled probe (3) with enteroviruses, live cell imaging of the fluorescent probe (4) in cells, and additional details of modeling of the hydrophobic pockets. See DOI: 10.1039/c5nr04139b

Two-color fluorescent (near-infrared and visible) triphasic perfluorocarbon nanoemulsions

PubMed Central

Patel, Sravan Kumar; Patrick, Michael J.; Pollock, John A.

2013-01-01

Abstract. Design and development of a new formulation as a unique assembly of distinct fluorescent reporters with nonoverlapping fluorescence spectra and a F19 magnetic resonance imaging agent into colloidally and optically stable triphasic nanoemulsion are reported. Specifically, a cyanine dye-perfluorocarbon (PFC) conjugate was introduced into the PFC phase of the nanoemulsion and a near-infrared dye was introduced into the hydrocarbon (HC) layer. To the best of our knowledge, this is the first report of a triphasic nanoemulsion system where each oil phase, HC, and PFC are fluorescently labeled and formulated into an optically and colloidally stable nanosystem. Having, each oil phase separately labeled by a fluorescent dye allows for improved correlation between in vivo imaging and histological data. Further, dual fluorescent labeling can improve intracellular tracking of the nanodroplets and help assess the fate of the nanoemulsion in biologically relevant media. The nanoemulsions were produced by high shear processing (microfluidization) and stabilized with biocompatible nonionic surfactants resulting in mono-modal size distribution with average droplet size less than 200 nm. Nanoemulsions demonstrate excellent colloidal stability and only moderate changes in the fluorescence signal for both dyes. Confocal fluorescence microscopy of macrophages exposed to nanoemulsions shows the presence of both fluorescence agents in the cytoplasm. PMID:23912666

Preclinical Comparison of Near-Infrared-Labeled Cetuximab and Panitumumab for Optical Imaging of Head and Neck Squamous Cell Carcinoma

PubMed Central

Day, Kristine E.; Sweeny, Larissa; Kulbersh, Brian; Zinn, Kurt R.; Rosenthal, Eben L.

2014-01-01

Purpose: Though various targets have been proposed and evaluated, no agent has yet been investigated in a clinical setting for head and neck cancer. The present study aimed to compare two fluorescently labeled anti-epidermal growth factor receptor (EGFR) antibodies for detection of head and neck squamous cell carcinoma (HNSCC). Procedures: Antigen specificities and in vitro imaging of the fluorescently labeled anti-EGFR antibodies were performed. Next, immunodeficient mice (n=22) bearing HNSCC (OSC-19 and SCC-1) tongue tumors received systemic injections of cetuximab-IRDye800CW, panitumumab-IRDye800CW, or IgG-IRDye800CW (a nonspecific control). Tumors were imaged and resected using two near-infrared imaging systems, SPY and Pearl. Fluorescent lymph nodes were also identified, and all resected tissues were sent for pathology. Results: Panitumumab-IRDye800CW and cetuximab-IRDye800CW had specific and high affinity binding for EGFR (KD=0.12 and 0.31 nM, respectively). Panitumumab-IRDye800CW demonstrated a 2-fold increase in fluorescence intensity compared to cetuximab-IRDye800CW in vitro. In vivo, both fluorescently labeled antibodies produced higher tumor-to-background ratios compared to IgG-IRDye800CW. However, there was no significant difference between the two in either cell line or imaging modality (OSC-19: p=0.08 SPY, p=0.48 Pearl; SCC-1: p=0.77 SPY, p=0.59 Pearl; paired t tests). Conclusions: There was no significant difference between the two fluorescently labeled anti-EGFR monoclonal antibodies in murine models of HNSCC. Both cetuximab and panitumumab can be considered suitable targeting agents for fluorescent intraoperative detection of HNSCC. PMID:23715932

High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid.

PubMed

Anumula, K R; Dhume, S T

1998-07-01

Facile labeling of oligosaccharides (acidic and neutral) in a nonselective manner was achieved with highly fluorescent anthranilic acid (AA, 2-aminobenzoic acid) (more than twice the intensity of 2-aminobenzamide, AB) for specific detection at very high sensitivity. Quantitative labeling in acetate-borate buffered methanol (approximately pH 5.0) at 80 degreesC for 60 min resulted in negligible or no desialylation of the oligosaccharides. A high resolution high performance liquid chromatographic method was developed for quantitative oligosaccharide mapping on a polymeric-NH2bonded (Astec) column operating under normal phase and anion exchange (NP-HPAEC) conditions. For isolation of oligosaccharides from the map by simple evaporation, the chromatographic conditions developed use volatile acetic acid-triethylamine buffer (approximately pH 4.0) systems. The mapping and characterization technology was developed using well characterized standard glycoproteins. The fluorescent oligosaccharide maps were similar to the maps obtained by the high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), except that the fluorescent maps contained more defined peaks. In the map, the oligosaccharides separated into groups based on charge, size, linkage, and overall structure in a manner similar to HPAEC-PAD with contribution of -COOH function from the label, anthranilic acid. However, selectivity of the column for sialic acid linkages was different. A second dimension normal phase HPLC (NP-HPLC) method was developed on an amide column (TSK Gel amide-80) for separation of the AA labeled neutral complex type and isomeric structures of high mannose type oligosaccharides. The oligosaccharides labeled with AA are compatible with biochemical and biophysical techniques, and use of matrix assisted laser desorption mass spectrometry for rapid determination of oligosaccharide mass map of glycoproteins is demonstrated. High resolution of NP-HPAEC and NP-HPLC methods combined with mass spectrometry (MALDI-TOF) can provide an effective technology for analyzing a wide repertoire of oligosaccharide structures and for determining the action of both transferases and glycosidases.

Mechanism of calmodulin recognition of the binding domain of isoform 1b of the plasma membrane Ca2+-ATPase: kinetic pathway and effects of methionine oxidation

PubMed Central

Slaughter, Brian D.; Bieber Urbauer, Ramona J.; Urbauer, Jeffrey L.; Johnson, Carey K.

2008-01-01

Calmodulin (CaM) binds to a domain near the C-terminus of the plasma-membrane Ca2+-ATPase (PMCA), causing the release of this domain and relief of its autoinhibitory function. We investigated the kinetics of dissociation and binding of Ca2+-CaM with a 28-residue peptide (C28W(1b)) corresponding to the CaM binding domain of isoform 1b of PMCA. CaM was labeled with a fluorescent probe on either the N-terminal domain at residue 34 or on the C-terminal domain at residue 110. Formation of complexes of CaM with C28W(1b) results in a decrease in the fluorescence yield of the fluorophore, allowing the kinetics of dissociation or binding to be detected. Using a maximum entropy method, we determined the minimum number and magnitudes of rate constants required to fit the data. Comparison of the fluorescence changes for CaM labeled on the C-terminal or N-terminal domain suggests sequential and ordered binding of the C-terminal and N-terminal domains of CaM with C28W(1b). For dissociation of C28W(1b) from CaM labeled on the N-terminal domain, we observed three time constants, indicating the presence of two intermediate states in the dissociation pathway. However, for CaM labeled on the C-terminal domain, we observed only two time constants, suggesting that the fluorescence label on the C-terminal domain was not sensitive to one of the kinetic steps. The results were modeled by a kinetic mechanism where an initial complex forms upon binding of the C-terminal domain of CaM to C28W(1b), followed by binding of the N-terminal domain, and then formation of a tight binding complex. Oxidation of methionine residues in CaM resulted in significant perturbations to the binding kinetics. The rate of formation of a tight binding complex was reduced, consistent with the lower effectiveness of oxidized CaM in activating the Ca2+ pump. PMID:17343368

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels.

PubMed

Zhu, Shengchao; Zhang, Qin; Guo, Liang-Hong

2008-08-22

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17beta-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17beta-estradiol is 1.9 pg mL(-1), which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.

Two-photon excited fluorescence emission from hemoglobin

NASA Astrophysics Data System (ADS)

Sun, Qiqi; Zeng, Yan; Zhang, Wei; Zheng, Wei; Luo, Yi; Qu, Jianan Y.

2015-03-01

Hemoglobin, one of the most important proteins in blood, is responsible for oxygen transportation in almost all vertebrates. Recently, we discovered two-photon excited hemoglobin fluorescence and achieved label-free microvascular imaging based on the hemoglobin fluorescence. However, the mechanism of its fluorescence emission still remains unknown. In this work, we studied the two-photon excited fluorescence properties of the hemoglobin subunits, heme/hemin (iron (II)/(III) protoporphyrin IX) and globin. We first studied the properties of heme and the similar spectral and temporal characteristics of heme and hemoglobin fluorescence provide strong evidence that heme is the fluorophore in hemoglobin. Then we studied the fluorescence properties of hemin, globin and methemoglobin, and found that the hemin may have the main effect on the methemoglobin fluorescence and that globin has tryptophan fluorescence like other proteins. Finally, since heme is a centrosymmetric molecule, that the Soret band fluorescence of heme and hemoglobin was not observed in the single photon process in the previous study may be due to the parity selection rule. The discovery of heme two-photon excited fluorescence may open a new window for heme biology research, since heme as a cofactor of hemoprotein has many functions, including chemical catalysis, electron transfer and diatomic gases transportation.

Cytochrome c conformations resolved by the photon counting histogram: Watching the alkaline transition with single-molecule sensitivity

PubMed Central

Perroud, Thomas D.; Bokoch, Michael P.; Zare, Richard N.

2005-01-01

We apply the photon counting histogram (PCH) model, a fluorescence technique with single-molecule sensitivity, to study pH-induced conformational changes of cytochrome c. PCH is able to distinguish different protein conformations based on the brightness of a fluorophore sensitive to its local environment. We label cytochrome c through its single free cysteine with tetramethylrhodamine-5-maleimide (TMR), a fluorophore with specific brightnesses that we associate with specific protein conformations. Ensemble measurements demonstrate two different fluorescence responses with increasing pH: (i) a decrease in fluorescence intensity caused by the alkaline transition of cytochrome c (pH 7.0–9.5), and (ii) an increase in intensity when the protein unfolds (pH 9.5–10.8). The magnitudes of these two responses depend strongly on the molar ratio of TMR used to label cytochrome c. Using PCH we determine that this effect arises from the proportion of a nonfunctional conformation in the sample, which can be differentiated from the functional conformation. We further determine the causes of each ensemble fluorescence response: (i) during the alkaline transition, the fluorophore enters a dark state and discrete conformations are observed, and (ii) as cytochrome c unfolds, the fluorophore incrementally brightens, but discrete conformations are no longer resolved. Moreover, we also show that functional TMR-cytochrome c undergoes a response of identical magnitude regardless of the proportion of nonfunctional protein in the sample. As expected for a technique with single-molecule sensitivity, we demonstrate that PCH can directly observe the most relevant conformation, unlike ensemble fluorometry. PMID:16314563

Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice.

PubMed

Feeks, James A; Hunter, Jennifer J

2017-05-01

In vivo cellular scale fluorescence lifetime imaging of the mouse retina has the potential to be a sensitive marker of retinal cell health. In this study, we demonstrate fluorescence lifetime imaging of extrinsic fluorophores using adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO). We recorded AOFLIO images of inner retinal cells labeled with enhanced green fluorescent protein (EGFP) and capillaries labeled with fluorescein. We demonstrate that AOFLIO can be used to differentiate spectrally overlapping fluorophores in the retina. With further refinements, AOFLIO could be used to assess retinal health in early stages of degeneration by utilizing lifetime-based sensors or even fluorophores native to the retina.

Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mulari, Mika T.K.; Centre for Military Medicine, Research Department, Lahti; Nars, Martin

2008-05-01

Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSDmore » to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-{beta}-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption.« less

FLUORESCENT IN SITU DETECTION OF ENCEPHALITOZOON HELLEM SPORES WITH A 6-CARBOXYFLUORESCEIN-LABELED RNA-TARGETED OLIGONUCLEOTIDE PROBE

EPA Science Inventory

A fluorescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem, in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligo...

Fluorescence Intensity- and Lifetime-Based Glucose Sensing Using Glucose/Galactose-Binding Protein

PubMed Central

Pickup, John C.; Khan, Faaizah; Zhi, Zheng-Liang; Coulter, Jonathan; Birch, David J. S.

2013-01-01

We review progress in our laboratories toward developing in vivo glucose sensors for diabetes that are based on fluorescence labeling of glucose/galactose-binding protein. Measurement strategies have included both monitoring glucose-induced changes in fluorescence resonance energy transfer and labeling with the environmentally sensitive fluorophore, badan. Measuring fluorescence lifetime rather than intensity has particular potential advantages for in vivo sensing. A prototype fiber-optic-based glucose sensor using this technology is being tested.Fluorescence technique is one of the major solutions for achieving the continuous and noninvasive glucose sensor for diabetes. In this article, a highly sensitive nanostructured sensor is developed to detect extremely small amounts of aqueous glucose by applying fluorescence energy transfer (FRET). A one-pot method is applied to produce the dextran-fluorescein isothiocyanate (FITC)-conjugating mesoporous silica nanoparticles (MSNs), which afterward interact with the tetramethylrhodamine isothiocyanate (TRITC)-labeled concanavalin A (Con A) to form the FRET nanoparticles (FITC-dextran-Con A-TRITC@MSNs). The nanostructured glucose sensor is then formed via the self-assembly of the FRET nanoparticles on a transparent, flexible, and biocompatible substrate, e.g., poly(dimethylsiloxane). Our results indicate the diameter of the MSNs is 60 ± 5 nm. The difference in the images before and after adding 20 μl of glucose (0.10 mmol/liter) on the FRET sensor can be detected in less than 2 min by the laser confocal laser scanning microscope. The correlation between the ratio of fluorescence intensity, I(donor)/I(acceptor), of the FRET sensor and the concentration of aqueous glucose in the range of 0.04–4 mmol/liter has been investigated; a linear relationship is found. Furthermore, the durability of the nanostructured FRET sensor is evaluated for 5 days. In addition, the recorded images can be converted to digital images by obtaining the pixels from the resulting matrix using Matlab image processing functions. We have also studied the in vitro cytotoxicity of the device. The nanostructured FRET sensor may provide an alternative method to help patients manage the disease continuously. PMID:23439161

Simultaneous detection of Escherichia coli O157:H7 and Salmonella Typhimurium using quantum dots as fluorescence labels.

PubMed

Yang, Liju; Li, Yanbin

2006-03-01

In this study, we explored the use of semiconductor quantum dots (QDs) as fluorescence labels in immunoassays for simultaneous detection of two species of foodborne pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium. QDs with different sizes can be excited with a single wavelength of light, resulting in different emission peaks that can be measured simultaneously. Highly fluorescent semiconductor quantum dots with different emission wavelengths (525 nm and 705 nm) were conjugated to anti-E. coli O157 and anti-Salmonella antibodies, respectively. Target bacteria were separated from samples by using specific antibody coated magnetic beads. The bead-cell complexes reacted with QD-antibody conjugates to form bead-cell-QD complexes. Fluorescent microscopic images of QD labeled E. coli and Salmonella cells demonstrated that QD-antibody conjugates could evenly and completely attach to the surface of bacterial cells, indicating that the conjugated QD molecules still retain their effective fluorescence, while the conjugated antibody molecules remain active and are able to recognize their specific target bacteria in a complex mixture. The intensities of fluorescence emission peaks at 525 nm and 705 nm of the final complexes were measured for quantitative detection of E. coli O157:H7 and S. Typhimurium simultaneously. The fluorescence intensity (FI) as a function of cell number (N) was found for Salmonella and E. coli, respectively. The regression models can be expressed as: FI = 60.6 log N- 250.9 with R(2) = 0.97 for S. Typhimurium, and FI = 77.8 log N- 245.2 with R(2) = 0.91 for E. coli O157:H7 in the range of cell numbers from 10(4) to 10(7) cfu ml(-1). The detection limit of this method was 10(4) cfu ml(-1). The detection could be completed within 2 hours. The principle of this method could be extended to detect multiple species of bacteria (3-4 species) simultaneously, depending on the availability of each type of QD-antibody conjugates with a unique emission peak and the antibody coated magnetic beads specific to each species of bacteria.

Fluorescent magnetic nanoparticles for cell labeling: flux synthesis of manganite particles and novel functionalization of silica shell.

PubMed

Kačenka, Michal; Kaman, Ondřej; Kikerlová, Soňa; Pavlů, Barbora; Jirák, Zdeněk; Jirák, Daniel; Herynek, Vít; Černý, Jan; Chaput, Frédéric; Laurent, Sophie; Lukeš, Ivan

2015-06-01

Novel synthetic approaches for the development of multimodal imaging agents with high chemical stability are demonstrated. The magnetic cores are based on La0.63Sr0.37MnO3 manganite prepared as individual grains using a flux method followed by additional thermal treatment in a protective silica shell allowing to enhance their magnetic properties. The cores are then isolated and covered de novo with a hybrid silica layer formed through the hydrolysis and polycondensation of tetraethoxysilane and a fluorescent silane synthesized from rhodamine, piperazine spacer, and 3-iodopropyltrimethoxysilane. The aminoalkyltrialkoxysilanes are strictly avoided and the resulting particles are hydrolytically stable and do not release dye. The high colloidal stability of the material and the long durability of the fluorescence are reinforced by an additional silica layer on the surface of the particles. Structural and magnetic studies of the products using XRD, TEM, and SQUID magnetometry confirm the importance of the thermal treatment and demonstrate that no mechanical treatment is required for the flux-synthesized manganite. Detailed cell viability tests show negligible or very low toxicity at concentrations at which excellent labeling is achieved. Predominant localization of nanoparticles in lysosomes is confirmed by immunofluorescence staining. Relaxometric and biological studies suggest that the functionalized nanoparticles are suitable for imaging applications. Copyright © 2015 Elsevier Inc. All rights reserved.

The design of a microfluidic biochip for the rapid, multiplexed detection of foodborne pathogens by surface plasmon resonance imaging

NASA Astrophysics Data System (ADS)

Zordan, Michael D.; Grafton, Meggie M. G.; Park, Kinam; Leary, James F.

2010-02-01

The rapid detection of foodborne pathogens is increasingly important due to the rising occurrence of contaminated food supplies. We have previously demonstrated the design of a hybrid optical device that has the capability to perform realtime surface plasmon resonance (SPR) and epi-fluorescence imaging. We now present the design of a microfluidic biochip consisting of a two-dimensional array of functionalized gold spots. The spots on the array have been functionalized with capture peptides that specifically bind E. coli O157:H7 or Salmonella enterica. This array is enclosed by a PDMS microfluidic flow cell. A magnetically pre-concentrated sample is injected into the biochip, and whole pathogens will bind to the capture array. The previously constructed optical device is being used to detect the presence and identity of captured pathogens using SPR imaging. This detection occurs in a label-free manner, and does not require the culture of bacterial samples. Molecular imaging can also be performed using the epi-fluorescence capabilities of the device to determine pathogen state, or to validate the identity of the captured pathogens using fluorescently labeled antibodies. We demonstrate the real-time screening of a sample for the presence of E. coli O157:H7 and Salmonella enterica. Additionally the mechanical properties of the microfluidic flow cell will be assessed. The effect of these properties on pathogen capture will be examined.

Early steps of supported bilayer formation probed by single vesicle fluorescence assays.

PubMed Central

Johnson, Joseph M; Ha, Taekjip; Chu, Steve; Boxer, Steven G

2002-01-01

We have developed a single vesicle assay to study the mechanisms of supported bilayer formation. Fluorescently labeled, unilamellar vesicles (30-100 nm diameter) were first adsorbed to a quartz surface at low enough surface concentrations to visualize single vesicles. Fusion and rupture events during the bilayer formation, induced by the subsequent addition of unlabeled vesicles, were detected by measuring two-color fluorescence signals simultaneously. Lipid-conjugated dyes monitored the membrane fusion while encapsulated dyes reported on the vesicle rupture. Four dominant pathways were observed, each exhibiting characteristic two-color fluorescence signatures: 1) primary fusion, in which an unlabeled vesicle fuses with a labeled vesicle on the surface, is signified by the dequenching of the lipid-conjugated dyes followed by rupture and final merging into the bilayer; 2) simultaneous fusion and rupture, in which a labeled vesicle on the surface ruptures simultaneously upon fusion with an unlabeled vesicle; 3) no dequenching, in which loss of fluorescence signal from both dyes occur simultaneously with the final merger into the bilayer; and 4) isolated rupture (pre-ruptured vesicles), in which a labeled vesicle on the surface spontaneously undergoes content loss, a process that occurs with high efficiency in the presence of a high concentration of Texas Red-labeled lipids. Vesicles that have undergone content loss appear to be more fusogenic than intact vesicles. PMID:12496104

Selection of fluorophore and quencher pairs for fluorescent nucleic acid hybridization probes.

PubMed

Marras, Salvatore A E

2006-01-01

With the introduction of simple and relatively inexpensive methods for labeling nucleic acids with nonradioactive labels, doors have been opened that enable nucleic acid hybridization probes to be used for research and development, as well as for clinical diagnostic applications. The use of fluorescent hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. The use of hybridization probes that bind to the amplification products in real-time markedly improves the ability to obtain quantitative results. Furthermore, real-time nucleic acid amplification assays can be carried out in sealed tubes, eliminating carryover contamination. Because fluorescent hybridization probes are available in a wide range of colors, multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. It is therefore important to carefully select the labels of hybridization probes, based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This chapter outlines different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers.

Fluorescently labeled chimeric anti-CEA antibody improves detection and resection of human colon cancer in a patient-derived orthotopic xenograft (PDOX) nude mouse model.

PubMed

Metildi, Cristina A; Kaushal, Sharmeela; Luiken, George A; Talamini, Mark A; Hoffman, Robert M; Bouvet, Michael

2014-04-01

The aim of this study was to evaluate a new fluorescently labeled chimeric anti-CEA antibody for improved detection and resection of colon cancer. Frozen tumor and normal human tissue samples were stained with chimeric and mouse antibody-fluorophore conjugates for comparison. Mice with patient-derived orthotopic xenografts (PDOX) of colon cancer underwent fluorescence-guided surgery (FGS) or bright-light surgery (BLS) 24 hr after tail vein injection of fluorophore-conjugated chimeric anti-CEA antibody. Resection completeness was assessed using postoperative images. Mice were followed for 6 months for recurrence. The fluorophore conjugation efficiency (dye/mole ratio) improved from 3-4 to >5.5 with the chimeric CEA antibody compared to mouse anti-CEA antibody. CEA-expressing tumors labeled with chimeric CEA antibody provided a brighter fluorescence signal on frozen human tumor tissues (P = 0.046) and demonstrated consistently lower fluorescence signals in normal human tissues compared to mouse antibody. Chimeric CEA antibody accurately labeled PDOX colon cancer in nude mice, enabling improved detection of tumor margins for more effective FGS. The R0 resection rate increased from 86% to 96% with FGS compared to BLS. Improved conjugating efficiency and labeling with chimeric fluorophore-conjugated antibody resulted in better detection and resection of human colon cancer in an orthotopic mouse model. © 2013 Wiley Periodicals, Inc.

Creating and virtually screening databases of fluorescently-labelled compounds for the discovery of target-specific molecular probes

NASA Astrophysics Data System (ADS)

Kamstra, Rhiannon L.; Dadgar, Saedeh; Wigg, John; Chowdhury, Morshed A.; Phenix, Christopher P.; Floriano, Wely B.

2014-11-01

Our group has recently demonstrated that virtual screening is a useful technique for the identification of target-specific molecular probes. In this paper, we discuss some of our proof-of-concept results involving two biologically relevant target proteins, and report the development of a computational script to generate large databases of fluorescence-labelled compounds for computer-assisted molecular design. The virtual screening of a small library of 1,153 fluorescently-labelled compounds against two targets, and the experimental testing of selected hits reveal that this approach is efficient at identifying molecular probes, and that the screening of a labelled library is preferred over the screening of base compounds followed by conjugation of confirmed hits. The automated script for library generation explores the known reactivity of commercially available dyes, such as NHS-esters, to create large virtual databases of fluorescence-tagged small molecules that can be easily synthesized in a laboratory. A database of 14,862 compounds, each tagged with the ATTO680 fluorophore was generated with the automated script reported here. This library is available for downloading and it is suitable for virtual ligand screening aiming at the identification of target-specific fluorescent molecular probes.

Different Approaches for Assaying Melanosome Transfer

PubMed Central

Berens, Werner; Van Den Bossche, Karolien; Yoon, Tae-Jin; Westbroek, Wendy; Valencia, Julio C.; Out, Coby J.; Naeyaert, Jean Marie; Hearing, Vincent J.; Lambert, Jo

2006-01-01

Summary Many approaches have been tried to establish assays for melanosome transfer to keratinocytes. In this report we describe and summarize various novel attempts to label melanosomes in search of a reliable, specific, reproducible and quantitative assay system. We tried to fluorescently label melanosomes by transfection of GFP-labeled melanosomal proteins and by incubation of melanocytes with fluorescent melanin intermediates or homologues. In most cases a weak cytoplasmic fluorescence was perceived, which was probably due to incorrect sorting or deficient incorporation of the fluorescent protein and different localisation. We were able to label melanosomes via incorporation of 14C-thiouracil into melanin. Consequently, we tried to develop an assay to separate keratinocytes with transferred radioactivity from melanocytes after co-culture. Differential trypsination and different magnetic bead separation techniques were tested with unsatisfactory results. An attempt was also made to incorporate fluorescent thiouracil, since this would allow cells to be separated by FACS. In conclusion, different methods to measure pigment transfer between donor melanocytes and acceptor keratinocytes were thoroughly examined. This information could give other researchers a head start in the search for a melanosome transfer assay with said qualities to better understand pigment transfer. PMID:16162177

Evaluation of nanoparticles as endocytic tracers in cellular microbiology

NASA Astrophysics Data System (ADS)

Zhang, Yuying; Hensel, Michael

2013-09-01

The study of pathogen interactions with eukaryotic host cells requires the introduction of fluorescent probes to visualize processes such as endocytosis, intracellular transport or host cell manipulation by the pathogen. Here, three types of fluorescent nanoparticles (NPs), i.e. Rhodamine-labeled polymethacrylate (PMA) NPs, silica NPs and gold NPs, were employed to label the host cellular endolysosomal system and monitor manipulations by the pathogen Salmonella enterica. Using live cell imaging, we investigated the performance of NPs in cellular uptake, labeling of endocytic vesicles and lysosomes, as well as interaction with the pathogen. We show that fluorescent gold and silica, but not PMA NPs appropriately label host cell structures and efficiently track rearrangements of the host endosomal system by the activities of intracellular Salmonella. Silica NPs slightly aggregated and located in Salmonella-induced compartments as isolated dots, while gold NPs distributed uniformly inside such structures. Both silica and gold NPs exhibited no adverse impact on either host cells or pathogens, and are versatile tools for infection biology.The study of pathogen interactions with eukaryotic host cells requires the introduction of fluorescent probes to visualize processes such as endocytosis, intracellular transport or host cell manipulation by the pathogen. Here, three types of fluorescent nanoparticles (NPs), i.e. Rhodamine-labeled polymethacrylate (PMA) NPs, silica NPs and gold NPs, were employed to label the host cellular endolysosomal system and monitor manipulations by the pathogen Salmonella enterica. Using live cell imaging, we investigated the performance of NPs in cellular uptake, labeling of endocytic vesicles and lysosomes, as well as interaction with the pathogen. We show that fluorescent gold and silica, but not PMA NPs appropriately label host cell structures and efficiently track rearrangements of the host endosomal system by the activities of intracellular Salmonella. Silica NPs slightly aggregated and located in Salmonella-induced compartments as isolated dots, while gold NPs distributed uniformly inside such structures. Both silica and gold NPs exhibited no adverse impact on either host cells or pathogens, and are versatile tools for infection biology. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr01550e

Artificial Photosynthesis of Alcohols by Multi-Functionalized Semiconductor Photocathodes.

PubMed

Zhang, Yuqian; Han, Bo; Xu, Yanjie; Zhao, Dongning; Jia, Yongjian; Nie, Rong; Zhu, Zhouhe; Chen, Fengjuan; Wang, Jianguo; Jing, Huanwang

2017-04-22

Novel artificial photosynthesis systems are devised as cells of dye/Pd/NR-MO x (M=Ti, Zn)∥CoPi/W:BiVO 4 that convert efficiently CO 2 to alcohols. The photocathodes are aminofunctionalized, palladium-deposited, and in situ sensitized nano-TiO 2 or ZnO/FTO (FTO: fluorine-doped tin oxide) electrodes that are characterized by X-ray photoelectron spectroscopy (XPS), TEM, XRD, UV/Vis spectra, and evaluated by electrochemical techniques. The cell of dye/Pd/S-TiO 2 ∥CoPi/W:BiVO 4 uniquely generates ethanol under irradiation of 200 mW cm -2 , reaching 0.56 % quantum efficiency (QE) at -0.56 V and 0.13 % QE without external electron supply. The cell of dye/Pd/ N-ZnO∥CoPi/W:BiVO 4 produces solely methanol at a rate of 42.8 μm h -1  cm -2 at -0.56 V of a Si solar cell, which is far less than the electrochemical voltage of water splitting (1.23 V). Its QE reaches to 0.38 %, which is equal to plants. The isotopic labeling experiments confirm the carbon source and oxygen releasing. The selectivity for alcohols of multi-functionalized semiconductors is discussed. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

AutoBD: Automated Bi-Level Description for Scalable Fine-Grained Visual Categorization.

PubMed

Yao, Hantao; Zhang, Shiliang; Yan, Chenggang; Zhang, Yongdong; Li, Jintao; Tian, Qi

Compared with traditional image classification, fine-grained visual categorization is a more challenging task, because it targets to classify objects belonging to the same species, e.g. , classify hundreds of birds or cars. In the past several years, researchers have made many achievements on this topic. However, most of them are heavily dependent on the artificial annotations, e.g., bounding boxes, part annotations, and so on . The requirement of artificial annotations largely hinders the scalability and application. Motivated to release such dependence, this paper proposes a robust and discriminative visual description named Automated Bi-level Description (AutoBD). "Bi-level" denotes two complementary part-level and object-level visual descriptions, respectively. AutoBD is "automated," because it only requires the image-level labels of training images and does not need any annotations for testing images. Compared with the part annotations labeled by the human, the image-level labels can be easily acquired, which thus makes AutoBD suitable for large-scale visual categorization. Specifically, the part-level description is extracted by identifying the local region saliently representing the visual distinctiveness. The object-level description is extracted from object bounding boxes generated with a co-localization algorithm. Although only using the image-level labels, AutoBD outperforms the recent studies on two public benchmark, i.e. , classification accuracy achieves 81.6% on CUB-200-2011 and 88.9% on Car-196, respectively. On the large-scale Birdsnap data set, AutoBD achieves the accuracy of 68%, which is currently the best performance to the best of our knowledge.Compared with traditional image classification, fine-grained visual categorization is a more challenging task, because it targets to classify objects belonging to the same species, e.g. , classify hundreds of birds or cars. In the past several years, researchers have made many achievements on this topic. However, most of them are heavily dependent on the artificial annotations, e.g., bounding boxes, part annotations, and so on . The requirement of artificial annotations largely hinders the scalability and application. Motivated to release such dependence, this paper proposes a robust and discriminative visual description named Automated Bi-level Description (AutoBD). "Bi-level" denotes two complementary part-level and object-level visual descriptions, respectively. AutoBD is "automated," because it only requires the image-level labels of training images and does not need any annotations for testing images. Compared with the part annotations labeled by the human, the image-level labels can be easily acquired, which thus makes AutoBD suitable for large-scale visual categorization. Specifically, the part-level description is extracted by identifying the local region saliently representing the visual distinctiveness. The object-level description is extracted from object bounding boxes generated with a co-localization algorithm. Although only using the image-level labels, AutoBD outperforms the recent studies on two public benchmark, i.e. , classification accuracy achieves 81.6% on CUB-200-2011 and 88.9% on Car-196, respectively. On the large-scale Birdsnap data set, AutoBD achieves the accuracy of 68%, which is currently the best performance to the best of our knowledge.

Preclinical Evaluation of Robotic-Assisted Sentinel Lymph Node Fluorescence Imaging

PubMed Central

Liss, Michael A.; Farshchi-Heydari, Salman; Qin, Zhengtao; Hickey, Sean A.; Hall, David J.; Kane, Christopher J.; Vera, David R.

2015-01-01

An ideal substance to provide convenient and accurate targeting for sentinel lymph node (SLN) mapping during robotic-assisted surgery has yet to be found. We used an animal model to determine the ability of the FireFly camera system to detect fluorescent SLNs after administration of a dual-labeled molecular imaging agent. Methods We injected the footpads of New Zealand White rabbits with 1.7 or 8.4 nmol of tilmanocept labeled with 99mTc and a near-infrared fluorophore, IRDye800CW. One and 36 h after injection, popliteal lymph nodes, representing the SLNs, were dissected with the assistance of the FireFly camera system, a fluorescence-capable endoscopic imaging system. After excision of the paraaortic lymph nodes, which represented non-SLNs, we assayed all lymph nodes for radioactivity and fluorescence intensity. Results Fluorescence within all popliteal lymph nodes was easily detected by the FireFly camera system. Fluorescence within the lymph channel could be imaged during the 1-h studies. When compared with the paraaortic lymph nodes, the popliteal lymph nodes retain greater than 95% of the radioactivity at both 1 and 36 h after injection. At both doses (1.7 and 8.4 nmol), the popliteal nodes had higher (P < 0.050) optical fluorescence intensity than the paraaortic nodes at the 1- and 36-h time points. Conclusion The FireFly camera system can easily detect tilmanocept labeled with a near-infrared fluorophore at least 36 h after administration. This ability will permit image acquisition and subsequent verification of fluorescence-labeled SLNs during robotic-assisted surgery. PMID:25024425

Preclinical evaluation of robotic-assisted sentinel lymph node fluorescence imaging.

PubMed

Liss, Michael A; Farshchi-Heydari, Salman; Qin, Zhengtao; Hickey, Sean A; Hall, David J; Kane, Christopher J; Vera, David R

2014-09-01

An ideal substance to provide convenient and accurate targeting for sentinel lymph node (SLN) mapping during robotic-assisted surgery has yet to be found. We used an animal model to determine the ability of the FireFly camera system to detect fluorescent SLNs after administration of a dual-labeled molecular imaging agent. We injected the footpads of New Zealand White rabbits with 1.7 or 8.4 nmol of tilmanocept labeled with (99m)Tc and a near-infrared fluorophore, IRDye800CW. One and 36 h after injection, popliteal lymph nodes, representing the SLNs, were dissected with the assistance of the FireFly camera system, a fluorescence-capable endoscopic imaging system. After excision of the paraaortic lymph nodes, which represented non-SLNs, we assayed all lymph nodes for radioactivity and fluorescence intensity. Fluorescence within all popliteal lymph nodes was easily detected by the FireFly camera system. Fluorescence within the lymph channel could be imaged during the 1-h studies. When compared with the paraaortic lymph nodes, the popliteal lymph nodes retain greater than 95% of the radioactivity at both 1 and 36 h after injection. At both doses (1.7 and 8.4 nmol), the popliteal nodes had higher (P < 0.050) optical fluorescence intensity than the paraaortic nodes at the 1- and 36-h time points. The FireFly camera system can easily detect tilmanocept labeled with a near-infrared fluorophore at least 36 h after administration. This ability will permit image acquisition and subsequent verification of fluorescence-labeled SLNs during robotic-assisted surgery. © 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Allelic divergence and cultivar-specific SSR alleles revealed by capillary electrophoresis using fluorescence-labeled SSR markers in sugarcane

USDA-ARS?s Scientific Manuscript database

Though sugarcane cultivars (Saccharum spp. hybrids) are complex aneu-polyploid hybrids, genetic evaluation and tracking of clone- or cultivar-specific alleles become possible due to capillary electrophoregrams (CE) using fluorescence-labeled SSR primer pairs. Twenty-four sugarcane cultivars, 12 each...

Towards label-free and site-specific probing of the local pH in proteins: pH-dependent deep UV Raman spectra of histidine and tyrosine

NASA Astrophysics Data System (ADS)

Bröermann, Andreas; Steinhoff, Heinz-Jürgen; Schlücker, Sebastian

2014-09-01

The site-specific pH is an experimental probe for assessing models of structural folding and function of a protein as well as protein-protein and protein-ligand interactions. It can be determined by various techniques such as NMR, FT-IR, fluorescence and EPR spectroscopy. The latter require the use of external labels, i.e., employ pH-dependent dyes and spin labels, respectively. In this contribution, we outline an approach to a label-free and site-specific method for determining the local pH using deep ultraviolet resonance Raman (UVRR) spectroscopic fingerprints of the aromatic amino acids histidine and tyrosine in combination with a robust algorithm that determines the pH value using three UVRR reference spectra and without prior knowledge of the pKa.

A fluorescent aptasensor for amplified label-free detection of adenosine triphosphate based on core-shell Ag@SiO2 nanoparticles.

PubMed

Song, Quanwei; Peng, Manshu; Wang, Le; He, Dacheng; Ouyang, Jin

2016-03-15

The novel, facile and universal aptamer-based methods for the highly sensitive and selective fluorescence detection of important biomolecules have attracted considerable interest. Here, we present a label-free aptasensor for adenosine triphosphate (ATP) detection in aqueous solutions by using an ultra-sensitive nucleic acid stain PicoGreen (PG) as a fluorescent indicator and core-shell Ag@SiO2 nanoparticles (NPs) as a metal-enhanced fluorescence (MEF) platform. In the presence of ATP, the complementary DNA (cDNA)/aptamer duplexes confined onto the Ag@SiO2 NPs surface can release their aptamers into the buffered solution, causing a significant reduction in fluorescence intensity. By virtue of the amplified fluorescence signal, this aptasensor toward ATP can achieve a detection limit of 14.2 nM with a wide linear range and exhibit a good assay performance in complex biological samples. This sensing approach is cost-effective and efficient because it avoids the fluorescence labeling process and the use of any enzymes. Hence, this method may offer an alternative tool for determining the concentrations of ATP in biochemical and biomedical research. Copyright © 2015 Elsevier B.V. All rights reserved.

Moving Toward the Light: Using New Technology to Answer Old Questions

PubMed Central

LUCITTI, JENNIFER L.; DICKINSON, MARY E.

2006-01-01

Fluorescence microscopy has become a principle methodology in the field of developmental biology. Recent technological advances have led to the design of high-speed and high-resolution confocal and multiphoton microscopes that enable researchers to obtain three- and four-dimensional information in living cells and whole embryos. Paralleling this progress, the development of stable and bright vital fluorescent probes has revolutionized the ability to track individual cells in vitro and in vivo and to visualize intercellular and subcellular molecular interactions in real time. Combining imaging modalities and labeling techniques that are increasingly unobtrusive to cell and whole animal function, our understanding of how proteins interact, tissues take form, and organs synchronize to create a functioning animal is reaching a whole new level. PMID:16690954

Europium containing red light-emitting fibers made by electrohydrodynamic casting

NASA Astrophysics Data System (ADS)

Gan, Yong X.; Panahi, Niousha; Yu, Christina; Gan, Jeremy B.; Cheng, Wanli

2018-05-01

Red light-emitting polymeric micro- and nanofibers were made by electrohydrodynamic co-casting of two fluids. One fluid contains a 10 wt% concentration europium (III) complex dissolved in a dimethylformamide (DMF) solvent. The europium complex, an Eu3+ compound with the nominal formula of Eu(BA)3phen/PAN, consists of polyacrylonitrile (PAN), 1,10-phenanthroline (phen), and benzoic acid (BA). The other fluid consists of iron metal oxide nanoparticles dispersed in a solution containing 10 wt% polyacrylonitrile polymer in DMF solvent. The two fluids were electrohydrodynamically co-cast onto a soft tissue paper using a stainless steel coaxial nozzle. The intensity of the electric field used for the co-casting was 1.5 kV/cm. Scanning electron microscopic observation on the fibers obtained from the co-casting was made. The size of the fibers ranges from several hundreds of nanometers to several microns. Energy dispersive X-ray spectroscopic analysis of the fibers confirmed that the major elements included C, O, Fe, and Eu. The fluorescence of the two types of fibers was tested under the excitation of a UV light source. It was found that when the europium complex-containing solution was the sheath fluid and the iron-containing solution was the core, the prepared fibers showed red light-emitting behavior under ultraviolet light. Time-dependent fluorescence shows the two-stage decaying behavior. The first stage lasts about 2000 s and the intensity of fluorescence decreases linearly. The second stage reveals the slow decaying behavior and it lasts longer than 3 h. Based on the bi-exponential data fitting using a processing MATLAB code, the fluorescence-related constants were extracted. A bi-exponential formula was proposed to describe the time-dependent fluorescence behavior of the fiber made by the europium complex-containing solution as the sheath fluid. The decaying in the fluorescence shows two different stages. The first stage lasts about 2000 s and it is characterized by a fast decaying model. The intensity of fluorescence decreases linearly. The second stage has a slow decaying feature. It takes over 3 h for the fluorescence to die out completely. Bi-exponential data fitting shows that the time constant for the decay of fluorescence is about 10,000 s.

Fluorescent Approaches to High Throughput Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Forsythe, Elizabeth; Achari, Aniruddha

2006-01-01

We have shown that by covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and the presence of the probe at low concentrations does not affect the X-ray data quality or the crystallization behavior. The presence of the trace fluorescent label gives a number of advantages when used with high throughput crystallizations. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a dark background. Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Brightly fluorescent crystals are readily found against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. We are now testing the use of high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that kinetics leading to non-structured phases may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Preliminary experiments with test proteins have resulted in the extraction of a number of crystallization conditions from screening outcomes based solely on the presence of bright fluorescent regions. Subsequent experiments will test this approach using a wider range of proteins. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons.

Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy

NASA Astrophysics Data System (ADS)

Alfonso-García, Alba; Smith, Tim D.; Datta, Rupsa; Luu, Thuy U.; Gratton, Enrico; Potma, Eric O.; Liu, Wendy F.

2016-04-01

Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.

Lateral microheterogeneity of diphenylhexatriene-labeled choline phospholipids in the erythrocyte ghost membrane as determined by time-resolved fluorescence spectroscopy.

PubMed

Prenner, E; Sommer, A; Maurer, N; Glatter, O; Gorges, R; Paltauf, F; Hermetter, A

2000-04-01

Choline phospholipids are the major constituents of the outer layer of the erythrocyte membrane. To investigate their lateral membrane organization we determined the fluorescence lifetime properties of diphenylhexatriene analogues of phosphatidylcholine, choline plasmalogen, (the respective enolether derivative), and sphingomyelin inserted into the outer layer of hemoglobin-free ghosts. Fluorescence lifetimes were recorded by time-resolved phase and modulation fluorometry and analyzed in terms of Continuous Lorentzian distributions. To assess the influence of membrane proteins on the fluorescence lifetime of the labeled lipids in the biomembrane, lipid vesicles were used as controls. In general, the lifetime distributions in the ghost membranes are broad compared to vesicles. Phosphatidylcholine and sphingomyelin exhibit very similar lifetime distributions in contrast to an increased plasmalogen lifetime heterogeneity in both systems. Orientational effects of side chain mobilities on the observed lifetimes can be excluded. Fluorescence anisotropies revealed identical values for all three labeled phospholipids in the biomembrane.

Two-photon microscopy and spectroscopy based on a compact confocal scanning head

NASA Astrophysics Data System (ADS)

Diaspro, Alberto; Chirico, Giberto; Federici, Federico; Cannone, Fabio; Beretta, Sabrina; Robello, Mauro; Olivini, Francesca; Ramoino, Paola

2001-07-01

We have combined a confocal laser scanning head modified for TPE (two-photon excitation) microscopy with some spectroscopic modules to study single molecules and molecular aggregates. The behavior of the TPE microscope unit has been characterized by means of point spread function measurements and of the demonstration of its micropatterning abilities. One-photon and two-photon mode can be simply accomplished by switching from a mono-mode optical fiber (one-photon) coupled to conventional laser sources to an optical module that allows IR laser beam (two- photon/TPE) delivery to the confocal laser scanning head. We have then described the characterization of the two-photon microscope for spectroscopic applications: fluorescence correlation, lifetime and fluorescence polarization anisotropy measurements. We describe the measurement of the response of the two-photon microscope to the light polarization and discuss fluorescence polarization anisotropy measurements on Rhodamine 6G as a function of the viscosity and on a globular protein, the Beta-lactoglobulin B labeled with Alexa 532 at very high dilutions. The average rotational and translational diffusion coefficients measured with fluorescence polarization anisotropy and fluorescence correlation methods are in good agreement with the protein size, therefore validating the use of the microscope for two-photon spectroscopy on biomolecules.

An automated segmentation methodology for quantifying immunoreactive puncta number and fluorescence intensity in tissue sections.

PubMed

Fish, Kenneth N; Sweet, Robert A; Deo, Anthony J; Lewis, David A

2008-11-13

A number of human brain diseases have been associated with disturbances in the structure and function of cortical synapses. Answering fundamental questions about the synaptic machinery in these disease states requires the ability to image and quantify small synaptic structures in tissue sections and to evaluate protein levels at these major sites of function. We developed a new automated segmentation imaging method specifically to answer such fundamental questions. The method takes advantage of advances in spinning disk confocal microscopy, and combines information from multiple iterations of a fluorescence intensity/morphological segmentation protocol to construct three-dimensional object masks of immunoreactive (IR) puncta. This new methodology is unique in that high- and low-fluorescing IR puncta are equally masked, allowing for quantification of the number of fluorescently-labeled puncta in tissue sections. In addition, the shape of the final object masks highly represents their corresponding original data. Thus, the object masks can be used to extract information about the IR puncta (e.g., average fluorescence intensity of proteins of interest). Importantly, the segmentation method presented can be easily adapted for use with most existing microscopy analysis packages.

Bismuth 213-labeled anti-CD45 radioimmunoconjugate to condition dogs for nonmyeloablative allogeneic marrow grafts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sandmaier, B M.; Bethge, W A.; Wilbur, D. Scott

To lower treatment-related mortality and toxicity of conventional marrow transplantation, a nonmyeloablative regimen using 200 cGy total-body irradiation (TBI) and mycophenolate mofetil (MMF) combined with cyclosporine (CSP) for postgrafting immunosuppression was developed. To circumvent possible toxic effects of external- beam gamma irradiation, strategies for targeted radiation therapy were investigated. We tested whether the short-lived (46 minutes) alpha-emitter Bi-213 conjugated to an anti-CD45 monoclonal antibody (mAb) could replace 200 cGy TBI and selectively target hematopoietic tissues in a canine model of nonmyeloablative DLA-identical marrow transplantation. Biodistribution studies using iodine 123-labeled anti-CD45 mAb showed uptake in blood, marrow, lymph nodes, spleen, andmore » liver. In a dose-escalation study, 7 dogs treated with the Bi-213-anti-CD45 conjugate (Bi-213 dose, 0.1-5.9 mCi/kg[3.7-218 MBq/kg]) without marrow grafts had no toxic effects other than a mild, reversible suppression of blood counts. On the basis of these studies, 3 dogs were treated with 0.5 mg/kg Bi-213-labeled anti-CD45 mAb (Bi-213 doses, 3.6, 4.6, and 8.8 mCi/kg[133, 170, and 326 MBq/kg]) given in 6 injections 3 and 2 days before grafting of marrow from DLA-identical littermates. The dogs also received MMF (10 mg/kg subcutaneously twice daily the day of transplantation until day 27 afterward) and CSP (15 mg/kg orally twice daily the day before transplantation until 35 days afterward). Therapy was well tolerated except for transient elevations in levels of transaminases in 3 dogs, followed by, in one dog, ascites. All dogs achieved prompt engraftment and stable mixed hematopoietic chimerism, with donor contributions ranging from 30% to 70% after more than 27 weeks of follow-up. These results form the basis for additional studies in animals and the design of clinical trials using Bi-213 as a nonmyeloablative conditioning regimen with minimal toxicity.« less

A novel application of the fluorescent dye bis-ANS for labeling neurons in acute brain slices.

PubMed

Mozes, Emese; Hunya, Akos; Toth, Aniko; Ayaydin, Ferhan; Penke, Botond; Datki, Zsolt L

2011-10-10

The cell-impermeant oligomer-(e.g. beta-amyloid-, or tubulin-) specific fluorescent dye, bis-ANS (4,4'-bis-1-anilinonaphtalene-8-sulfonate), was successfully used for labeling mechanically damaged but still viable neuron bodies, neurites and neurite cross sections in acute brain slices. Acute hippocampal brain slices of rats were co-stained with bis-ANS and the cell-impermeant, DNA-specific dye propidium iodide (PI) and were then analyzed using fluorescence and confocal microscopes. Both the neuron bodies and the neurites were found to exhibit increased fluorescence intensities, suggesting that using this method they can be detected more easily. In addition, bis-ANS showed good region - but not cell specific co-localization with the neuron-specific fluorescent dye Dil (1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate). These two dyes label different neuronal structures: Dil binds specifically to intact cell membranes while bis-ANS can enter cells with compromised cell membranes and then stain the microtubules in the cytoplasm. For a quick (10min) staining of acute brain slices with bis-ANS both HEPES and NaHCO(3) were needed in order to achieve high signal intensity. Labeling with bis-ANS fluorescent dye is an easy method for imaging the neuronal structures on the surface of acute brain slices. Copyright © 2011 Elsevier Inc. All rights reserved.

A general approach for chemical labeling and rapid, spatially controlled protein inactivation

PubMed Central

Marks, Kevin M.; Braun, Patrick D.; Nolan, Garry P.

2004-01-01

Chemical labeling of proteins inside of living cells can enable studies of the location, movement, and function of proteins in vivo. Here we demonstrate an approach for chemical labeling of proteins that uses the high-affinity interaction between an FKBP12 mutant (F36V) and a synthetic, engineered ligand (SLF′). A fluorescein conjugate to the engineered ligand (FL-SLF′) retained binding to FKBP12(F36V) and possessed similar fluorescence properties as parental fluorescein. FL-SLF′ labeled FKBP12(F36V) fusion proteins in live mammalian cells, and was used to monitor the subcellular localization of a membrane targeted FKBP12(F36V) construct. Chemical labeling of FKBP12(F36V) fusion proteins with FL-SLF′ was readily detectable at low expression levels of the FKBP12(F36V) fusion, and the level of fluorescent staining with FL-SLF′ was proportional to the FKBP12(F36V) expression level. This FL-SLF′-FKBP12(F36V) labeling technique was tested in fluorophore assisted laser inactivation (FALI), a light-mediated technique to rapidly inactivate fluorophore-labeled target proteins. FL-SLF′ mediated FALI of a β-galactosidase-FKBP12(F36V) fusion protein, causing rapid inactivation of >90% of enzyme activity upon irradiation in vitro. FL-SLF′ also mediated FALI of a β-galactosidase fusion expressed in living NIH 3T3 cells, where β-galactosidase activity was reduced in 15 s. Thus, FL-SLF′ can be used to monitor proteins in vivo and to target rapid, spatially and temporally defined inactivation of target proteins in living cells in a process that we call FK-FALI. PMID:15218100

Short communication: Labeling Listeria with anaerobic fluorescent protein for food safety studies.

PubMed

Landete, José M; Peirotén, Ángela; Medina, Margarita; Arqués, Juan L

2017-01-01

Many food safety-related studies require the tracking of inoculated food-borne pathogens to monitor their fate in food complex environments. In the current study, we demonstrate the potential of plasmids containing the fluorescence protein gene evoglow-Pp1 (Evocatal, Dusseldorf, Germany) as a real-time reporter system for Listeria strains. This anaerobic fluorescent protein provides an easily detectable phenotype of microorganisms for food safety studies. This work is the first to report a reliable method to identify fluorescently labeled Listeria strains in food ecosystems. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Fluorescent triplex-forming DNA oligonucleotides labeled with a thiazole orange dimer unit

PubMed Central

Ikeda, Shuji; Yanagisawa, Hiroyuki; Yuki, Mizue; Okamoto, Akimitsu

2013-01-01

Fluorescent probes for the detection of a double-stranded DNA were prepared by labeling a triplex-forming DNA oligonucleotide with a thiazole orange (TO) dimer unit. They belong to ECHO (exciton-controlled hybridization-sensitive fluorescent oligonucleotide) probes which we have previously reported. The excitonic interaction between the two TO molecules was expected to effectively suppress the background fluorescence of the probes. The applicability of the ECHO probes for the detection of double-stranded DNA was confirmed by examining the thermal stability and photophysical and kinetic properties of the DNA triplexes formed by the ECHO probes. PMID:23445822

Detection of Naja atra Cardiotoxin Using Adenosine-Based Molecular Beacon.

PubMed

Shi, Yi-Jun; Chen, Ying-Jung; Hu, Wan-Ping; Chang, Long-Sen

2017-01-07

This study presents an adenosine (A)-based molecular beacon (MB) for selective detection of Naja atra cardiotoxin (CTX) that functions by utilizing the competitive binding between CTX and the poly(A) stem of MB to coralyne. The 5'- and 3'-end of MB were labeled with a reporter fluorophore and a non-fluorescent quencher, respectively. Coralyne induced formation of the stem-loop MB structure through A₂-coralyne-A₂ coordination, causing fluorescence signal turn-off due to fluorescence resonance energy transfer between the fluorophore and quencher. CTX3 could bind to coralyne. Moreover, CTX3 alone induced the folding of MB structure and quenching of MB fluorescence. Unlike that of snake venom α-neurotoxins, the fluorescence signal of coralyne-MB complexes produced a bell-shaped concentration-dependent curve in the presence of CTX3 and CTX isotoxins; a turn-on fluorescence signal was noted when CTX concentration was ≤80 nM, while a turn-off fluorescence signal was noted with a further increase in toxin concentrations. The fluorescence signal of coralyne-MB complexes yielded a bell-shaped curve in response to varying concentrations of N. atra crude venom but not those of Bungarus multicinctus and Protobothrops mucrosquamatus venoms. Moreover, N. nigricollis venom also functioned as N. atra venom to yield a bell-shaped concentration-dependent curve of MB fluorescence signal, again supporting that the hairpin-shaped MB could detect crude venoms containing CTXs. Taken together, our data validate that a platform composed of coralyne-induced stem-loop MB structure selectively detects CTXs.

Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice

PubMed Central

Feeks, James A.; Hunter, Jennifer J.

2017-01-01

In vivo cellular scale fluorescence lifetime imaging of the mouse retina has the potential to be a sensitive marker of retinal cell health. In this study, we demonstrate fluorescence lifetime imaging of extrinsic fluorophores using adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO). We recorded AOFLIO images of inner retinal cells labeled with enhanced green fluorescent protein (EGFP) and capillaries labeled with fluorescein. We demonstrate that AOFLIO can be used to differentiate spectrally overlapping fluorophores in the retina. With further refinements, AOFLIO could be used to assess retinal health in early stages of degeneration by utilizing lifetime-based sensors or even fluorophores native to the retina. PMID:28663886

Bone marrow mesenchymal stem cells repair the hippocampal neurons and increase the expression of IGF-1 after cardiac arrest in rats.

PubMed

Tang, Xiahong; Chen, Feng; Lin, Qinming; You, Yan; Ke, Jun; Zhao, Shen

2017-11-01

The present study aimed to investigate the beneficial effects and underlying mechanisms of bone marrow mesenchymal stem cells (BMSCs) on global ischemic hypoxic brain injury. Cells collected from the femurs and tibias of male Sprague Dawley rats were used to generate BMSCs following three culture passages. A rate model of cardiac arrest (CA) was induced by asphyxia. One hour following return of spontaneous circulation (ROSC), BMSCs were transplanted through injection into the tail vein. Neurological status was assessed using modified neurological severity score (mNSS) tests 1, 3 and 7 days following ROSC. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical staining were used to detect insulin-like growth factor 1 (IGF-1) expression in the hippocampus. Furthermore, double-fluorescent labeling of green fluorescent protein (GFP) and IGF-1 was used to detect the IGF-1 expression in transplanted BMSCs. Serum levels of protein S100-B were examined using ELISA. GFP-labeled BMSCs were observed in the hippocampus at 1, 3 and 7 days post transplantation through fluorescent microscopy. BMSC transplantation resulted in reduced protein S100-B levels. The mNSS of the BMSC-treatment group was significantly reduced compared with that of the CA group. The RT-qPCR analysis and immunohistochemistry results demonstrated that BMSC treatment significantly increased IGF-1 expression in the hippocampus. In addition, the double-fluorescent labeling results demonstrated that transplanted BMSCs expressed IGF-1 in the hippocampus. The results of the present study suggest that BMSC treatment promotes the recovery of cerebral function following CA in rats possibly through the secretion of IGF-1.

Fluorescent probes for tracking the transfer of iron–sulfur cluster and other metal cofactors in biosynthetic reaction pathways

DOE PAGES

Vranish, James N.; Russell, William K.; Yu, Lusa E.; ...

2014-12-05

Iron–sulfur (Fe–S) clusters are protein cofactors that are constructed and delivered to target proteins by elaborate biosynthetic machinery. Mechanistic insights into these processes have been limited by the lack of sensitive probes for tracking Fe–S cluster synthesis and transfer reactions. Here we present fusion protein- and intein-based fluorescent labeling strategies that can probe Fe–S cluster binding. The fluorescence is sensitive to different cluster types ([2Fe–2S] and [4Fe–4S] clusters), ligand environments ([2Fe–2S] clusters on Rieske, ferredoxin (Fdx), and glutaredoxin), and cluster oxidation states. The power of this approach is highlighted with an extreme example in which the kinetics of Fe–S clustermore » transfer reactions are monitored between two Fdx molecules that have identical Fe–S spectroscopic properties. This exchange reaction between labeled and unlabeled Fdx is catalyzed by dithiothreitol (DTT), a result that was confirmed by mass spectrometry. DTT likely functions in a ligand substitution reaction that generates a [2Fe–2S]–DTT species, which can transfer the cluster to either labeled or unlabeled Fdx. The ability to monitor this challenging cluster exchange reaction indicates that real-time Fe–S cluster incorporation can be tracked for a specific labeled protein in multicomponent assays that include several unlabeled Fe–S binding proteins or other chromophores. Such advanced kinetic experiments are required to untangle the intricate networks of transfer pathways and the factors affecting flux through branch points. High sensitivity and suitability with high-throughput methodology are additional benefits of this approach. Lastly, we anticipate that this cluster detection methodology will transform the study of Fe–S cluster pathways and potentially other metal cofactor biosynthetic pathways.« less

Protein sorption on polymer surfaces measured by fluorescence labels.

PubMed

Brynda, E; Drobník, J; Vacík, J; Kálal, J

1978-01-01

Fluorescence labeling can be used in studying protein sorption on various surfaces with a sensitivity of about 10(-8) g/cm2, commensurate with radioactive labeling. Fluorescamine proved to be the most suitable compound for studying protein sorption on hydrophilic gels, because, unlike fluoresceine isothiocyanate and dansylchloride, free fluorochrome does not interfere with measurements. Sorption properties of labeled serum albumin were tested on poly(2-hydroxyethyl methacrylate), on the copolymer of 2-hydroxyethyl methacrylate with methyl methacrylate, and on polyethylene. Labeling does not cause aggregation of the protein, but, as expected, it shifts and somewhat broadens its electrophoretic band while at the same time slightly raising its affinity toward hydrophobic surfaces.

Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging.

PubMed

Valm, Alex M; Mark Welch, Jessica L; Rieken, Christopher W; Hasegawa, Yuko; Sogin, Mitchell L; Oldenbourg, Rudolf; Dewhirst, Floyd E; Borisy, Gary G

2011-03-08

Microbes in nature frequently function as members of complex multitaxon communities, but the structural organization of these communities at the micrometer level is poorly understood because of limitations in labeling and imaging technology. We report here a combinatorial labeling strategy coupled with spectral image acquisition and analysis that greatly expands the number of fluorescent signatures distinguishable in a single image. As an imaging proof of principle, we first demonstrated visualization of Escherichia coli labeled by fluorescence in situ hybridization (FISH) with 28 different binary combinations of eight fluorophores. As a biological proof of principle, we then applied this Combinatorial Labeling and Spectral Imaging FISH (CLASI-FISH) strategy using genus- and family-specific probes to visualize simultaneously and differentiate 15 different phylotypes in an artificial mixture of laboratory-grown microbes. We then illustrated the utility of our method for the structural analysis of a natural microbial community, namely, human dental plaque, a microbial biofilm. We demonstrate that 15 taxa in the plaque community can be imaged simultaneously and analyzed and that this community was dominated by early colonizers, including species of Streptococcus, Prevotella, Actinomyces, and Veillonella. Proximity analysis was used to determine the frequency of inter- and intrataxon cell-to-cell associations which revealed statistically significant intertaxon pairings. Cells of the genera Prevotella and Actinomyces showed the most interspecies associations, suggesting a central role for these genera in establishing and maintaining biofilm complexity. The results provide an initial systems-level structural analysis of biofilm organization.

Seeing the electroporative uptake of cell-membrane impermeable fluorescent molecules and nanoparticles

NASA Astrophysics Data System (ADS)

Kim, Kisoo; Kim, Jeong Ah; Lee, Soon-Geul; Lee, Won Gu

2012-07-01

This paper presents direct visualization of uptake directionality for cell-membrane impermeant fluorescent molecules and fluorescence-doped nanoparticles at a single-cell level during electroporation. To observe directly the uptake direction, we used microchannel-type electroporation that can generate a relatively symmetric and uniform electric field. For all the image frames during electroporation, fluorescence intensities that occurred at cell membranes in both uptake directions toward the electrodes have been sequentially recorded and quantitatively analyzed pixel by pixel. In our experiments, we found that fluorescent molecules, even not labeled to target biomolecules, had their own uptake direction with different intensities. It is also observed that the uptake intensity toward the cell membrane had a maximal value at a certain electric voltage, not at the highest value of voltages applied. The results also imply that the uptake direction of fluorescence-doped nanoparticles can be determined by a net surface charge of uptake materials and sizes in the electroporative environments. In summary, we performed a quantitative screening and direct visualization of uptake directionality for a set of fluorescent molecules and fluorescence-doped nanoparticles using electric-pulsation. Taking a closer look at the uptake direction of exogenous materials will help researchers to understand an unknown uptake phenomenon in which way foreign materials are inclined to move, and furthermore to design functional nanoparticles for electroporative gene delivery.This paper presents direct visualization of uptake directionality for cell-membrane impermeant fluorescent molecules and fluorescence-doped nanoparticles at a single-cell level during electroporation. To observe directly the uptake direction, we used microchannel-type electroporation that can generate a relatively symmetric and uniform electric field. For all the image frames during electroporation, fluorescence intensities that occurred at cell membranes in both uptake directions toward the electrodes have been sequentially recorded and quantitatively analyzed pixel by pixel. In our experiments, we found that fluorescent molecules, even not labeled to target biomolecules, had their own uptake direction with different intensities. It is also observed that the uptake intensity toward the cell membrane had a maximal value at a certain electric voltage, not at the highest value of voltages applied. The results also imply that the uptake direction of fluorescence-doped nanoparticles can be determined by a net surface charge of uptake materials and sizes in the electroporative environments. In summary, we performed a quantitative screening and direct visualization of uptake directionality for a set of fluorescent molecules and fluorescence-doped nanoparticles using electric-pulsation. Taking a closer look at the uptake direction of exogenous materials will help researchers to understand an unknown uptake phenomenon in which way foreign materials are inclined to move, and furthermore to design functional nanoparticles for electroporative gene delivery. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr30578j