Concentration‐mediated multicolor fluorescence polymer carbon dots

Polymer dots (PDs) showing concentration‐mediated multicolor fluorescence were first prepared from sulfuric acid‐treated dehydration of Pluronic® F‐127 in a single step. Pluronic‐based PDs (P‐PDs) showed high dispersion stability in solvent media and exhibited a fluorescence emission that was widely tunable from red to blue by adjusting both the excitation wavelengths and the P‐PD concentration in an aqueous solution. This unique fluorescence behavior of P‐PDs might be a result of cross‐talk in the fluorophores of the poly(propylene glycol)‐rich core inside the P‐PD through either energy transfer or charge transfer. Reconstruction of the surface energy traps of the P‐PDs mediated through aggregation may lead to a new generation of carbon‐based nanomaterials possessing a fluorescence emission and tunable by adjusting the concentration. These structures may be useful in the design of multifunctional carbon nanomaterials with tunable emission properties according to a variety of internal or external stimuli. Copyright © 2015 John Wiley & Sons, Ltd.     

Altered near‐infrared spectroscopy response to breath‐holding in patients with fibromyalgia

Fibromyalgia (FM) is a complex syndrome characterized by chronic widespread pain and a heightened response to pressure. Most medical researches pointed out that FM patients with endothelial dysfunction and arterial stiffness. A continuous‐wave near‐infrared spectroscopy (NIRS) system is used in present study to measure the hemodynamic changes elicited by breath‐holding task in patients with FM. Each patient completed a questionnaire survey including demographics, characteristics of body pain, associated symptoms, headache profiles and Hospital Anxiety and Depression Scale. A total of 27 FM patients and 26 health controls were enrolled. In comparison with healthy controls, patients with FM showed lower maximal and averaged change of oxyhemoglobin concentration in both the left (1.634 ±0.890 and 0.810 ±0.525 μM) and the right (1.576 ±0.897 and 0.811 ±0.601 μM) prefrontal cortex than healthy controls (P < .05 for both sides) during the breath‐holding task. In conclusion, FM is associated with altered cerebrovascular reactivity measured by NIRS and breath‐holding task, which may reflect endothelial dysfunction or arterial stiffness. Oxygenated hemoglobin concentration changes of healthy controls and FM patients.      

Investigation of HIV‐1 Assembly and Release Using Modern Fluorescence Imaging Techniques

The replication of HIV‐1, like that of all viruses, is intimately connected with cellular structures and pathways. For many years, bulk biochemical and cell biological methods were the main approaches employed to investigate interactions between HIV‐1 and its host cell. However, during the past decade advancements in fluorescence imaging technologies opened new possibilities for the direct visualization of individual steps occurring throughout the viral replication cycle. Electron microscopy (EM) methods, which have traditionally been employed for the study of viruses, are complemented by fluorescence microscopy (FM) techniques that allow us to follow the dynamics of virus–cell interaction. Subdiffraction fluorescence microscopy, as well as correlative EM/FM approaches, are narrowing the fundamental gap between the high structural resolution provided by EM and the high temporal resolution and throughput accomplished by FM. The application of modern microscopy to the study of HIV‐1–host cell interactions has provided insights into the biology of the virus which could not easily, or not at all, have been gained by other methods. Here, we review how modern fluorescence imaging techniques enhanced our knowledge of the dynamic and structural changes involved in HIV‐1 particle formation.      

Precision and Accuracy of Dual‐Energy X‐ray Absorptiometry for Determining in Vivo Body Composition of Mice

Objective: To evaluate the precision and accuracy of dual‐energy X‐ray absorptiometry (DXA) for the measurement of total‐bone mineral density (TBMD), total‐body bone mineral (TBBM), fat mass (FM), and bone‐free lean tissue mass (LTM) in mice. Research Methods and Procedures: Twenty‐five male C57BL/6J mice (6 to 11 weeks old; 19 to 29 g) were anesthetized and scanned three times (with repositioning between scans) using a peripheral densitometer (Lunar PIXImus). Gravimetric and chemical extraction techniques (Soxhlet) were used as the criterion method for the determination of body composition; ash content was determined by burning at 600°C for 8 hours. Results: The mean intraindividual coefficients of variation (CV) for the repeated DXA analyses were: TBMD, 0.84%; TBBM, 1.60%; FM, 2.20%; and LTM, 0.86%. Accuracy was determined by comparing the DXA‐derived data from the first scan with the chemical carcass analysis data. DXA accurately measured bone ash content (p = 0.942), underestimated LTM (0.59 ± 0.05g, p < 0.001), and overestimated FM (2.19 ± 0.06g, p < 0.001). Thus, DXA estimated 100% of bone ash content, 97% of carcass LTM, and 209% of carcass FM. DXA‐derived values were then used to predict chemical values of FM and LTM. Chemically extracted FM was best predicted by DXA FM and DXA LTM [FM = ?0.50 + 1.09(DXA FM) ? 0.11(DXA LTM), model r² = 0.86, root mean square error (RMSE) = 0.233 g] and chemically determined LTM by DXA LTM [LTM = ?0.14 + 1.04(DXA LTM), r² = 0.99, RMSE = 0.238 g]. Discussion: These data show that the precision of DXA for measuring TBMD, TBBM, FM, and LTM in mice ranges from a low of 0.84% to a high of 2.20% (CV). DXA accurately measured bone ash content but overestimated carcass FM and underestimated LTM. However, because of the close relationship between DXA‐derived data and chemical carcass analysis for FM and LTM, prediction equations can be derived to more accurately predict body composition.     

A new filtering technique for removing anti‐Stokes emission background in gated CW‐STED microscopy

Stimulated emission depletion (STED) microscopy is a prominent approach of super‐resolution optical microscopy, which allows cellular imaging with so far unprecedented unlimited spatial resolution. The introduction of time‐gated detection in STED microscopy significantly reduces the (instantaneous) intensity required to obtain sub‐diffraction spatial resolution. If the time‐gating is combined with a STED beam operating in continuous wave (CW), a cheap and low labour demand implementation is obtained, the so called gated CW‐STED microscope. However, time‐gating also reduces the fluorescence signal which forms the image. Thereby, background sources such as fluorescence emission excited by the STED laser (anti‐Stokes fluorescence) can reduce the effective resolution of the system. We propose a straightforward method for subtraction of anti‐Stokes background. The method hinges on the uncorrelated nature of the anti‐Stokes emission background with respect to the wanted fluorescence signal. The specific importance of the method towards the combination of two‐photon‐excitation with gated CW‐STED microscopy is demonstrated. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The color of lactotroph secretory granules stained with FM1-43 depends on dye concentration

When pituitary lactotroph granules undergo exocytosis in the presence of FM1-43, their cores absorb dye and fluoresce brightly. We report that different granules fluoresce with different colors, despite being stained with a single fluorescent dye; emission spectra from individual granules show up to a 25 nm difference between the greenest and reddest granules. We found a correlation between granule color and average fluorescence intensity, suggesting that granule color depends upon dye concentration. We confirmed this in two ways: by increasing FM dye concentration in granules, which red shifted granule color, and by partially photobleaching the FM dye in granules, which green shifted granule color. Increasing stimulation intensity (by increasing KCl concentration) increased the proportion of red granules, indicating that granules exocytosing during intense stimulation bound more dye. This, perhaps, reflects differences in granule core maturation and condensation in which mature granules with condensed cores bind more FM dye but require more intense stimulation to be released. Concentration-dependent color shifts of FM dyes may be useful for monitoring aggregation processes occurring on a size scale smaller than the optical limit.     

Second harmonic imaging of plants tissues and cell implosion using two‐photon process in ZnO nanoparticles

The optical properties of colloidal ZnO nanoparticle (NP) solutions, with size ranging from several nm to around 200 nm, have been tailored to have high optical nonlinearity for bioimaging with no auto‐fluorescence above 750 nm and minimal auto‐fluorescence below 750 nm. The high second harmonic conversion efficiency enables selective tissue imaging and cell tracking using tunable near‐infrared femtosecond laser source ranging from 750‐980 nm. For laser energies exceeding the two‐photon energy of the bandgap of ZnO (half of 3.34 eV), the SHG signal greatly decreases and the two‐photon emission becomes the dominant signal. The heat generated due to two‐photon absorption within the ZnO NPs enable selective cell or localized tissue destruction using excitation wavelength ranging from 710–750 nm. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Simultaneous determination of piroxicam and norfloxacin in biological fluids by high‐performance liquid chromatography with fluorescence detection at zero‐order emission mode

A new highly sensitive high‐performance liquid chromatographic method with fluorescence detection (HPLC–FLD) in zero‐order emission mode was developed for the first time for the simultaneous determination of piroxicam (PRX) and norfloxacin (NRF) in biological fluids. The fluorescence detector wavelengths were set at 278 nm for excitation and zero‐order mode for emission. The zero‐order emission mode produced greater sensitivity for the measurement of both drugs than a fixed emission wavelength (446 nm). The new developed method was validated according to International Conference of Harmonization (ICH) guidelines. Linearity was found to be over concentration ranges 0.001–20 μg/ml and 0.00003–0.035 μg/ml for PRX and NRF, respectively. The limits of detection were 4.87 × 10^?4 and 1.32 × 10^?5 μg/ml for PRX and NRF, and the limits of quantitation were 1.47 × 10^?3 and 4.01 × 10^?5 μg/ml, respectively. The current fluorescence method was found to be more sensitive than most commonly used analytical methods and was successfully applied for simultaneous determination of PRX and NRF in biological fluids (serum and urine) with recoveries ranging from 91.67% to 100.36% for PRX and from 96.00% to 101.43% for NRF.     

Hyperspectral imaging microscopy for identification and quantitative analysis of fluorescently‐labeled cells in highly autofluorescent tissue

Standard fluorescence microscopy approaches rely on measurements at single excitation and emission bands to identify specific fluorophores and the setting of thresholds to quantify fluorophore intensity. This is often insufficient to reliably resolve and quantify fluorescent labels in tissues due to high autofluorescence. Here we describe the use of hyperspectral analysis techniques to resolve and quantify fluorescently labeled cells in highly autofluorescent lung tissue. This approach allowed accurate detection of green fluorescent protein (GFP) emission spectra, even when GFP intensity was as little as 15% of the autofluorescence intensity. GFP‐expressing cells were readily quantified with zero false positives detected. In contrast, when the same images were analyzed using standard (single‐band) thresholding approaches, either few GFP cells (high thresholds) or substantial false positives (intermediate and low thresholds) were detected. These results demonstrate that hyperspectral analysis approaches uniquely offer accurate and precise detection and quantification of fluorescence signals in highly autofluorescent tissues. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Fluorescence and photochemical properties of phytochromes in wild-type wheat and a transgenic line overexpressing an oat phytochrome A (PHYA) gene: functional implications

Etiolated seedlings of wild‐type wheat and a transgenic line overexpressing an oat PHYA gene were investigated by the use of in situ low‐temperature fluorescence spectroscopy. The red‐absorbing phytochrome form, Pr, was characterized by (1) fluorescence emission spectrum; (2) total phytochrome content, and (3) by the extent of the Pr → lumi‐R photoconversion at low temperature (γ₁), and of the Pr → Pfr photoconversion at ambient temperature (γ₂) as derived from emission data. All the characteristics were shown to be variable and to depend on (1) organ and tissue used; (2) seedling age; (3) transgenic wheat modification, and (4) continuous far‐red irradiation of seedlings during their growth. These variations were interpreted in terms of the existence in wheat seedlings of the two phenomenological Pr types: (a), Pr′– major longer wavelength (687/673 nm, emission/absorption maxima) variable and light‐labile with γ₁ ≈ 0·5; and (b), Pr′′– minor, shorter wavelength (682/668 nm), relatively constant with its concentration not changing significantly with the increase of total phytochrome content in tissues and light‐stable with γ₁ ≤ 0·05–0·1. Overexpression of oat phyA increases primarily the content of Pr′ suggesting that it is comprised of phyA (phyA′) whereas Pr′′ is believed to consist of the minor phyA fraction (phyA′′) and phyB. The transgenic wheat line has been demonstrated to have a modified phenotype – the appearance of the far‐red high irradiance reaction (FR‐HIR) (Shlumukov et al. Plant, Cell and Environment 24, 703–712). The increased content of phyA′ in the transgenic line, whereas the total [phyA′′ + phyB] remains the same as in the wild type, indicates that the phyA′ pool is primarily responsible for the observed modification of the phenotype and suggests that even in wild‐type plants the phyA′ component of the phyA pool may mediate the FR‐HIR.     

A simple and sensitive spectrofluorimetric method for the determination of furosemide using zinc(II)–1,4‐bis(imidazol‐1‐ylmethyl)benzene complexes

A novel spectrofluorometric method for the determination of furosemide (FUR) is described. The method is based on enhancement of fluorescence emission of FUR in the presence of zinc (II) complexes of 1,4‐bis(imidazol‐1‐ylmethyl)benzene. Under optimum conditions, the enhanced fluorescence intensity is linearly related to the concentration of FUR. The proposed method has been successfully applied to the determination of FUR in pharmaceutical preparations. The possible mechanism of this reaction is discussed briefly based on data from fluorescence spectroscopy, UV–vis absorption and infrared spectroscopy. Copyright © 2012 John Wiley & Sons, Ltd.     

Systematic colocalization errors between acridine orange and EGFP in astrocyte vesicular organelles

Dual-color imaging of acridine orange (AO) and EGFP fused to a vesicular glutamate transporter or the vesicle-associated membrane proteins 2 or 3 has been used to visualize a supposedly well-defined subpopulation of glutamatergic astrocytic secretory vesicles undergoing regulated exocytosis. However, AO metachromasy results in the concomitant emission of green and red fluorescence from AO-stained tissue. Therefore, the question arises whether AO and EGFP fluorescence can be distinguished reliably. We used evanescent-field imaging with spectral fluorescence detection as well as fluorescence lifetime imaging microscopy to demonstrate that green fluorescent AO monomers inevitably coexist with red fluorescing AO dimers, at the level of single astroglial vesicles. The green monomer emission spectrally overlaps with that of EGFP and produces a false apparent colocalization on dual-color images. On fluorophore abundance maps calculated from spectrally resolved and unmixed single-vesicle spectral image stacks, EGFP is obscured by the strong green monomer fluorescence, precluding the detection of EGFP. Hence, extreme caution is required when deriving quantitative colocalization information from images of dim fluorescing EGFP-tagged organelles colabeled with bright and broadly emitting dyes like AO. We finally introduce FM4-64/EGFP dual-color imaging as a remedy for imaging a distinct population of astroglial fusion-competent secretory vesicles.     

High-Resolution Ultrasound-Switchable Fluorescence Imaging in Centimeter-Deep Tissue Phantoms with High Signal-To-Noise Ratio and High Sensitivity via Novel Contrast Agents

For many years, investigators have sought after high-resolution fluorescence imaging in centimeter-deep tissue because many interesting in vivo phenomena—such as the presence of immune system cells, tumor angiogenesis, and metastasis—may be located deep in tissue. Previously, we developed a new imaging technique to achieve high spatial resolution in sub-centimeter deep tissue phantoms named continuous-wave ultrasound-switchable fluorescence (CW-USF). The principle is to use a focused ultrasound wave to externally and locally switch on and off the fluorophore emission from a small volume (close to ultrasound focal volume). By making improvements in three aspects of this technique: excellent near-infrared USF contrast agents, a sensitive frequency-domain USF imaging system, and an effective signal processing algorithm, for the first time this study has achieved high spatial resolution (~ 900 μm) in 3-centimeter-deep tissue phantoms with high signal-to-noise ratio (SNR) and high sensitivity (3.4 picomoles of fluorophore in a volume of 68 nanoliters can be detected). We have achieved these results in both tissue-mimic phantoms and porcine muscle tissues. We have also demonstrated multi-color USF to image and distinguish two fluorophores with different wavelengths, which might be very useful for simultaneously imaging of multiple targets and observing their interactions in the future. This work has opened the door for future studies of high-resolution centimeter-deep tissue fluorescence imaging.     

Imaging Escherichia coli using functionalized core/shell CdSe/CdS quantum dots

The internalization of a series of water-soluble CdSe/CdS quantum dots (QDs) stabilized by citrate, isocitrate, succinate, and malate by Escherichia coli is established by epifluorescence and confocal fluorescence scanning microscopy, fluorimetry, and UV–vis spectroscopy on whole and lysed bacterial cells. The organic-acid-stabilized QDs span a range in size from 3.8±1.1 to 6.0±2.4 nm with emission wavelengths from 540 to 630 nm. QDs of different sizes (i.e., 3.8–6 nm) can enter the bacterium and be detected on different fluorescence channels with little interference from other QDs as a result of the distinct emission profiles (i.e., 540–630 nm, respectively). Costaining QD-labeled E. coli with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) demonstrates that the QDs and DAPI are colocalized within E. coli, whereas costaining QD-labeled E. coli with membrane dye FM4-64 shows that the FM4-64 is localized in the outer bacterial membrane and that the QDs are inside.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Study of the vesicular cycle in nerve structures in somatic muscle of earthworm (<Emphasis Type="Italic">Lumbricus terrestris</Emphasis>)

In the muscle wall of the earthworm Lumbricus terrestris, with the aid of fluorescent endocytotic dyes FM1-43, FM2-10, and FM4-64, there are revealed fluorescent spots 1–2 μm in diameter that represent clusters of “synaptic boutons.” Application takes place onto ganglia of the abdominal nerve chain of the Dil membrane probe capable of translocation by axoplasmic transport; the subsequent (next day) staining of nerve structures with the endocytotic marker FM4-64 showed the complete superposition of fluorescence of these dyes fluorescing in different specter areas. The fluorescent marker DiBAC₄(3) revealed an enhancement of fluorescence of nerve elements with increase of K⁺ concentration in the extracellular medium. Use of FM2-10 showed that, the higher the K⁺ content in solution and, accordingly, the nerve cell depolarization, the faster the release of the marker and, on the contrary, the slower the process in the absence of K⁺ in the medium. In the Ca²⁺-free solution and in the presence of the Ca²⁺ chelator BAPTA or BAPTA-AM, there uptake and release of FM2-10 are blocked, but only after preliminary 40-min incubation in such solution. In clusters of synaptic boutons, exo- and endocytosis processes take place that are also preserved under conditions of rest. This vesicular cycle depends on the membrane potential of nerve structures and on the content of K⁺ and Ca²⁺ in the medium, the calcium sensor working most likely by the “all or nothing” principle.     

A ratiometric fluorescent probe for bisulphite anion,employing intramolecular charge transfer

4‐(1H‐benzimidazol‐2‐yl)benzaldehyde (1) has been developed as a new ratiometric fluorescent probe for bisulphite, based on the modulation of intramolecular charge transfer (ICT). Upon mixing with bisulphite in aqueous ethanol, an aldehyde–bisulphite adduct was formed and the ICT of the probe was switched off, which resulted in a ratiometric fluorescence response with an enhancement of the ratios of emission intensities at 368 and 498 nm. The detection range of the probe for bisulphite is in the 2.0–200 µmol/L concentration range and the detection limit is 0.4 µmol/L. Probe 1 produces a ratiometric fluorescent response to bisulphite with a marked emission wavelength shift (130 nm) and displays high selectivity for bisulphite over other anions. Copyright © 2012 John Wiley & Sons, Ltd.     

Micelle‐enhanced direct spectrofluorimetric method for the determination of linifanib: Application to stability studies

A new simple stability‐indicating spectrofluorimetric method has been developed and validated for the determination of the tyrosine kinase inhibitor, linifanib (LNF). The proposed method makes use of the native fluorescence characteristics of LNF in a micellar system. Compared with aqueous solutions, the fluorescence intensity of LNF was greatly enhanced upon the addition of Tween‐80. The relative fluorescence intensity of LNF was measured in a diluting solvent composed of 2% Tween‐80: phosphate buffer pH 8.0 (20: 80, v/v) using excitation and emission wavelengths of 290 and 450 nm, respectively. The proposed method was fully validated as per the ICH guidelines. The recorded fluorescence intensity of LNF was rectilinear over a concentration range of 0.3–2 μg/ml with a high correlation coefficient (r = 0.9990) and low limits of detection (0.091 μg/ml) and quantitation (0.275 μg/ml). The applicability of the method was extended to study the inherent stability of LNF under different stress degradation conditions including, alkaline, acidic, oxidative, photolytic and thermal degradation. Moreover, the method was utilized to study the kinetics of the alkaline and oxidative degradation of LNF. The pseudo‐first order rate constants and half‐lives were calculated.     

Two‐channel near‐infrared fluorescence Ag+ ion sensing of a new star‐shaped dendrimer

A new near‐infrared fluorescence sensor PDI‐PD for Ag⁺ ions was successfully prepared and its structure characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR and high‐resolution mass spectrometry; matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (HRMS MALDI‐TOF). The probe exhibited rapid, sensitive, and selective two‐channel fluorescence responses towards Ag⁺ ions and protons. The probe has a marked high binding affinity and high sensitivity for Ag⁺, with a detection limit of 1.4 × 10^?6 M. An approximately five‐fold enhanced core emission at 784 nm was attributed to fluorescence resonance energy transfer (FRET). The enhanced core emission of the probe with Ag⁺ ions based on photo‐induced electron transfer and FRET is discussed. In addition, the probe presented a visible colour change. All experimental results demonstrated that PDI‐PD is an efficient tool for the selective, sensitive and rapid detection of Ag⁺ ions and protons using two‐channel fluorescence responses.