Use Of Fluorescent Probes Research Articles

Use of a fluorescence-based approach to assess short-term responses of the alga Pseudokirchneriella subcapitata to metal stress

This work explores the use of fluorescent probes to evaluate the responses of the green alga Pseudokirchneriella subcapitata to the action of three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II) for a short time (6 h). The toxic effect of the metals on algal cells was monitored using the fluorochromes SYTOX Green (SG, membrane integrity), fluorescein diacetate (FDA, esterase activity) and rhodamine 123 (Rh123, mitochondrial membrane potential). The impact of metals on chlorophyll a (Chl a) autofluorescence was also evaluated. Esterase activity was the most sensitive parameter. At the concentrations studied, all metals induced the loss of esterase activity. SG could be used to effectively detect the loss of membrane integrity in algal cells exposed to 0.32 or 1.3 μmol L−1 Cu(II). Rh123 revealed a decrease in the mitochondrial membrane potential of algal cells exposed to 0.32 and 1.3 μmol L−1 Cu(II), indicating that mitochondrial activity was compromised. Chl a autofluorescence was also affected by the presence of Cr(VI) and Cu(II), suggesting perturbation of photosynthesis. In conclusion, the fluorescence-based approach was useful for detecting the disturbance of specific cellular characteristics. Fluorescent probes are a useful diagnostic tool for the assessment of the impact of toxicants on specific targets of P. subcapitata algal cells.

Silencing the radicals improves Click Chemistry.

Modern fluorescence imaging microscopy in living and fixed material makes use of fluorescent probes to label targeted entities. Common labelling approaches include classical immunocytochemistry, expression of chimerically tagged fluorescent protein domains, and chemical affinity-binding or covalent labelling. Of these methods, the so-called "Click Chemistry", is emerging as one of the most influential labelling chemistries introduced in recent times, offering enormous utility for bio-orthoganol attachment of fluorescent probes to biological target entities. In this issue of Biotechnology Journal, Löschberger, Niehörster and Sauer report "ClickOx", a Click Chemistry protocol that uses an enzymatic oxygen scavenger system to reduce concurrent ROS-associated damage during Click labeling.

Use of fluorescent probes to estimate solvent polarity

A method is proposed for estimating the polarity of a number of solvents using the fluorescent probes prodan (6-propionyl-2-(N,N-dimethyl)aminonaphthalene) and laurdan (6-dodecanoyl-2-(N,N-dimethyl) aminonaphthalene). The dependence of the fluorescence band shift on various polarity parameters of the solvents (E N T , SA, and SDP) is examined. The parameters E N T and SDP are determined for triton X-100. Using methods of quantum chemistry, the dipole moments of the ground state and the excited state are determined, and also the specific solvation centers. It is shown that the change in the dipole moment during a transition from the ground state to a lower excited state of ππ* type is not definitive for manifesting the reasons for the significant shift of the fluorescence band of prodan and laurdan when the solvent is replaced by one with different polarity.

Abstract LB-88: A new rapid diagnostic test to identify epidermal growth factor receptor mutations in non-small cell lung cancer.

Abstract An epidermal growth factor receptor (EGFR) mutation is the best marker of sensitivity to the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Polymerase chain reaction (PCR)-based methods combined with the use of fluorescence probes, such as the Scorpion Amplification Refractory Mutation System (ARMS) or the PCR-Invader method, are frequently used to detect EGFR mutations before therapy. The sensitivities and specificities of these methods are satisfactory; however, there is potential to further improve the cost and detection time. We developed a new method for the rapid and reliable detection of EGFR mutations by using a combination of mutation-specific primers and the newly developed ultra-rapid real-time PCR (Hyper-PCR) amplification method that can be accomplished in <7 min. We designed specific ARMS primers for the common EGFR del E746-A750 mutations, and optimized the reaction conditions for the PCR machine that contained a thin disc-type reaction vessel, the temperature of which could be quickly altered by rotating it on thermal sources. We used Hyper-PCR to analyze the common EGFR mutations del E746-A750 and L858R in lung cancer samples from 143 non-small cell lung cancer (NSCLC) patients. The results were compared with those obtained by direct sequencing, conventional PCR, and the PCR-Invader method. Using the direct sequencing method, 13 (9.1%) and 15 (10.5%) NSCLC samples were positive for the del E746-A750 and L858R mutations, respectively, while the Hyper-PCR method revealed that 15 (10.5%) and 18 (12.6%) samples were positive for the del E746-A750 and L858R mutations, respectively. Five samples, whose results were discordant between the Hyper-PCR and direct sequencing methods, were analyzed by conventional PCR and the PCR-Invader method, and the results were found to be consistent with those obtained by Hyper-PCR. These results suggest that the novel Hyper-PCR method is an inexpensive, rapid, and reliable diagnostic test for EGFR mutations. Citation Format: Hiroki Chikumi, Miyako Takata, Keiji Matsunami, Shingo Matsumoto, Masahiro Kotani, Tomohiro Sakamoto, Hirokazu Touge, Kosuke Yamaguchi, Jun Kurai, Naomi Miyake, Masaki Nakamoto, Tadashi Igishi, Eiji Shimizu. A new rapid diagnostic test to identify epidermal growth factor receptor mutations in non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-88. doi:10.1158/1538-7445.AM2013-LB-88

Abstract 728: Fluorescence-guided surgery for human-patient colon cancer tumorgrafts using a fluorophore-conjugated anti-CEA antibody.

Abstract One of the most important prognostic factors for overall survival in colorectal cancer patients is the complete resection of tumor. We have previously demonstrated the effective fluorescence-guided surgery of primary and metastatic cancer with the use of fluorescent probes in mouse models. In this study, we determined the effectiveness of fluorescence-guided surgery using a fluorophore-conjugated anti-CEA antibody for human-patient colon cancer tumorgrafts in nude mouse models. Colon-cancer-patient tumor specimens were initially established subcutaneously in NOD/SCID mice immediately after surgery. The patient tumors were then harvested from NOD/SCID mice and passed orthotopically in nude mice. Eight weeks after orthotopic implantation of human colon cancer tumorgraft, a monoclonal anti-CEA antibody conjugated with Alexa 488 was delivered to tumor-bearing mice as a single intravenous dose 24 hours before laparotomy. Using fluorescence imaging, the primary tumor was clearly visible at laparotomy. The tumor was completely resected under fluorescence navigation. Histologic evaluation of the resected specimen revealed there were no cancer cells in the margin. The cancer cells were strongly stained by anti-CEA antibody conjugated with Alexa488. Fluorescence-guided surgery using a fluorophore-conjugated anti-CEA antibody should improve intraoperative staging and efficacy of resection for CEA-positive colorectal cancer. Citation Format: Yukihiko Hiroshima, Ali Maawy, Yong Zhang, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. Fluorescence-guided surgery for human-patient colon cancer tumorgrafts using a fluorophore-conjugated anti-CEA antibody. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 728. doi:10.1158/1538-7445.AM2013-728

Direct translocation of cell-penetrating peptides in liposomes: A combined mass spectrometry quantification and fluorescence detection study

Cell-penetrating peptides (CPPs) can cross cell membranes in a receptor-independent manner. Two main routes for their cellular uptake have been proposed: endocytosis and direct translocation through the cell membrane. The ability of a peptide to enter cells through direct translocation can be assessed by evaluating the amount of peptide crossing the membrane of liposomes. Most methods reported so far rely on the use of fluorescent probes, which, when attached to a CPP, often alter its physical/chemical properties. Herein, a matrix-assisted laser desorption/ionization time-of-flight MS-based method is described to quantify the amount of CPP taken up into lipid vesicles and to distinguish it from the amount that is bound or inserted in the membrane. For comparison, visualization of the uptake of the same, but fluorophore-labeled, peptides into giant vesicles and cells by fluorescence microscopy is also reported. We show that membrane charge density is an important factor for direct translocation. We also show that fluorophore-labeled peptides have a different translocation behavior and that they are more toxic to cells. Alternative methods to fluorescence, such as the one reported herein, should be favored when investigating the uptake mechanism of CPPs, as fluorescent dyes can alter short peptides’ physical/chemical properties and their internalization capacities.

Development of Novel Fluorescent Probes for the Analysis of Protein Interactions under Physiological Conditions with Medical Devices

In this article, a method to analyze protein adsorption on porous, clinically relevant samples under physiologically relevant conditions is described. The use of fluorescent probes was identified as a methodology that would facilitate analysis under a range of conditions including fully competitive conditions where a protein of interest may be labeled in isolation and then allowed to compete with unlabeled proteins on samples that require no specialized surface pretreatment. As a first step, this article describes the covalent labeling of isolated bovine serum albumin (BSA) with fluorescent fluoresceinthioureidoaminocaproic acid, FTCA, giving FTCA-BSA. The fluorescence intensity of FTCA-BSA was then used to monitor the adsorption and desorption of the protein under noncompetitive conditions with two forms of hydroxyapatite discs (silicate-substituted, SA and stoichiometric, HA) in phosphate-buffered saline (PBS) and minimum essential Eagles' medium (MEM). Noncompetitive conditions were used to facilitate the validation of the technique in which data obtained from these experiments were corroborated against data obtained using an established total protein assay method (Quant-IT kit, Invitrogen). These experiments demonstrated that the FTCA-BSA probe had several advantages including a greater sensitivity at lower concentrations and a considerably longer lifetime. The results also demonstrated that the interaction of BSA with SA and HA was also highly temperature- and media-dependent. Under the most physiologically relevant conditions of MEM at 37 °C, BSA was more readily adsorbed to SA with significant differences between biomaterials, but no differences were observed during the desorption process. The use of this method to analyze adsorption under competitive conditions will be the subject of further investigations.

Immunofluorescence and its application in dermatopathology with oral manifestations: Revisited

The use of fluorescence probes the field of cell and molecular biology. Immunofluorescence is a molecular method for detection of antigen or antibody in a tissue section/serum sample. Although histopathology remains gold standard for most of auto immune, immune mediated and vesiculo - bullous diseases. Immunofluorescence is an ancillary investigation, which are used to support clinical diagnosis more evidently. This method provides additional diagnostic, prognostic information and remains more specific diagnostic aid for vesiculo - bullous diseases of skin. This review article discusses about immunofluorescence techniques and its application in dermatopathology with oral manifestations.

How Different Lipidic Mimetic Systems of E. COLI Membranes Affect Membrane Protein Reconstitution: A Steady-State Fluorescence Anisotropy Study

Outer membrane proteins (Omp) in bacteria, porins, have an important role on antibiotic translocation trough bacterial membranes. OmpF is one of the most important E. coli porins in the permeation of beta-lactam and quinolone antibiotics. Moreover, these antibiotics might penetrate the bacterial outer membrane through porins or through the lipid/protein interface. We focused on the characterization of E. coli membranes, once this knowledge is crucial for studies of antibiotic translocation and useful to understand and counteract bacterial antibiotic resistance, one of the biggest concerns of nowadays. Steady-state fluorescence anisotropy is a good technique to detect changes in lipid environments through the use of fluorescent probes. We used two fluorescent probes, DPH and TMA-DPH (which provide information from the core and from the interface regions of the bilayer, respectively), and determined the temperature dependence of these two anisotropy probes, incubated or incorporated into POPE/POPG (0.75:0.25), POPE/POPG/Cardiolipin (0.67:0.23:0.10) and E. coli total lipid extract liposomes (LUVs) and OmpF proteoliposomes. POPE/POPG and POPE/POPG/Cardiolipin mixtures are conventionally used to mimic the E. coli membranes, while the E. coli total lipid extract is a natural system. In this study we determine the phase transition temperatures characteristics of each membrane mimetic system and still prove the correct insertion of the protein in the membrane. When comparing the transition temperatures obtained for each mimetic system, our study suggest that the ternary lipid mixture is a better system to mimic the E. coli membranes than the binary system.

Use of Fluorescent Probes: Their Effect on Cell Biology and Limitations

Use of Fluorescent Probes: Their Effect on Cell Biology and Limitations

A brilliant new addition to the fluorescent probe toolbox

A brilliant new addition to the fluorescent probe toolbox

Study of apoptosis induction using fluorescent and higher harmonic generation microscopy techniques in Acartia tonsa nauplii exposed to chronic concentrations of nickel

In this study, we applied different fluorescent techniques to analyse the induction of apoptosis in the copepod Acartia tonsa nauplii exposed to low concentrations of nickel chloride (NiCl2). Newly hatched and later naupliar stages were exposed to increasing concentrations of NiCl2 (0.016, 0.025 and 0.063 mg·L−1) for 7 days and then stained with annexin V–FITC, a vital fluorescent probe commonly used to visualise apoptotic cells in live samples. Nauplii were also stained with TUNEL, a non-vital fluorescent probe used to detect apoptosis in fixed copepods. Moreover, we used for the first time, two-photon fluorescence (2PF) microscopy and higher harmonic generation microscopy (second SHG and third THG harmonic generation) to study apoptosis induction in A. tonsa nauplii without the use of fluorescent probes. 2PF and THG intensity increased in samples exposed to higher Ni concentrations, with respect to the control, whereas SHG signals were similar in all treated samples and visualised muscles. Future perspectives on the use of these new technologies to reveal apoptosis are discussed.

Generation of hydrogen peroxide in the developing rat heart: the role of elastin metabolism

Reports describing production of reactive oxygen species in neonatal heart are missing. As lysyl oxidase is potentially important source of H(2)O(2), we studied its role during ontogenic development of rat heart. H(2)O(2) was detected in thin sections of developing rat heart by fluorescence microscopy with the use of fluorescence probe 2'-7'-dichlorofluorescin. The experimental design comprised foetuses 21 days after conception, and then the animals sampled on the 1st, 4th, 7th, 10th, 15th, 30th and 60th day after birth. We also used 7-month-old animals as an example of ageing effects. Since the day 4 on, H(2)O(2) was produced only extracellularly up to the day 15, between days 30 and 60 intracellular production was detected as well, and in 7-month-old animals only extracellular production was observed. The specific inhibitors of lysyl oxidase almost completely quenched the H(2)O(2)-dependent fluorescence. Starting from day 7, blue autofluorescence specific to oxidized proteins developed in the vessel wall. Intracellular blue autofluorescence specific to autoxidation products developed after day 30. Chloroform extraction diminished the intracellular blue fluorescence, leaving the extracellular fluorescence intact. This confirmed the protein nature of the fluorophores. Lysyl oxidase is significant source of H(2)O(2) in the heart vessel wall during development and H(2)O(2) oxidatively modifies elastin producing protein blue autofluorescence.

Polymer Vesicles as Microreactors for Bioinspired Calcium Carbonate Precipitation

Giant polymer vesicles made by electroformation have been shown to encapsulate salts up to concentrations of about 10 mM. The impermeability of these "polymersomes" to calcium ions is demonstrated by the use of fluorescent probes dedicated to calcium analysis. Permeability to calcium ions can be triggered by the addition of calcimycin, an ionophore molecule that is able to transport cations selectively through the membrane. As a result, we show that the mineralization of calcium carbonate can be induced within the polymersomes, which were previously loaded with carbonate ions. This is a further step toward the use of polymersomes as microreactors and the study of mineralization schemes, including biomimetic ones, in confined environments.

The Use of Fluorescent Probes for Monitoring the Water Absorption in Corrosion Protection Sol-Gel Coatings

not Available.

A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies

BackgroundSome plants inhabiting saline environment remove salts via the salt glands embedded in the epidermal tissues. Cytological studies of salt glands will provide valuable information to our understanding of the secretory process. Previous studies on salt gland histology relied mainly on two-dimensional microscopic observations of microtome sections. Optical sectioning properties of confocal laser scanning microscope offer alternative approach for obtaining three-dimensional structural information of salt glands. Difficulty in light penetration through intact leaves and interference from neighbouring leaf cells, however, impede the acquiring of good optical salt gland sections and limit its applications in salt gland imaging. Freeing the glands from adjacent leaf tissues will allow better manipulations for three-dimensional imaging through confocal laser scanning microscopy.ResultsHere, we present a simple and fast method for the isolation of individual salt glands released from the interference of neighbouring cells. About 100-200 salt glands could be isolated from just one cm2 of Avicennia officinalis leaf within hours and microscopic visualization of isolated salt glands was made possible within a day. Using these isolated glands, confocal laser scanning microscopic techniques could be applied and better resolution salt gland images could be achieved. By making use of their intrinsic fluorescent properties, optical sections of the gland cells could be acquired without the use of fluorescent probes and the corresponding three-dimensional images constructed. Useful cytological information of the salt gland cells could also be obtained through the applications of fluorescent dyes (e.g., LysoTracker® Red, FM®4-64, Texas Red®).ConclusionsThe study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures. Preparation of materials for subsequent microscopic observations of salt glands could be achieved within a day. Potential applications of confocal fluorescence microscopic techniques could also be performed using these isolated glands. Experiments designed and targeted directly at the salt glands were explored and cytological information obtained herein could be further incorporated towards the understanding of the mechanism underlying secretion in plant salt glands.

Fluorescence spectroscopy as a non invasive tool to follow in situ the polymerization in miniemulsion

AbstractMiniemulsion polymerization is a powerful technique to produce a whole variety of latexes with controlled colloidal and chemical properties, which has gained an increasing importance in the past years. For many applications, the following of the kinetics of polymerization is of crucial importance. In this study, we developed a technique based on the use of fluorescent probes that could be a very promising noninvasive method to follow the reaction in situ. It was possible to follow qualitatively the kinetics of methyl methacrylate (MMA) polymerization by measuring the photophysical properties of fluorescent probes sensitive to the modification of the viscosity of the microenvironment along the reaction course. Two fluorescent sensors have been considered: 1,1‐dicyano‐4‐(4′‐dimethylaminophenyl)‐1,3‐butadiene) and pyrene. In this article, we will show that using these two different fluorescent probes allow the detection of MMA polymerization in miniemulsion. The results will be compared with a classical NMR method. This method could be easily adapted to different monomers. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Low micromolar zinc exerts cytotoxic action under H 2O 2-induced oxidative stress: Excessive increase in intracellular Zn 2+ concentration

The ability of zinc to retard oxidative processes has been recognized for many years. However, zinc is cytotoxic under certain oxidative stress. In this study, we investigated the effect of H 2O 2 on intracellular Zn 2+ concentration of rat thymocytes and its relation to the cytotoxicity. Experiments were cytometrically performed by the use of fluorescent probes, propidium iodide, FluoZin-3-AM, and 5-chloromethylfluorescein diacetate. ZnCl 2 potentiated cytotoxicity of H 2O 2 while TPEN, a chelator for intracellular Zn 2+, attenuated it. Results suggested an involvement of intracellular Zn 2+ in the cytotoxicity of H 2O 2. H 2O 2 at concentrations of 30 μM or more (up to 1000 μM) significantly increased intracellular Zn 2+ concentration. There were two mechanisms. (1) H 2O 2 decreased cellular content of nonprotein thiols, possibly resulting in release of Zn 2+ from thiols as cellular Zn 2+ binding sites. (2) H 2O 2 increased membrane Zn 2+ permeability because external ZnCl 2 application further elevated intracellular Zn 2+ concentration. Micromolar H 2O 2 may induce excessive elevation of intracellular Zn 2+ concentration that is harmful to cellular functions. However, the incubation with micromolar ZnCl 2 alone increased cellular content of nonprotein thiols, one of the factors protecting cells against oxidative stress. Though zinc is generally considered to be protective with its antioxidant property, this study reveals the toxic effect of zinc even in micromolar range under oxidative stress induced by H 2O 2.

Polyscale technology for developing near infrared fluorescence bioimaging system based on novel synthese approaches for rare-earth doped nanophspors

Fluorescence bioimaging is one of the key technologies for biomedical science and engineering. Major issues of the current fluorescence bioimaiging are colour fading of the fluorescent probes, phototoxicity and strong light scattering due to the use of a short wavelength excitation light, an ultraviolet or visible light. The use of fluorescent probes to be excited by longer wavelength excitation light, i.e. near infrared light, should solve all of the above issues. In this paper, materials and system developments for the fluorescence bioimaging system with near infrared excitation are reviewed. The development of the system is based on 'polyscale technology,' a post-nanotechnology by cooperative works of the technologies in various scales. The concept and example of the 'polyscale technology' are also described.

On the use of fluorescence probes for detecting reactive oxygen and nitrogen species associated with photodynamic therapy

Fluorescent probes are frequently employed for the detection of different reactive oxygen and nitrogen species formed during the irradiation of photosensitized cells and tissues. Investigators often interpret the results in terms of information provided with the different probes without examining specificity or determinants of fluorogenic reactions. We examine five fluorescent probes in a cell-free system: reduced 2',7'-dichlorofluorescein, dihydroethidine, dihydrorhodamine, 3'-(p aminophenyl) fluorescein (APF), and 4',5'-diaminofluorescein. Of these, only APF demonstrates a high degree of specificity for a single reactive species. There is a substantial influence of peroxidase activity on all fluorogenic interactions. The fluorescence of the photosensitizing agent also must be taken into account in evaluating results.