Light-emitting Fluorescence Research Articles

Dispersion and Orientation of Zeolite ZSM‐5 Crystallites within a Fluid Catalytic Cracking Catalyst Particle

Confocal fluorescence microscopy was employed to selectively visualize the dispersion and orientation of zeolite ZSM-5 domains inside a single industrially applied fluid catalytic cracking (FCC) catalyst particle. Large ZSM-5 crystals served as a model system together with the acid-catalyzed fluorostyrene oligomerization reaction to study the interaction of plane-polarized light with these anisotropic zeolite crystals. The distinction between zeolite and binder material, such as alumina, silica, and clay, within an individual FCC particle was achieved by utilizing the anisotropic nature of emitted fluorescence light arising from the entrapped fluorostyrene-derived carbocations inside the zeolite channels. This characterization approach provides a non-invasive way for post-synthesis characterization of an individual FCC catalyst particle in which the size, distribution, orientation, and amount of zeolite ZSM-5 aggregates can be determined. It was found that the amount of detected fluorescence light originating from the stained ZSM-5 aggregates corresponds to about 15 wt %. Furthermore, a statistical analysis of the emitted fluorescence light indicated that a large number of the ZSM-5 domains appeared in small sizes of about 0.015-0.25 μm(2), representing single zeolite crystallites or small aggregates thereof. This observation illustrated a fairly high degree of zeolite dispersion within the FCC binder material. However, the highest amount of crystalline material was aggregated into larger domains (ca. 1-5 μm(2)) with more or less similarly oriented zeolite crystallites. It is clear that this visualization approach may serve as a post-synthesis quality control on the dispersion of zeolite ZSM-5 crystallites within FCC particles.

Regulation of red fluorescent light emission in a cryptic marine fish

IntroductionAnimal colouration is a trade-off between being seen by intended, intra- or inter-specific receivers while not being seen by the unintended. Many fishes solve this problem by adaptive colouration. Here, we investigate whether this also holds for fluorescent pigments. In those aquatic environments in which the ambient light is dominated by bluish light, red fluorescence can generate high-contrast signals. The marine, cryptic fish Tripterygion delaisi inhabits such environments and has a bright red-fluorescent iris that can be rapidly up- and down-regulated. Here, we described the physiological and cellular mechanism of this phenomenon using a neurostimulation treatment with KCl and histology.ResultsKCl-treatment revealed that eye fluorescence regulation is achieved through dispersal and aggregation of black-pigmented melanosomes within melanophores. Histology showed that globular, fluorescent iridophores on the anterior side of the iris are grouped and each group is encased by finger-like extensions of a single posterior melanophore. Together they form a so-called chromatophore unit. By dispersal and aggregation of melanosomes into and out of the peripheral membranous extensions of the melanophore, the fluorescent iridophores are covered or revealed on the anterior (outside) of the iris.ConclusionT. delaisi possesses a well-developed mechanism to control the fluorescent emission from its eyes, which may be advantageous given its cryptic lifestyle. This is the first time chromatophore units are found to control fluorescent emission in marine teleost fishes. We expect other fluorescent fish species to use similar mechanisms in the iris or elsewhere in the body. In contrast to a previously described mechanism based on dendritic fluorescent chromatophores, chromatophore units control fluorescent emission through the cooperation between two chromatophore types: an emitting and an occluding type. The discovery of a second mechanism for fluorescence modulation strengthens our view that fluorescence is a relevant and adaptive component of fish colouration.

Novel metallo-therapeutics of the NSAID naproxen. Interaction with intracellular components that leads the cells to apoptosis

Two new mixed ligand-silver(I) complexes of the anti-inflammatory drug naproxen (naprH) and triphenylphosphine (tpp) or tri(p-tolyl)phosphine (tptp) of formulae {[Ag(tpp)3(napr)](H2O)} (1) and [Ag(tptp)2(napr)] (2) have been synthesized and characterized by m.p., vibrational spectroscopy (mid-FT-IR), Raman, (1)H-NMR, UV-Vis, ESI-MS spectroscopic techniques and X-ray crystallography. The complexes show high photo-sensitivity to UVC light. Photolysis of 1-2 was studied and the results showed monotonic degradation of the complexes with simultaneous triarylphosphine oxide formation. The complexes 1-2 were tested for their antiproliferative activity against human breast adenocarcinoma (MCF-7) cells. Complexes 1-2 were more active than cisplatin against cells. UVC light increases the effectiveness of complexes 1-2 on MCF-7 cells by 13% and 38% respectively. Due to the morphology of the MCF-7 cells, which were incubated with the complexes 1-2, the cell death was ascribed to apoptosis. Electrophoresis to genomic DNA of MCF-7 cells confirmed the apoptosis through DNA fragmentation. The binding affinity of 1-2 towards the intracellular molecules CT-DNA and lipoxygenase (LOX) was studied for the evaluation of the mechanism of cell death. Thus, the binding constants (K(b)) of 1-2 towards CT-DNA calculated by UV-Vis spectra are 32.8 ± 8.5 × 10(4) (1) and 4.7 ± 1.8 × 10(4) (2) M(-1), respectively. Changes in fluorescent emission light of ethidium bromide (EB) in the presence of DNA suggest intercalation or electrostatic interactions into DNA of both complexes 1-2 in the minor groove. The corresponding apparent binding constants (K(app)) of 1-2 towards CT-DNA calculated through fluorescence spectra are 2.9 ± 0.3 × 10(4) (1) and 1.6 ± 0.4 × 10(4) (2) M(-1) respectively. Docking studies on DNA-complexes interactions show the binding of 1 in the major groove and the corresponding one of 2 in the minor one. Moreover, the influence of complexes 1-2 on the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX) was kinetically and theoretically studied. Only 1 inhibits lipoxygenase activity (IC50 = 5.1 (1), >30 (2) μM).

Tailoring the directivity of both excitation and emission of dipole simultaneously with two-colored plasmonic antenna

We report a two-colored plasmonic antenna which can control the directivity of the excitation and emission light independently and simultaneously. By carefully tuning the phase difference of the constituting elements of the antenna, unidirectional fluorescence emission and laser light scattering can be obtained. In particular, the direction of the maximum emission and minimum scattering can be tailored in the same direction resulting improvement of signal to noise ratio in single molecule experiment. A two-dipole model is applied to describe the phenomena. The radiation and scattering pattern can be further tuned by varying the antenna structure.

Empowering Low-Cost CMOS Cameras by Image Processing to Reach Comparable Results with Costly CCDs

Despite the huge research effort to improve the performance of the complementary metal oxide semiconductor (CMOS) image sensors, charge-coupled devices (CCDs) still dominate the cell biology-related conventional fluorescence microscopic imaging market where low or ultra-low noise imaging is required. A detailed comparison of the sensor specifications and performance is usually not provided by the manufacturers which leads the end users not to go out of the habitude and choose a CCD camera instead of a CMOS one. However, depending on the application, CMOS cameras, when empowered by image processing algorithms, can become cost-efficient solutions for conventional fluorescence microscopy. In this paper, we introduce an application-based comparative study between the default CCD camera of an inverted microscope (Nikon Ti-S Eclipse) and a custom-designed CMOS camera and apply efficient image processing algorithms to improve the performance of CMOS cameras. Quantum micro-bead samples (emitting fluorescence light at different intensity levels), breast cancer diagnostic tissue cell samples, and Caco-2 cell samples are imaged by both CMOS and CCD cameras. The results are provided to show the reliability of CMOS camera processed images and finally to be of assistance when scientists select their cameras for desired applications.

Fixed eruption due to quinine in tonic water: A case report with high‐performance liquid chromatography and ultraviolet A analyses

Fixed drug eruption is a common cutaneous adverse reaction in young patients with a characteristic clinical appearance. However, the diagnosis and identification of the substance may be difficult if food or food additives provoke the fixed eruption. A 26-year-old man had a history of two episodes of cutaneous erythema with residual pigmentation. Close examination of the history including his diet in addition to an oral challenge test and patch testing led to the diagnosis of fixed eruption secondary to quinine in tonic water. We examined for the presence of quinine in commercially available brands of tonic water using ultraviolet A and irradiation and high-performance liquid chromatography. Both Schweppes and CANADA DRY brands of tonic water emitted fluorescent light upon ultraviolet A irradiation, and contained quinine at concentrations of 67.9 and 61.3mg/L, respectively. Quinine contained in some tonic waters may trigger fixed eruption.

Compact surface plasmon-enhanced fluorescence biochip

A new concept of compact biochip for surface plasmon-enhanced fluorescence assays is reported. It takes advantage of the amplification of fluorescence signal through the coupling of fluorophore labels with confined and strongly enhanced field intensity of surface plasmons. In order to efficiently excite and collect the emitted fluorescence light via surface plasmons on a metallic sensor surface, (reverse) Kretschmann configuration is combined with diffractive optical elements embedded on the chip surface. These include a concentric relief grating for the imaging of highly directional surface plasmon-coupled emission to a detector. Additional linear grating is used for the generating of surface plasmons at the excitation wavelength on the sensor surface in order to increase the fluorescence excitation rate. The reported approach offers the increased intensity of fluorescence signal, reduced background, and compatibility with nanoimprint lithography for cost-effective preparation of sensor chip. The presented approach was implemented for biosensing in a model immunoassay experiment in which the limit of detection of 11 pM was achieved.

Photoluminescence and thermoanalytical studies of complexes based on 5-Cl-8-hydroxyquinoline and calix[4]arene ligands

A innovative 5-Cl-8-oxyquinolinepropoxycalix[4]arene ligand (2) have been prepared, exhibiting, at room temperature, blue fluorescent light emission and resulting in shift band to green fluorescent light (fluorescence mode) in the presence of coordinated Eu(III) and Tb(III) ions. Terbium complex presented phosphorescence emission as noted by typical bands at 490nm, 545nm and 585nm. TG/DTG data exhibited typical thermal behavior for these compounds, however DSC curves showed the melting temperature near 300°C for the samples, demonstrating an unusual thermal stability when quinoline derivatives are attached to calix[4]arene matrix. This fact strongly suggests an effective approach to preparing the photoluminescent compound associating high chemical and thermal stability.

Supramolecular hybrid assemblies based on gold nanoparticles, amphiphilic cyclodextrin and porphyrins with combined phototherapeutic action

Gold nanoparticles (AuNPs), amphiphilic cyclodextrin (SC6NH2) and anionic porphyrin (TPPS4) spontaneously self-assemble in aqueous basic solution forming nanostructures with controlled size and shape. Spectroscopic techniques such UV-Vis, fluorescence emission and Dynamic Light Scattering (DLS) demonstrate that AuNPs are covered by SC6NH2, and TPPS4 is strongly entrapped within, forming assemblies with size of 4 and 50 nm. Scanning Near-Field Optical Microscopy (SNOM) and Scanning Near-Field Optical Luminescence (SNOL) assess the presence of luminescent vescicular structures incorporating porphyrin with dimensions of about 150 nm. Transmission electron microscopy (TEM) indicates AuNPs with sizes of 20–30 nm can be located inside the largest cyclodextrin/porphyrin aggregates (≅150 nm) or interact externally with the smallest cyclodextrin/porphyrin vesicles (≅ 40 nm). The generation of an appreciable quantum yield of singlet oxygen (ΦΔ = 0.25) guarantees the photodynamic action excluding mostly strong interactions between gold and porphyrin. These hybrid systems are fairly stable at physiological conditions (pH = 7.4) and can be taken up by HeLa cancer cells which, upon treatment, show co-localization of gold and porphyrins in the cytoplasm. After intracellular delivery, these assemblies induce low dark toxicity and a combined photothermal–photodynamic effect upon proper irradiation, showing promising properties as dual-action phototherapeutics.

Measurements of the longitudinal shower development with the Pierre Auger Observatory

In this work we explain how the Pierre Auger Observatory measures and reconstructs the longitudinal development of air showers. The measurement of the energy deposit in the atmosphere by the detection of the emitted fluorescence light is going to be briefly reviewed and the reconstruction procedure is going to be explained in detail. The two main outputs of this analysis are: a) the depth at which the shower reaches its maximum (Xmax) and b) the shower energy. In this work we concentrate on the analysis techniques developed with the aim to evaluate the unbiased Xmax distributions. We show how using measured events it is possible to estimate the range of the true Xmax distribution. Then we select events with geometries that allow an unbiased measurement of Xmax anywhere along its estimated range. The analysis procedure explained here is a fundamental step in the measurements of cosmic ray mass composition and proton-air cross section.

69. All that’s needed is inside the cell: New method to measure permeability of red blood cell membrane using intrinsic fluorescence

To design effective cryopreservation procedures for cells and tissues, it is critical to know permeability of cell membrane to water and solutes. To determine the latter, one needs to measure the rate of cell volume changes when placed in anisotonic environment. Red blood cells (RBCs) respond very quickly to changes in extracellular solutes concentration, which makes it difficult to measure the rate of RBC volume change using traditional methods. Stopped-flow method captures rapid changes of RBC volume and is a common method to measure RBC osmotic permeability. There are two types of stopped-flow methods: the first measures RBC volume based on changes in the intensity of light scattered by RBCs (Sidel and Solomon, 1957), the second measures it based on the self-quenching of fluorescent dye entrapped inside RBCs (Chen et al., 1988). However, both techniques have flaws. A number of factors other than cell volume influence the intensity of scattered light (Chen et al., 1988). Absorbance of a significant portion of emitted fluorescent light by the broad spectrum of hemoglobin complicates the fluorescent quenching technique. Literature shows that RBC hemoglobin has autofluorescent properties (Hirstch, 2003). This work explored the possibility of using intrinsic fluorescence of intracellular hemoglobin to measure RBC permeability to water and solutes. Network Center for Applied Development in Vancouver provided adult RBCs for this study. We used SX20 stopped-flow reaction analyser (Applied Photophysics) to measure changes in RBC autofluorescence when mixed with sodium chloride or glycerol solutions of different concentration (excitation 280 nm, emission 314 nm). Equilibrium volumes of RBCs in sodium chloride solutions were measured on Coulter Counter Model ZBI (Coulter Electronics, Inc.). Lysed RBCs served as a negative control to verify that changes in autofluorescence intensity are caused by the changes in volume of intact RBCs. We also measured absolute values of autofluorescence as a function of hemoglobin concentration in the solution using SpectraMax GEMINI EM dual-scanning microplate spectrofluorometer (Molecular devices). Our results showed that the intensity of RBC autofluorescence and RBC volume measured on Coulter Counter were directly related. Specifically, mixing RBCs with hypotonic solution caused increase in cell volume and gradual increase in autofluorescence; cell shrinking in hypertonic solution resulted in autofluorescence decrease. On the contrary, tonicity of solution had no effect on autofluorescence of lysed RBCs. When RBCs were injected in glycerol solution, autofluorescence first dropped, then gradually increased, and finally reached a plateau. This response is similar to the well-known pattern of cell volume changes upon mixing with permeable cryoprotectant. We also found that at high (intracellular) hemoglobin concentrations fluorescence intensity and hemoglobin concentration are inversely related. Combined, our data shows that RBC autofluorescence is directly translated to RBC volume, and self-quenching of intracellular hemoglobin is responsible for the cell volume driven changes in RBC autofluorescence. Based on these findings, we are working on developing a new method to determine osmotic permeability of RBCs, which will significantly help with future design and optimization of cryopreservation protocols.

Development of an indirect method of microalgal lipid quantification using a lysochrome dye, Nile red

Earlier studies showed that the lipophilic dye, Nile red (9-diethylamino 5H-benzo[α]phenoxazine-5-one) can be used to measure the lipid content of microalgae by cellular in vivo fluorescence. It was observed that a higher amount of lipid present in lipid droplets of microalgal cells would result in higher degree of emitted fluorescent light. In this present study, the feasibility of using Nile red, a fluorescent dye specific for intracellular lipid droplets, as an indirect method of lipid quantifications was investigated. Following cellular staining of different microalgal species with nile red, the in vivo fluorescence of the whole cell was visualized by fluorescence microscopy (excitation: 450 to 590 nm and emission: 520 nm). Intensity of the relative in vivo fluorescence was measured using a fluorescence spectrophotometer at excitation and emission wavelengths of 485 and 590 nm, respectively. Lipid content was determined gravimetrically and the fluorescence of the extract was measured using the microemulsion method at emission and excitation wavelengths of 540 and 617 nm. The equivalent oil content of the extracted lipid was correlated to the fluorescence of pure olive oil using the microemulsion method. Cellular in vivo fluorescence of stained cells (ex: 485 nm and em: 590 nm), fluorescence of extracted lipid (ex: 540 nm and em: 617 nm) and gravimetrically determined lipid were linearly correlated. This suggests that Nile red can serve as a vital stain which allows a relatively rapid method of determining the lipid content of microalgal samples and is as good as the gravimetric method used for lipid determination, eliminating the requirement for the toxic solvents and timeconsuming manipulations. Keywords: Nile red, microalgae, lipid, fluorescence

Microelectromechanical systems scanning-mirror-based handheld probe for fluorescence molecular tomography

A novel handheld probe based on a microelectromechanical systems (MEMS) scanning mirror for three-dimensional (3D) fluorescence molecular tomography (FMT) is described. The miniaturized probe consists of a MEMS mirror for delivering an excitation light beam to multiple preselected points at the tissue surface and an optical fiber array for collecting the fluorescent emission light from the tissue. Several phantom experiments based on indocyanine green, an FDA approved near-infrared (NIR) fluorescent dye, were conducted to assess the imaging ability of this device. Tumor-bearing mice with systematically injected tumor-targeted NIR fluorescent probes were scanned to further demonstrate the ability of this MEMS-based FMT for imaging small animals.

Dual Role of the Molybdenum Cofactor Biosynthesis Protein MOCS3 in tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Humans

We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are β-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.

Interactions between cyclodextrins and fluorescent T-2 and HT-2 toxin derivatives: a physico-chemical study

T-2 and HT-2 toxins are mycotoxins produced by several Fusarium species that are commonly found in various cereal grains, including oats, barley, wheat and maize. Intake estimates indicate that the presence of these mycotoxins in the diet can be of concern for public health. In this work, the inclusion processes occurring between fluorescent anthracene-derivatives of T-2 and HT-2 toxins and different cyclodextrin (CD) molecules were investigated in aqueous solutions by means of UV–Vis absorption, fluorescence emission and dynamic light scattering. Binding constant values and chemico-physical parameters were calculated. It was found that β-CDs give stronger inclusion reactions with both T-2 and HT-2 derivatives, as stated by important emission intensity increments. Such interactions were found to be fundamentally enthalpy-driven. Among β-CDs, the effect of the methylation at hydroxyl groups was tested: as a result, the di-methyl form of β-CD was found to induce the best fluorescence intensity enhancements.

Automation Highlights from the Literature

Automation Highlights from the Literature

Sound-evoked network calcium transients in mouse auditory cortexin vivo

Population calcium signals generated by the action potential activity of local clusters of neurons have been recorded in the auditory cortex of mice using an optical fibre-based approach. These network calcium transients (NCaTs) occurred spontaneously as well as in response to sound stimulation. Two-photon calcium imaging experiments suggest that neurons and neuropil contribute about equally to the NCaT. Sound-evoked calcium signals had two components: an early, fast increase in calcium concentration, which corresponds to the short-latency spiking responses observed in electrophysiological experiments, and a late, slow calcium transient which lasted for at least 1 s. The slow calcium transients evoked by sound were essentially identical to spontaneous NCaTs. Their sizes were dependent on the spontaneous activity level at sound onset, suggesting that spontaneous and sensory-evoked NCaTs excluded each other. When using pure tones as stimulus, the early evoked calcium transients were more narrowly tuned than the slow NCaTs. The slow NCaTs were correlated with global ‘up states' recorded with epidural potentials, and sound presented during an epidural ‘down state' triggered a calcium transient that was associated with an epidural ‘up state'. Essentially indistinguishable calcium transients were evoked by optogenetic activation of local clusters of layer 5 pyramidal neurons in the auditory cortex, indicating that these neurons play an important role in the generation of the calcium signal. Taken together, our results identify sound-evoked slow NCaTs as an integral component of neuronal signalling in the mouse auditory cortex, reflecting the prolonged neuronal activity of local clusters of neurons that can be activated even by brief stimuli.

Quantum dot enabled detection of Escherichia coli using a cell-phone.

We report a cell-phone based Escherichia coli (E. coli) detection platform for screening of liquid samples. In this compact and cost-effective design attached to a cell-phone, we utilize anti-E. coli O157:H7 antibody functionalized glass capillaries as solid substrates to perform a quantum dot based sandwich assay for specific detection of E. coli O157:H7 in liquid samples. Using battery-powered inexpensive light-emitting-diodes (LEDs) we excite/pump these labelled E. coli particles captured on the capillary surface, where the emission from the quantum dots is then imaged using the cell-phone camera unit through an additional lens that is inserted between the capillary and the cell-phone. By quantifying the fluorescent light emission from each capillary tube, the concentration of E. coli in the sample is determined. We experimentally confirmed the detection limit of this cell-phone based fluorescent imaging and sensing platform as ∼5 to 10 cfu mL(-1) in buffer solution. We also tested the specificity of this E. coli detection platform by spiking samples with different species (e.g., Salmonella) to confirm that non-specific binding/detection is negligible. We further demonstrated the proof-of-concept of our approach in a complex food matrix, e.g., fat-free milk, where a similar detection limit of ∼5 to 10 cfu mL(-1) was achieved despite challenges associated with the density of proteins that exist in milk. Our results reveal the promising potential of this cell-phone enabled field-portable and cost-effective E. coli detection platform for e.g., screening of water and food samples even in resource limited environments. The presented platform can also be applicable to other pathogens of interest through the use of different antibodies.

Multistage morphological segmentation of bright-field and fluorescent microscopy images

AbstractThis paper describes the multistage morphological segmentation method (MSMA) for microscopic cell images. The proposed method enables us to study the cell behaviour by using a sequence of two types of microscopic images: bright field images and/or fluorescent images. The proposed method is based on two types of information: the cell texture coming from the bright field images and intensity of light emission, done by fluorescent markers. The method is dedicated to the image sequences segmentation and it is based on mathematical morphology methods supported by other image processing techniques. The method allows for detecting cells in image independently from a degree of their flattening and from presenting structures which produce the texture. It makes use of some synergic information from the fluorescent light emission image as the support information. The MSMA method has been applied to images acquired during the experiments on neural stem cells as well as to artificial images. In order to validate the method, two types of errors have been considered: the error of cell area detection and the error of cell position using artificial images as the “gold standard”.

Investigation of Fluorescent Shape Memory Polyurethanes Grafted with Various Dyes

Shape memory polyurethane (SMPU), grafted with a fluorescent dye (Rhodamine, Mehylene violet, or Fluorescein) through an allophanate linking, was tested for the fluorescence and the shape recovery effect. The main chain of SMPU was composed of 4,4'-methylenebis(phenylisocyanate) (MDI), poly(tetramethyleneglycol) (PTMG), and 1,4-butanediol (BD), and a fluorescent dye was connected through a second MDI linked to the carbamate moiety of the main chain. Three series of SMPU, differing according to their dye content, were prepared to compare their shape recovery and fluorescence properties. In tensile mechanical property, maximum stress increased up to 350% compared to the linear SMPU, and strain remained above 2000%. Shape recovery went to as high as 97%, and remained almost same after repetitive shape recovery test cycles. Finally, the fluorescence emission of SMPU was demonstrated in the luminescence spectrum and fluorescent light emission pictures. In addition, the response of SMPU to external stimuli such as metal ions was investigated.