Analysis Of Fluorescence Data Research Articles

Robust reconstruction of gene expression profiles from reporter gene data using linear inversion.

Motivation: Time-series observations from reporter gene experiments are commonly used for inferring and analyzing dynamical models of regulatory networks. The robust estimation of promoter activities and protein concentrations from primary data is a difficult problem due to measurement noise and the indirect relation between the measurements and quantities of biological interest.Results: We propose a general approach based on regularized linear inversion to solve a range of estimation problems in the analysis of reporter gene data, notably the inference of growth rate, promoter activity, and protein concentration profiles. We evaluate the validity of the approach using in silico simulation studies, and observe that the methods are more robust and less biased than indirect approaches usually encountered in the experimental literature based on smoothing and subsequent processing of the primary data. We apply the methods to the analysis of fluorescent reporter gene data acquired in kinetic experiments with Escherichia coli. The methods are capable of reliably reconstructing time-course profiles of growth rate, promoter activity and protein concentration from weak and noisy signals at low population volumes. Moreover, they capture critical features of those profiles, notably rapid changes in gene expression during growth transitions.Availability and implementation: The methods described in this article are made available as a Python package (LGPL license) and also accessible through a web interface. For more information, see https://team.inria.fr/ibis/wellinverter.Contact: Hidde.de-Jong@inria.frSupplementary information: Supplementary data are available at Bioinformatics online.

Fluorescent polymeric aggregates induced by Eu3+ ions and their surface morphologies

A novel method to prepare fluorescent polymeric aggregates via Eu3+-induced self-assembly is described in this research contribution. The amphiphilic polymeric ligand, polystyrene-block-poly(N-vinylpyrrolidone) (PS-b-PNVP) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. Eu3+ ions that function as coordination crosslinking agents were added to the reactive solution to induce self-assembly within the PNVP block. Eu3+ ions preferentially coordinate to PNVP segments, yielding coordinatively cross-linked networks with reduced solubility in the solvent. Polystyrene segments retain their solubility in solution due to the amphiphilic character of the block copolymer. Mechanisms for self-assembly and reversible network formation are discussed. Due to the unique optical properties of Eu3+ ions, in-depth analysis of fluorescent data was performed. These aggregates were drip-coated onto the surface of silicon wafers, with subsequent investigation of polymer morphology near the interface with silicon. This represents the initial step in the development of opto-electronic and photovoltaic applications for these self-assembled fluorescent polymeric aggregates.

Time-resolved fluorescence methods (IUPAC Technical Report)

Abstract This IUPAC Technical Report describes and compares the currently applied methods for measuring and analyzing time-resolved fluorescence traces using phase-modulation fluorometry as well as pulse fluorometry (direct emission decay measurements, single-photon timing, streak camera measurements, fluorescence upconversion, and optical Kerr gating). The paper starts with a brief description of the basic principles for time and frequency domain fluorescence spectroscopy. The fundamental equations are given, and recommendations for adequate use are emphasized. The up-to-date, commonly employed excitation sources and photodetectors are described in detail. The analysis of time-resolved fluorescence data is discussed. Attention is paid to possible artifacts, and remedies are presented on how to avoid them or to account for them. Finally, fluorescence lifetime standards for the nanosecond and picosecond timescales are collected.

Soluble cyanine dye/β-cyclodextrin derivatives: Potential carriers for drug delivery and optical imaging

Two soluble cyanine/β-cyclodextrin derivatives have been synthesized under simultaneous ultrasound/microwave irradiation. UV–Vis, steady-state, time-resolved fluorescence and circular dichroism spectroscopies were used to evaluate their photophysical properties, as well as to study their complexation with the anticancer drug doxorubicin. Titration experiments were performed by monitoring corrected emission intensity. The analysis of fluorescence data provided stability constants for doxorubicin complexes with cyanine/β-cyclodextrins which are 4 orders of magnitude greater than those reported for its complexation with native β-cyclodextrin and one order greater than its association with DNA. The complexation has also been studied using Molecular Mechanics and Molecular Dynamics simulations. Both electrostatic and van de Waals binding energy contributions are important to system stabilization. The potential use of these systems as carriers has been evaluated via in vitro experiments on HeLa cells and by monitoring cell entrance via confocal laser scanning microscopy.

Phasor approaches simplify the analysis of tryptophan fluorescence data in protein denaturation studies

The intrinsic fluorescence of tryptophan is frequently used to investigate the structure of proteins. The analysis of tryptophan fluorescence data is challenging: fluorescence (anisotropy) decays typically have multiple lifetime (correlation time) components and fluorescence spectra are broad and exhibit only minor shifts. In this work, we show that phasor approaches can substantially simplify tryptophan fluorescence analysis. To demonstrate this, we re-analyse previously recorded datasets of the denaturant (guanidinium hydrochloride, GuHCl) induced unfolding of a single-tryptophan-containing variant of apoflavodoxin from Azotobacter vinelandii. For three methods—(1) time-resolved fluorescence, (2) time-resolved fluorescence anisotropy and (3) steady-state fluorescence spectroscopy—we show that the phasor analysis can readily identify the presence of a folding intermediate. Moreover, the fractional contributions of protein states at various stages of unfolding and the values of the free energy difference of the unfolding process are obtained. The outcomes are compared to the global analysis results published previously.

Phasor approaches simplify the analysis of tryptophan fluorescence data in protein denaturation studies

Phasor approaches simplify the analysis of tryptophan fluorescence data in protein denaturation studies

Studies of the interaction between FNC and human hemoglobin: A spectroscopic analysis and molecular docking

FNC (2′-deoxy-2′-bfluoro-4′-azidocytidine) is a novel nucleoside analogue with pharmacologic effects on several human diseases. In this work, the binding of FNC to human hemoglobin (HHb) have been investigated by absorption spectroscopy, fluorescence quenching technique, synchronous fluorescence, three-dimensional fluorescence and molecular modeling methods. Analysis of fluorescence data showed that the binding of FNC to HHb occurred via a static quenching mechanism. Thermodynamic analysis and molecular modeling suggest that hydrogen bond and van der Waals force are the mainly binding force in the binding of FNC to HHb.

Spectroscopic investigation on the interaction of some polymer–cobalt(III) complexes with serum albumins

The interaction between the polymer–cobalt(III) complexes, [Co(dien)(phen)BPEI]Br3·2H2O and [Co(phen)2(BPEI)Cl]Cl2·4H2O (where phen is 1,10- phenanthroline, dien is diethylenetriamine, BPEI is branched polyethyleneimine) and serum albumins is studied using steady state fluorescence, lifetime measurement and circular dichroism spectroscopy techniques under physiological condition. The analysis of fluorescence data indicates the presence of static quenching mechanism in the binding process. Various binding parameterss including thermodynamic parameters ΔH°, ΔS° and ΔG° have been evaluated. The quantitative analysis of CD spectra reveals information on the changes in the content of the α-helix of the HSA/BSA upon binding.

Advanced Multi-Color Fluorescence Imaging System for Detection of Biotic and Abiotic Stresses in Leaves

The autofluorescence of a sample is a highly sensitive and selective optical property and gives the possibility to establish non-destructive techniques of the investigation of plants, like detecting the chlorophyll fluorescence related to stress phenomena. In this study, an advanced multi-color fluorescence imaging system and data analysis were presented. The advantage of an imaging system is the additional receiving of spatial information over a sample area, this is a strong improvement compared to spot measurements commonly used. The purpose was to demonstrate the possibility of the detection and characterization of stress symptoms using this system. Specific fluorescence ratios were identified to characterize the stress status over the whole leaf, here shown on barley grown under different nitrogen supply (abiotic stress). Due to the changes, it is possible to make conclusions about leaf pigments (chlorophylls and phenolics) related to stress response. The second aim was to use the shape of local symptoms (biotic stress) as a criterion. For this purpose, three structural different kinds of fungal symptoms were analyzed using shape descriptors. It shows that an additional image shape analysis can be very useful for extracting further information, in this case the successful discrimination of fungal infections.

Multi-Dimensional Analysis of Fluorescence Fluctuation Spectroscopy of Lipid Anchored Proteins in Live Cells Reveal Complex Organization in the Plasma Membrane

Membrane organization of lipid anchored proteins plays a fundamental role in proper cell signaling behavior. Several important signaling proteins, such as members of the Src family of kinases, are anchored to the membrane by covalent acyl, alkyl, and glycosylphosphatidylinositol (GPI) moieties. A convenient approach to studying this lipid anchor organization in cells is to image genetically encoded lipidation motifs of various signaling proteins fused to a fluorescent protein. However, previous efforts to reduce this lateral organization to a simple model often ignore the complexity inherent in cell membranes and have contributed to an inconsistent picture of membrane organization. We use two-color fluorescence fluctuation spectroscopy to study the spatial organization of fluorescently tagged lipid anchors in live cell membranes. In order to retain the complexity of this organization, we simultaneously observe several modes of lipid anchor dynamics by employing a multi-dimensional analysis of the time-resolved fluorescence data. Time-correlated single photon counting (TCSPC) data is gathered on a 3-detector system optimized for simultaneous polarization and two-color fluorescence correlation spectroscopy (FCS). The degree of dynamic colocalization of eGFP- and mCherry-fused lipid anchors can be quantified by pulse-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS), while single color FCS evaluates the translational mobility of the lipid anchors. The photon counting method also allows for time-resolved fluorescence anisotropy (TRFA) measurements, evaluating the rotational diffusion of the fluorescent protein, as well as fluorescence lifetime measurements to determine energy transfer. Taken together, this simultaneous multi-dimensional analysis of time-resolved fluorescence data offers a powerful approach towards determining the complex organizations of lipid anchors in live cell membranes.

Probing the Site-Selective Binding of an Antiretroviral Drug, Stavudine to Calf Thymus Dna†

The interaction of an anti-HIV drug, stavudine (STV) with calf thymus deoxyribonucleic acid (DNA) was investigated employing acridine orange (AO) as a fluorescence probe. Spectroscopic investigations revealed the intercalative mode of binding of STV to DNA. The analysis of fluorescence data indicated the presence of static quenching mechanism between STV and DNA. Thermodynamic parameters indicated the presence of van der Waals forces in addition to intercalative mode of binding. CD data revealed the partial B→A conformational transition of DNA upon intercalative mode of binding with STV.

Single-Molecule Analysis of Cytochrome c Folding by Monitoring the Lifetime of an Attached Fluorescent Probe

Conformational dynamics of proteins are important for function. However, obtaining information about specific conformations is difficult for samples displaying heterogeneity. Here, time-resolved fluorescence resonance energy transfer is used to characterize the folding of single cytochrome c molecules. In particular, measurements of the fluorescence lifetimes of individual cytochrome c molecules labeled with a single dye that is quenched by energy transfer to the heme were used to monitor conformational transitions of the protein under partially denaturing conditions. These studies indicate significantly more conformational heterogeneity than has been described previously. Importantly, the use of a purified singly-labeled sample made a direct comparison to ensemble data possible. The distribution of lifetimes of single-proteins was compared to the distribution of lifetimes determined from analysis of ensemble lifetime fluorescence data. The results show broad agreement between single-molecule and ensemble data, with a similar range of lifetimes. However, the single-molecule data reveal greater conformational heterogeneity.

Analysis of steady-state Förster resonance energy transfer data by avoiding pitfalls: Interaction of JAK2 tyrosine kinase with N-methylanthraniloyl nucleotides

Förster resonance energy transfer (FRET) between the fluorescent ATP analogue 2′/3′-(N-methyl-anthraniloyl)-adenosine-5′-triphosphate (MANT–ATP) and enzymes is widely used to determine affinities for ATP–protein binding. However, in analysis of FRET fluorescence data, several important parameters are often ignored, resulting in poor accuracy of the calculated dissociation constant (Kd). In this study, we systematically analyze factors that interfere with Kd determination and describe methods for correction of primary and secondary inner filter effects that extend the use of the FRET method to higher MANT nucleotide concentrations. The interactions of the fluorescent nucleotide analogues MANT–ATP, MANT–ADP [2′/3′-O-(N-methylanthraniloyl) adenosine diphosphate], and MANT–AMP [2′/3′-O-(N-methylanthraniloyl) adenosine monophosphate] with the JAK2 tyrosine kinase domain are characterized. Taking all interfering factors into consideration, we found that JAK2 binds MANT–ATP tightly with a Kd of 15 to 25nM and excluded the presence of a second binding site. The affinity for MANT–ADP is also tight with a Kd of 50 to 80nM, whereas MANT–AMP does not bind. Titrations of JAK2 JH1 with nonhydrolyzable ATP analogue MANT–ATP-γ-S [2′/3′-O-(N-methylanthraniloyl) adenosine-5′-(thio)- triphosphate] yielded a Kd of 30 to 50nM. The methods demonstrated here are applicable to other enzyme–fluorophore combinations and are expected to help improve the analysis of steady-state FRET data in MANT nucleotide binding studies and to obtain more accurate results for the affinities of nucleotide binding proteins.

Application of the wavelet transform for feature extraction in the analysis of hyperspectral laser-induced fluorescence data

Remote-sensing approaches for environmental protection and exploration have evolved rapidly in the last decade. Among the new operational tools, hyperspectral Fluorescent LiDAR System (FLS®) lidar has demonstrated a high sensitivity and the ability to function in complex environments for real-time, robust oil-spill monitoring on airborne or ship-borne analytical platforms. The capabilities of such analytical platforms include real-time analysis of laser-induced fluorescence (LIF) data. Although numerous examples of the application of signal theory to the analysis of hyperspectral data appear in the remote-sensing literature, the conventional data analysis strategies are not well adapted to the practical issues of the LIF applications. The aim of this article is to provide a new approach for LIF lidar analytical platforms, which is focused on the specifics of hyperspectral LIF data. The approach is based on structural data analysis and interpretation, through which more detailed spectral matching is performed. This article is based on a simulated experiment in which the spectra of actual seawater and well-known types of petroleum products were combined to demonstrate the wavelet-transform-based analysis of LIF data. The final part of the article demonstrates the application of the wavelet transform to the structural analysis of LIF data from field experiments for the detection and identification of oil products in difficult environmental conditions.

Pyrazinoporphyrazines with Externally Appended Pyridine Rings. 13. Structure, UV–Visible Spectral Features, and Noncovalent Interaction with DNA of a Positively Charged Binuclear (ZnII/PtII) Macrocycle with Multimodal Anticancer Potentialities

We investigated with spectroscopic techniques the noncovalent interaction of a bimetallic water-soluble (Zn(II)/Pt(II)) porphyrazine hexacation, [(PtCl(2))(CH(3))(6)LZn](6+), and its octacationic analogue [(CH(3))(8)LZn](8+), lacking the cis-platin-like functionality, with a 21-mer double strand (ds) 5'-d[GGG(TTAGGG)(3)]-3'/3'-d[CCC(AATCCC)(3)]-5', as model for B-DNA. Both hexacation and octacation tend to aggregate in water. The structure as well as the ground and excited-state electronic properties of the Zn(II)/Pt(II) hexacation [(PtCl(2))(CH(3))(6)LZn](6+) in water solution were investigated using density functional theory (DFT) and time-dependent DFT (TDDFT) methods. TDDFT calculations of the lowest excited states of [(PtCl(2))(CH(3))(6)LZn](6+) in water provided an accurate description of the Q-band spectral region. In particular, the calculated optical spectra were in agreement with the experimental ones, obtained in the presence of micelles favoring complete disruption of the aggregates. The model for dsDNA binding that emerges from the analysis of UV-vis absorption and time-resolved fluorescence data shows the presence of complexes of 1 dsDNA molecule with 1, 2, and 4 macrocycles. Comparing the results for the hexacation [(PtCl(2))(CH(3))(6)LZn](6+) with those for the [(CH(3))(8)LZn](8+)octacation, we observed a higher degree of monomerization for the [(PtCl(2))(CH(3))(6)LZn](6+) derivative.

Monitoring of harmful algal blooms in the era of diminishing resources: A case study of the U.S. West Coast

Spatial and temporal decorrelation scales in phytoplankton bloom magnitudes are reviewed with the goal of informing the design of efficient and informative observing networks for monitoring of potentially harmful algal blooms (HABs) along the U.S. West Coast. Our analysis of historic MODIS Fluorescent Line Height data shows that, unlike several previous studies, seasonal timing of phytoplankton blooms off the U.S. West Coast propagates from South to North. In situ data show that temporal decorrelation scales are shortest off Northern California (∼4 days) and longest in the Southern California Bight (∼17 days). In a cross-shore direction, we find that variability in the near-shore biomass is decoupled from biomass further offshore (∼2–4km).Our review of the cloud cover gap statistics suggests that satellite data provide reasonably inexpensive information about bloom events, particularly at seasonal to inter-annual scales, but is insufficient to capture many event-scale blooms. Absent adequate satellite data, in situ monitoring becomes essential. Existing networks of automated monitoring sites from piers and moorings off the California coast is insufficient to describe regional variability in blooms, but is likely informative of bloom magnitudes in the immediate proximity of observation stations. We suggest that a more effective network will have a combination of shore-based stations and a few (5–10) offshore moorings. Shore-based stations should be located in proximity to sensitive HAB targets. This would provide monitoring of existing conditions and guide decision-making about beach closures and aquaculture management practices. Offshore stations can serve as proxy for regional conditions and can be used to issue early warnings of potential HAB conditions developing in a specific region.

Comparison of fluorescence spectroscopy and FTIR in differentiation of plant pollens

Spectroscopic techniques are under investigation on possibility of differentiation of airborne particles. This paper describes pollen discrimination among others bio-particles in laboratory conditions. Pollen samples were characterized with UV–Vis fluorescence, drift and KBr pellet techniques of infrared spectroscopy. Principal Component Analysis of UV–Vis fluorescence and FTIR spectra revealed that pollens can be distinguished from other bio-materials with use of these methods. Both methods resulted in similar classification capability. Combined FTIR and fluorescence data analysis did not improve the discrimination between pollen allergens and other airborne biological materials.

PARAFAC analysis of front-face fluorescence data: Absorption and scattering effects assessed by means of Monte Carlo simulations

Three-way fluorescence data originating from mixtures of fluorophores embedded in turbid media such as biological media get strongly modulated by wavelength dependent absorption and scattering phenomena. Thus the consistent resolution and quantitative determination of the mixture becomes a difficult task. In this study two chemometric methodologies frequently used to deal with this type of data were applied to fluorescence simulated data sets qualitatively similar to those measured in biological samples: Parallel Factor Analysis (PARAFAC) that does require the fulfillment of trilinearity, and multivariate curve resolution‐alternating least squares (MCR‐ALS) which decomposes the data according to a model lacking this structure. Monte Carlo simulations were used to simulate fluorescence excitation–emission matrices (EEMs) of known fluorescent mixtures under separated and simultaneous variations of the absorption parameter μa and the scattering parameter μs. PARAFAC and constrained MCR-ALS models were then fitted to the simulated data. Both algorithms failed the recover the true profiles. The results obtained with PARAFAC and MCR-ALS models are similar and the recovered profiles exhibit severe distortions due to the absorption and scattering effects. Finally, qualitative and quantitative effects of the absorption and scattering on the fluorescence data were assessed and discussed.

Molecular binding of self-assembling peptide EAK16-II with anticancer agent EPT and its implication in cancer cell inhibition

The current drug delivery techniques involve encapsulation, targeting and controlled release of the drug with various molecules or nanoparticles, but rarely has the drug molecular state or form been investigated. It is necessary to deliver a drug with a prescribed molecular state in order to maximize drug therapeutic effects. Here we present two facile methods to characterize molecular states of the anticancer drug ellipticine (EPT) encapsulated in the self-assembling peptide EAK, and relate the different molecular states of EPT to their respective cancer inhibition efficacies. The first method is UV-based, where drug loading capacity of a particular molecular state was determined. The experimental data corroborated a molecular binding model, where peptide–drug interaction was assumed to be electrostatic in nature. The developed model could elucidate a unique pH effect on protonated EPT loading capacity. The second method is based on fluorescence characteristics of EPT, which could differentiate the two molecular states: protonated and crystalline of EPT in situ. The inner filter effect was, however, found with this method, presenting an ineluctable obstacle in quantitative analysis of fluorescence data. A correction method for the inner filter effect was thus developed. With this approach, concentrations of EPT at different molecular states in its peptide complex solutions were determined. In vitro cytotoxicity assay was applied to evaluate the efficacy of the two molecular states of EPT, showing that protonated EPT was more efficient at killing cancer cells than crystalline EPT. The molecular binding model and two characterization methods for EAK–EPT complexation could be extended to other carrier-drug systems.

A novel method for fast and robust estimation of fluorescence decay dynamics using constrained least-squares deconvolution with Laguerre expansion

We report a novel method for estimating fluorescence impulse response function (fIRF) from noise-corrupted time-domain fluorescence measurements of biological tissue. This method is based on the use of high-order Laguerre basis functions and a constrained least-squares approach that addresses the problem of overfitting due to increased model complexity. The new method was extensively evaluated on fluorescence data from simulation, fluorescent standard dyes, ex vivo tissue samples of atherosclerotic plaques and in vivo oral carcinoma. Current results demonstrate that this method allows for rapid and accurate deconvolution of multiple channel fluorescence decays without adaptively adjusting the Laguerre scale parameter. The appropriate choice of the scale parameter is essential for accurate estimation of the fIRF. The method described here is anticipated to play an important role in the development of computational techniques for real-time analysis of time-resolved fluorescence data from biological tissues and to support the advancement of fluorescence lifetime instrumentation for biomedical diagnostics by providing a means for on-line robust analysis of fluorescence decay.