Observed Fluorescence Intensity Research Articles

Unveiling the multifaceted potential of (E)-N-(4-Chlorophenyl)-1-(thiophen-2-yl) methanimine with special reference to solution-state fluorescence, synthesis, electronic structure, antimicrobial, antibiofilm, larvicidal activities, toxicity, docking and dynamics

Schiff bases exhibit distinctive characteristics in the realm of chemistry, physics, and medicine, rendering them highly efficacious in the field of biology. A Schiff base was synthesized by reacting 4-chloroaniline with thiophene-2-carboxaldehyde, and its structure was subsequently optimized using density functional theory (DFT). We conducted experimental simulations and validations of the IR, UV, and NMR spectra. The compound formation was confirmed through the utilization of FTIR, UV–VIS, 1HNMR, and 13CNMR analyses. The observed fluorescence intensities at wavelengths of 438.28 and 666.83 nm provided indications of its inherent fluorescence properties. A conspicuous alteration in the thermogravimetric analysis (TGA) plot within the temperature range of 250 °C to 315 °C was indicative of a chemical metamorphosis. The compound exhibited an energy gap of 3.97 electron volts (eV). In the MEP graph, the blue regions denoted electron-deficient areas, whereas the red regions represented electron-abundant regions. In the context of natural population analysis, the hydrogen atoms exhibit positive charges, which are associated with electron transfer phenomena. The optimized compound exhibited nitrogen and chlorine atoms with negative charges, functioning as electron donors. The utilization of NBO (Natural Bond Orbital) analysis and spectral analysis unveiled the occurrence of electron transfers within the molecular framework. The ELF diagrams illustrate the presence of hydrogen by utilizing the colour red, while carbon and nitrogen are represented by the colour blue. The carbon atoms exhibited a blue hue in the LOL diagram, indicating a state of electron deficiency. Non-covalent interactions were exemplified in RDG maps. It exhibited superior antimicrobial efficacy against both bacteria and fungi, surpassing that of amoxicillin The compound exhibited a moderate level of antibiofilm activity, with an IC50 value of 158.9 μg/ml. Additionally, it demonstrated larvicidal effects that were dependent on the concentration. The antimicrobial properties of the compound were confirmed through molecular docking and dynamic simulation. The insilico assessments conducted against toxicity models have revealed a notable absence of toxicity and a commendable level of tolerance.

Quantifying Localized Stresses in the Matrix of a Fiber‐Reinforced Composite via Mechanophores

AbstractUnderstanding the stress distribution within fiber‐reinforced polymers (FRPs) is critical to extending their operational lifespan. The integration of mechanoresponsive molecular force probes, referred to as mechanophores, presents a potential solution by enabling direct monitoring of stress concentrations. In this study, spiropyran (SP) mechanophores (MPs) are embedded within a polydimethylsiloxane (PDMS) matrix to visualize stress localization during loading within a single fiber‐reinforced framework. The SP mechanophore undergoes a transition from a non‐fluorescent state to an active state (merocyanine) through isomerization in response to mechanical forces. Using a single fiber mounted axially within the matrix, the fundamental failure modes observed in conventional fiber‐reinforced composites are replicated. Samples are strained under uniaxial tensile loading along the fiber direction and the localization of stresses is observed via MP activation. Stresses are concentrated in the matrix near the fiber region that gradually decreases away from the fiber surface. Confocal microscopy is used to visualize mechanophore activation and quantitatively assess fluorescence intensity. Finite element modeling is used to develop a calibration to quantify the stresses based on the observed fluorescence intensity. These outcomes underscore the viability of employing these mechanoresponsive molecules as a potential means to visualize real‐time stress distribution, thereby facilitating the design of high‐performance composites.

Kinetics of macroautophagy in lung adenocarcinoma (A549) cells exposed to nanoparticles

Macroautophagy is an intracellular process helping to maintain normal cellular function. Dysregulation of autophagy is recognized in certain diseases, but it is unknown how maintenance of cellular homeostasis might be affected by the kinetics of autophagy in response to various stimuli. In this study, we measured autophagy kinetics in A549 cells exposed to nanoparticles (NP) (including ambient ultrafine particles (UFP)) and/or Rapamycin. A549 cells were apically exposed (t=0-24hrs) in four experimental settings: (1) control exposure only to culture fluid; (2) exposure to polystyrene NP (PNP), UFP or Rapamycin; (3) concurrent exposure to PNP and Rapamycin or UFP and Rapamycin; and, (4) sequential (in both orders) exposure to a first agent (PNP, UFP or Rapamycin) at t=0-5hrs, followed by exposure to an additional agent (Rapamycin after PNP (or UFP) and PNP (or UFP) after Rapamycin) at t=5-24hrs. Autophagic activity was assessed using fluorescence intensity of DAPRed (a marker for autophagosomes and autolysosomes; 0.1 μM; Dojindo; Rockville, MD) or BacMam RFP-GFP-LC3B (Thermo Fisher; Waltham, MA) in the presence or absence of 40 μM chloroquine measured over the entire cell volume of live single cells using confocal laser scanning microscopy. Autophagic flux was calculated as the difference of observed fluorescence intensity of DAPRed or RFP-GFP-LC3B in the presence and absence of chloroquine. Autophagic flux increased in all experimental modalities, including both single agent and double agent exposures, and reached steady state in all cases ~2 times control from ~8 to 24 hrs, suggesting the presence of an upper limit to autophagic activity. Rapamycin exposure alone induced a rapid rise in autophagic flux, peaking at ~3hrs post exposure, followed by a lowered steady state autophagic flux for up to 24hrs. Exposure to PNP or UFP alone also increased autophagic flux, gradually reaching steady state at ~10-24hrs. During concurrent double exposure to Rapamycin and PNP or UFP at t=0, the steady state level of autophagic flux remained comparable to that observed with single exposures to Rapamycin, PNP or UFP for up to 24 hrs. Under sequential exposure conditions, the steady state level of autophagic flux did not exceed the steady state level observed in single or concurrent exposures. In summary, exposure to nanoparticles activates macroautophagy in a time-dependent manner in A549 cells. Autophagic activity demonstrated comparable steady state levels after exposure to single or dual stimuli, indicating an effective upper limit of autophagic flux in A549 cells under our experimental conditions. These findings suggest that environmental stressors might exert their harmful effects, at least in part, by limiting available autophagic capacity, thereby making NP-exposed cells more susceptible to secondary injury due to autophagic overload. Funding: NIH; WRMPPF This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Simultaneous analyses of fluorescence decay and anisotropy decay in green fluorescent protein dimer from jellyfish Clytia gregaria: FRET and molecular dynamics simulation

Structural and dynamic behaviors of the green fluorescent protein dimer from jellyfish Clytia gregaria (cgGFP) were investigated by means of molecular dynamics (MD) simulation. Both neutral and ionic forms of the chromophore, p-hydroxybenzylideneimidazolinone (GYS) were considered. The partial atomic charges of the chromophore were derived by BCC and RESP approaches. The structures were compared between the anionic and neutral cgGFP, and between the two subunits (Sub A and Sub B) of the protein dimer. The observed fluorescence intensity and anisotropy decays were further analyzed with theoretical expressions by employing the atomic coordinates of neutral cgGFP obtained by MD simulation. It was assumed that the fluorescence quenching of GYSA and GYSB is ascribed to HB formations between heteroatoms of GYSs and nearby amino acids. Excellent agreement between the observed and calculated intensity decays, and the observed and calculated anisotropy decays were obtained with RESP1 model. The agreements were better in RESP model than those in BCC one. Mean quenching constants of GYSA and GYSB were 0.27 and 0.59 ns−1 overall MD snapshots with RESP1. Mean value of square of direction cosine between the two transition moments of GYSs was 0.74, and that of square of orientation factor was 0.53, and the FRET rates from GYSA to GYSB, and from GYSB to GYSA were 0.87 and 1.87 ns−1.

Effects of Trichoderma asperellum and its siderophores on endogenous auxin in Arabidopsis thaliana under iron-deficiency stress.

Iron (Fe) deficiency is one of the major limiting factors affecting crop yields. Trichoderma asperellum Q1, a biocontrol and plant growth promoting fungus, can produce the siderophore which has a high affinity to Fe3+ in the absence of iron. In this study, Trichoderma asperellum Q1 was found to be able to promote growth of Arabidopsis thaliana in an iron-deficient or insoluble iron-containing (Fe2O3) medium. It also can produce more siderophore and indole-3-acetic acid (IAA) as the concentration of iron ions decreased. However, it is unclear that the relationship between siderophore and IAA in promoting plant growth. Both Trichoderma asperellum Q1 and siderophore promotes not only the DR5::GFP transgenic Arabidopsis thaliana seedlings, in which the root IAA is labeled by green fluorescent protein gene, but also increases the content of endogenous IAA in the roots, which was shown by the fluorescence study. The strongest fluorescence was observed in the treated group inoculated with Trichoderma asperellum Q1 under the condition of insoluble iron. In the case of iron-free medium, adding siderophore also increased the observed fluorescence intensity. These results suggest that the siderophores produced by Trichoderma asperellum Q1 increased the content of IAA in Arabidopsis roots by enhancing the conversion of poorly soluble iron or by the siderophore itself.

Micellar-based spectrofluorimetric method for the selective determination of ledipasvir in the presence of sofosbuvir: application to dosage forms and human plasma.

A fast, low-cost, sensitive, and selective spectrofluorimetric method for the determination of ledipasvir was developed and validated. The method is based on an enhancement in the native fluorescence intensity of ledipasvir by 500% of its original value by the formation of hydrogen bonds between the cited drug and Tween-20 in the micellar system (pH = 5.0). All fluorescence measurements were carried out at 425 nm and 340 nm for emission and excitation wavelengths, respectively. A linear relationship between the concentration of ledipasvir and the observed fluorescence intensity was achieved in the range of 0.1-2.0 μg ml-1 with 0.028, 0.084 μg ml-1 , for detection and quantitation limits, respectively. The acquired selectivity and sensitivity using the proposed method facilitate the analysis of ledipasvir in spiked human plasma with sufficient percentage recovery (95.36-99.30%). The proposed method was developed and validated according to International Council for Harmonisation (ICH) guidelines. Moreover, the cited drug was successfully determined in its pharmaceutical dosage form using the proposed method. In addition, the validity of the proposed results was statistically confirmed using Student's t-test, variance ratio F-test, and interval hypothesis test.

Microenvironment improvement protocol for the sensitive spectrofluorimetric determination of an hepatitis C virus antiviral (Simeprevir): application to human plasma.

Simeprevir (SPV) is a powerful antihepatitis C virus agent that was newly introduced into the pharmaceutical market. We here established and validated an easy, simple, and sensitive spectrofluorimetric method for its estimation at λem 427 nm (λex 337 nm). The suggested procedure was based on two times enhancement in the original emission of SPV through modifying its microenvironment in buffered aqueous solution by adding Triton X-100. The relationship between the concentration of SPV and the observed fluorescence intensity was linear in the range 0.06-1.0 μg ml-1 with a correlation coefficient of 0.9997. The limits of detection and quantitation were 21 and 64 ng ml-1 , respectively. The present method was effectively applied to quantify SPV content in pharmaceutical tablets and human plasma spiked with the drug with no interference from tablet excipients or plasma components.

Derived Absorbance Spectral Parameter as a Tool for Sensitive Fluorescence Measurements of Optically Dense Systems.

The present study proposes an approach to use the derived absorbance spectral parameter (Dabs) as a tool for sensitive fluorescence measurement of optically dense multifluorophoric systems. Mishra and group have demonstrated the use of Dabs in obtaining optimum offset wavelength for sensitive fluorescence measurement in synchronous fluorescence spectra (SFS) (Anal. Chim. Acta 2016, 940, 113-119; Anal. Chim. Acta 2008, 630 (1), 47-56). The Dabs is obtained with the knowledge of UV-vis absorbance spectral data of the system under study and fluorescence observation filed (FOF) parameters of the instrument. Dabs is proportional to the observed fluorescence intensity of the system. Excitation of an optically dense sample at Dabs maximum leads to maximum fluorescence intensity obtainable for that system even in the presence of considerable inner filter effect (IFE). The earlier studies were applicable to SFS at right-angle fluorescence measurement geometry and for samples showing only primary IFE. In the present study, the work has been expanded to general fluorescence measurements, even when both primary and secondary IFE is present and for different fluorescence measurement geometries (right-angle, 45° and front-face). The procedure for obtaining Dabs is simple and can be applied to other analyte-specific/unconventional fluorescence measurement strategies reported in the literature.

Fluorescence lifetime imaging reveals heterogeneous functional distribution of eGFP expressed in Xenopus oocytes

The oocytes from Xenopus laevis are well known for their polarity, presenting a distinct animal and vegetal pole. Other heterogeneities are less known. To study the heterogeneity of the Xenopus oocyte, we expressed eGFP and analyzed the protein distribution with fluorescence lifetime microscopy. The vegetal pole exhibited higher levels of fluorescence, than the animal pole. However, the fluorescence lifetimes between the two areas were indistinguishable, suggesting similar environments. In contrast, we observed a substantial and gradual decrease in the fluorescence lifetime from 2.9 ns to 2.6 ns as slices approached the periphery. This has an important implication for future oocyte studies as it demonstrates the environment inside the oocyte is not uniform and might affect the fluorescence intensity. As a result, it cannot be assumed that the observed fluorescence intensity reflects the expression of the proteins but might reflect the environment within the oocyte.

Dihydroxy-Substituted Coumarins as Fluorescent Probes for Nanomolar-Level Detection of the 4-Amino-TEMPO Spin Label.

This paper reports on dihydroxycoumarins as fluorescent probes suitable for the detection and determination of the nitroxide radical, namely 4-amino-TEMPO. Since 4-amino-TEMPO is used as a spin label for the detection of various radicals and damage caused by these species, its determination under physiological conditions might help us to understand the mechanism of the oxidative stress. Among different coumarins studied, only dihydroxy-substituted derivatives show high sensitivity, specificity, and selectivity for the nitroxide radical. In this assay, dihydroxy-substituted coumarins under the action of 4-amino-TEMPO show a very fast and significant increase in fluorescence intensity and lifetime. Among them 6,7-dihydroxycoumarin (esculetin) exhibits the strongest fluorescence enhancement (up to 40 times), with an estimated limit of detection equal to 16.7 nM—a significantly lower value when compared with UV-Vis or electron paramagnetic resonance (EPR) spectroscopy. The method is characterized by an easy procedure of sample preparation and very short time of analysis. The mechanism of the interaction between 6,7-dihydroxycoumarin and 4-amino-TEMPO has been examined with the use of a series of complementary techniques, such as steady-state and time-resolved fluorescence spectroscopy, UV-Vis spectroscopy, electron paramagnetic resonance spectroscopy, potentiometric titration, and high-performance liquid chromatography. It has been proven that the only route of the reaction in the system studied is a proton transfer from the molecule of esculetin to the amino group of the nitroxide. Biological studies performed on prostate cancer cells, breast cancer cells, and normal skin fibroblasts revealed significant anticancer properties of 6,7-dihydroxycoumarin, which caused a considerable decrease in the viability and number of cancer cells, and affected their morphology, contrary to normal fibroblasts. Furthermore, the experiment performed on prostate cancer cells showed that fluorescence emission of esculetin is closely related to intracellular pH—the higher pH, the higher observed fluorescence intensity (in accordance with a chemical experiment). On the other hand, the studies performed in different pH levels revealed that when pH of the solution increases, the observed fluorescence intensity enhancement under the action of 4-amino-TEMPO decreases (better sensing properties of esculetin towards the nitroxide in lower pH).

Excimer Disaggregation Enhanced Emission: A Fluorescence "Turn-On" Approach to Oxoanion Recognition.

A new approach to anion sensing that involves excimer disaggregation induced emission (EDIE) is reported. It involves the anion-mediated disaggregation of the excimer formed from a cationic macrocycle. This leads to an increase in the observed fluorescence intensity. The macrocycle in question, cyclo[1] N2, N6-dimethyl- N2, N6-bis(6-(1 H-imidazolium-1-yl)pyridin-2-yl)pyridine-2,6-diamine[1]1,4-dimethylbenzene (12+; prepared as its PF6- salt), is obtained in ca. 70% yield via a simple cyclization. X-ray diffraction analyses of single crystals revealed that, as prepared, this macrocycle exists in a supramolecular polymeric form in the solid state. Macrocycle 12+ is weakly fluorescent in acetonitrile. The emission intensity is concentration dependent, with the maximum intensity being observed at [12+] ≈ 0.020 mM. This finding is ascribed to formation of an excimer, followed possibly by higher order aggregates as the concentration of 12+ is increased. Addition of tetrabutylammonium pyrophosphate (HP2O73-) to 12+ (0.020 mM in acetonitrile) produces a ca. 200-fold enhancement in the emission intensity (λex = 334 nm; λem = 390-650 nm). These findings are rationalized in terms of the HP2O73- serving to break up essentially non-fluorescent excited-state dimers of 12+ through formation of a highly fluorescent anion-bound monomeric complex, 12+·HP2O73-. A turn-on in the fluorescence intensity is also seen for H2PO4- and, to a lesser extent, HCO3-. Little (HSO4-, NO3-) or essentially no (N3-, SCN-, F-, Cl-, Br- and I-) response is seen for other anions. Solid-state structural analysis of single crystals obtained after treating 12+ with HP2O73- in the presence of water revealed a salt form wherein a H2P2O72- anion sits above the cone-like macrocycle.

Study on the dependence of fluorescence intensity on optical density of solutions: the use of fluorescence observation field for inner filter effect corrections.

In this study, we report the identification of absorbance value of an analyte at the excitation wavelength that corresponds to the maximum of the observed fluorescence intensity obtainable for a certain instrument operating with right-angle fluorescence measurement (). The value depends on the fluorescence observation field (FOF) dimensions of the concerned spectrofluorometer. As the FOF varies from instrument to instrument, this study presents a simple method for obtaining FOF dimensions. Using the knowledge of FOF, absorbance of analyte at the excitation wavelength (Aλex) and emission wavelength (Aλem), we deduced a derived absorbance spectral parameter (Dabs). The observed fluorescence intensity of an analyte is proportional to the Dabs. While differentiating Dabs w.r.t. Aλex, the value of for the concentred spectrofluorometer was obtained and subsequently could be used for maximizing fluorescence sensitivity. It was observed that when the FOF was a point at the centre of a 1 cm path-length cuvette, the value was 0.87 with a progressive widening of FOF, the value increased gradually till ∼1.0. The proposed methodology was established using two well-known inner filter effect (IFE) correction models (Parker and Lakowicz model). The Dabs obtained from the Parker model corresponded well with the observed fluorescence data; however, the Dabs obtained using the Lakowicz model overestimated the loss of fluorescence because of IFE. Using equations derived from the Parker model, the correction of observed fluorescence intensity for IFE could be achieved. Furthermore, it is demonstrated that the commonly used the Lakowicz model loses its correction efficiency at absorbance values of ≥0.7.

Diketopyrrolopyrrole-based fluorescence probes for the imaging of lysosomal Zn2+ and identification of prostate cancer in human tissue.

A series of diketopyrrolopyrrole-based fluorescent probes (DPP-C2, LysoDPP-C2, LysoDPP-C3, and LysoDPP-C4) have been developed for the detection of low pH and Zn2+ in an AND logic fashion. The chelation of Zn2+ or the protonation of a morpholine moiety within these probes results in a partial increase in the fluorescence intensity, an effect ascribed to suppression of one possible photo-induced electron transfer (PET) pathway. In contrast, a large increase in the observed fluorescence intensity is observed at low pH and in the presence of Zn2+; this is rationalized in terms of both possible PET pathways within the probes being blocked. Job plots, fluorescence titration curves, and isothermal titration calorimetry proved consistent with a 1 : 1 Zn2+ complexation stoichiometry. Each probe demonstrated an excellent selectivity towards Zn2+ and the resulting Zn2+ complexes demonstrated pH sensitivity over the 3.5-9 pH range. Fluorescence imaging experiments confirmed that LysoDPP-C4 was capable of imaging lysosomal Zn2+ in live cells. Little evidence of cytotoxicity was seen. LysoDPP-C4 was successfully applied to the bioimaging of nude mice, wherein it was shown capable of imaging the prostate. Histological studies using a human sample revealed that LysoDPP-C4 can discriminate cancerous prostate tissue from healthy prostate tissue.

Microscale Objects via Restructuring of Large, Double-Stranded DNA Molecules.

As the interest in DNA nanotechnology increases, so does the need for larger and more complex DNA structures. In this work, we describe two methods of using large, double-stranded (ds) DNA to self-assemble sequence-specific, nonrepetitive microscale structures. A model system restructures T7 DNA (40 kb) through sequence-specific biotinylation followed by intramolecular binding to a 40 nm diameter neutravidin bead to create T7 "rosettes". This model system informed the creation of "nodal DNA" where "nodes" with single-stranded DNA flaps are attached to a large dsDNA insert so that a complementary oligonucleotide "strap" bridges the two nodes for restructuring to form a DNA "bolo". To do this in high yield, several methodologies were developed, including a protection/deprotection scheme using RNA/RNase H and dialysis chambers, which remove excess straps while retaining large DNA molecules. To assess these restructuring processes, the DNA was adsorbed onto supported lipid bilayers, allowing for a visual assay of their structure using single-molecule fluorescence microscopy. Good agreement between the expected and observed fluorescence intensity measurements of the individual features of restructured DNA for both the DNA rosettes and bolos gives us a high degree of confidence that both processes give sequence-specific restructuring of large, dsDNA molecules to create microscale objects.

Orientation selection of broad-spectrum aptamers against lipopolysaccharides based on capture-SELEX by using magnetic nanoparticles

The authors describe a new kind of selection method (referred to as orientation selection) for improved screening for broad-spectrum lipopolysaccharides (LPSs) using unlabeled ssDNA aptamers. The method is based on the capture-SELEX technique using magnetic nanoparticles. LPS from Salmonella enterica serotype typhimurium was chosen as an exemplary target. Once the ssDNA library is preconcentrated to a certain degree, two Gram-negative bacteria were used as orientation molecules in the subsequent selection process. Using this strategy, one optimal aptamer ('EA7') was captured that has a high affinity (Kd = 102 ± 17 nM) for LPS and it was also confirmed that it can recognize three other bacterial LPSs. It is presumed that EA7 binds to the lipid A region of LPS. When using carboxyfluorescein labeled EA7, the observed fluorescence intensity and concentrations of four types of LPSs in drinking water are linearly correlated. The lower detection limit of the LPS is 3 ng·mL−1. Compared to multi-target mixed selection and conventional SELEX methods, the new orientation selection strategy produces results that are less uncertain.

Recovering fluorophore concentration profiles from confocal images near lateral refractive index step changes.

Optical aberrations due to refractive index mismatches occur in various types of microscopy due to refractive differences between the sample and the immersion fluid or within the sample. We study the effects of lateral refractive index differences by fluorescence confocal laser scanning microscopy due to glass or polydimethylsiloxane cuboids and glass cylinders immersed in aqueous fluorescent solution, thereby mimicking realistic imaging situations in the proximity of these materials. The reduction in fluorescence intensity near the embedded objects was found to depend on the geometry and the refractive index difference between the object and the surrounding solution. The observed fluorescence intensity gradients do not reflect the fluorophore concentration in the solution. It is suggested to apply a Gaussian fit or smoothing to the observed fluorescence intensity gradient and use this as a basis to recover the fluorophore concentration in the proximity of the refractive index step change. The method requires that the reference and sample objects have the same geometry and refractive index. The best results were obtained when the sample objects were also used for reference since small differences such as uneven surfaces will result in a different extent of aberration.

Enhancement of Curcumin Fluorescence by Ascorbic Acid in Bicontinuous Microemulsion.

Steady-state fluorescence spectro-photometric technique is used in this work to determine the chemical parameters of the complex formed between curcumin and ascorbic acid in bicontinuous microemuslion (Bμen). The Bμen liquid used is made up of a four-components system (water-oil-surfactant and co-surfactant (1-pentanol)) in the ratio of 42.11:13.7:21.34:22.85 % w/w. The oil and surfactant used are tetradecane and cetyltrimethylammonium bromide. Curcumin is known to have low solubility in water, but liberally soluble in Bμen, hence the use of Bμen in this study. The observed fluorescence intensity of curcumin was enhanced by introduction of ascorbic acid to the curcumin solution. The increase in the fluorescence intensity showed a very good linearity with a regression coefficient of 0.9974. The association constant, Ka, that resulted between curcumin and ascorbic acid was calculated as 2.15 × 10(4) with the free energy of association, ∆Ga, of -24.71 kJ/mol. The ratio of the complex that was formed by these two molecules was determined as 1:1.

Gold nanoparticle-mediated fluorescence enhancement by two-photon polymerized 3D microstructures

Fluorescence enhancement achieved by functionalized microstructures made by two-photon polymerization (TPP) is reported for the first time. Microstructures of various shapes made of SU-8 photoresist were prepared and coated with gold nanoparticles (NP) of 80nm. Localized fluorescence enhancement was demonstrated by microstructures equipped with tips of sub-micron dimensions. The enhancement was realized by positioning the NP-coated structures over fluorescent protein layers. Two fluorophores with their absorption in the red and in the green region of the VIS spectrum were used. Laser scanning confocal microscopy was used to quantify the enhancement. The enhancement factor was as high as 6 in areas of several square-micrometers and more than 3 in the case of local enhancement, comparable with literature values for similar nanoparticles. The structured pattern of the observed fluorescence intensity indicates a classic enhancement mechanism realized by standing waves over reflecting surfaces. With further development mobile microtools made by TPP and functionalized by metal NPs can be actuated by optical tweezers and position to any fluorescent micro-object, such as single cells to realize localized, targeted fluorescence enhancement.

Optimization of protein production by Micrococcus luteus for exploring pollutant-degrading uncultured bacteria

The screening of pollutant-degrading bacteria are limited due to most of bacteria in the natural environment cannot be cultivated. For the purpose of resuscitating and stimulating “viable but non-culturable” (VBNC) or uncultured bacteria, Micrococcus luteus proteins are more convenient and cost-effective than purified resuscitation-promoting factor (Rpf) protein. In this study, medium composition and culture conditions were optimized by using statistical experimental design and analysis to enhance protein production by M. luteus. The most important variables influencing protein production were determined using the Plackett-Burman design (PBD) and then central composite design (CCD) was adopted to optimize medium composition and culture conditions to achieve maximum protein yield. Results showed that the maximum protein yield of 25.13 mg/L (vs. 25.66 mg/L predicted) was obtained when the mineral solution, Lithium L-lactate, initial pH and incubation time were set at 1.5 ml/L, 8.75 g/L, 7.5 and 48 h, respectively. The predicated values calculated with the model were very close to the experimental values. Protein production was obviously increased with optimization fitting well with the observed fluorescence intensity. These results verified the feasibility and accuracy of this optimization strategy. This study provides promising information for exploring highly desirable pollutant-degrading microorganisms.

Maximum Likelihood Estimation of FRET Efficiency

FRET is sensitive technique for the determination of protein clustering in living cells. It is often applied in fluorescence microscopy where ratiometric measurements are easy to carry out. Pixel-by-pixel determination of FRET efficiency is expected to reveal potential heterogeneity in protein association states. However, a systematic evaluation of how reliable pixel-by-pixel FRET calculations are in the presence of low photon numbers, significant background fluorescence or outlier pixels has not been reported. Here, we have carried out such an analysis and conclude that the mean of pixel-by-pixel FRET efficiencies is often a biased and unreliable estimator of FRET efficiency. In addition, we have developed a method to determine the FRET efficiency based on maximum likelihood estimation. The Poisson statistics of photon numbers detected in the donor, FRET and acceptor channels is taken in to account to maximize the likelihood of the observed fluorescence intensities measured using a photon counting detector. Our calculations and simulations show that the maximum-likelihood-based method is a reliable method to estimate the FRET efficiency and outperforms both pixel-by-pixel FRET calculations and FRET efficiency calculated from summed intensities.