Measured Fluorescence Signals Research Articles

Enhancement in the Sensitivity of Microfluidic Enzyme-Linked Immunosorbent Assays through Analyte Preconcentration

In this Article, we describe a microfluidic enzyme-linked immunosorbent assay (ELISA) method whose sensitivity can be substantially enhanced through preconcentration of the target analyte around a semipermeable membrane. The reported preconcentration has been accomplished in our current work via electrokinetic means allowing a significant increase in the amount of captured analyte relative to nonspecific binding in the trapping/detection zone. Upon introduction of an enzyme substrate into this region, the rate of generation of the ELISA reaction product (resorufin) was observed to increase by over a factor of 200 for the sample and 2 for the corresponding blank compared to similar assays without analyte trapping. Interestingly, in spite of nonuniformities in the amount of captured analyte along the surface of our analysis channel, the measured fluorescence signal in the preconcentration zone increased linearly with time over an enzyme reaction period of 30 min and at a rate that was proportional to the analyte concentration in the bulk sample. In our current study, the reported technique has been shown to reduce the smallest detectable concentration of the tumor marker CA 19-9 and Blue Tongue Viral antibody by over 2 orders of magnitude compared to immunoassays without analyte preconcentration. When compared to microwell based ELISAs, the reported microfluidic approach not only yielded a similar improvement in the smallest detectable analyte concentration but also reduced the sample consumption in the assay by a factor of 20 (5 μL versus 100 μL).

The statistics of protein expression ratios for cellular fluorescence studies

Fluorescence studies of cellular protein-protein interactions commonly employ transient cotransfection to express two proteins carrying distinct fluorescent labels. Because transiently transfected cells differ significantly in their expression level, the concentration ratio of the two expressed proteins varies, which in turn influences the measured fluorescence signal. Knowledge of the statistics of protein expression ratios is of considerable interest both from a fundamental point of view and for cellular fluorescence studies. Despite the perceived randomness of transient transfection, we were able to develop a quantitative model that describes the average and distribution of the protein expression ratio from a cell population. We show that the expression ratio is proportional to the molar plasmid ratio and relate the distribution to the finite number of active plasmids in the cell. The process of cationic lipid-mediated transfection is explored in more detail. Specifically, the influence of lipoplexes on the statistics of the expression ratio is examined. We further demonstrate that the transfection model provides a quantitative description of fluorescence fluctuation experiments, where only a fraction of the proteins are labeled.

Quantifying Ternary Protein Expression Fractions using Three-Channel Fluorescence Fluctuation Spectroscopy

Fluorescence studies of cellular protein-protein interactions commonly employ transient transfection to express proteins carrying distinct fluorescent labels. Because transiently transfected cells differ significantly in their expression level, the concentration ratio of the expressed proteins varies, which in turn influences the measured fluorescence signal. Knowledge of the statistics of protein expression ratios, which currently is not well understood, is of considerable interest both from a fundamental point of view and for cellular fluorescence studies. Despite the perceived randomness of transient transfection, we recently succeeded in developing a model that describes the distribution and the average of the protein expression ratio for the coexpression of two proteins. Here we extend our previous work to the simultaneous coexpression of three proteins, where each protein species is labeled with a distinctly colored fluorescent protein. All protein species are simultaneously excited using two-photon absorption. The fluorescence of the protein mixture is split according to color into three detection channels. Brightness measurements of the individual proteins combined with intensity ratio data between detection channels is used to identify the protein expression ratios of all three proteins. We examine the relationship between DNA-plasmid fractions and protein expression fractions. In addition, we quantify the cell-to-cell scatter in the protein expression ratios for different transfection protocols in order to identify the best conditions for three-color fluorescence fluctuation experiments. This work was supported by grants from the National Institutes of Health (GM64589) and the National Science Foundation (PHY 0346782).

Portable system for measuring fluorescence signals of biological tissue

In this issue the development of a portable system for measuring fluorescence and reflectance signals of biological tissue is presented. It includes a mini-spectrometer HR4000CG-UV-NIR, with a working range from 200 to 1100 nm and a resolution of 0.5 nm. A light emitting diode at 365 nm with maximum radiant power of 230 mW was used as an excitaton source. A bifurcated fiber optic with six 400 um diameter irradiation fibers and one 400 um collection fiber is used. The system is powered and controlled by a laptop, avoiding the use of an external power supply. A program to control the light source and to measure the spectra in real time was developed using LabVIEW. Preliminary results of ex vivo and in vivo fluorescence measurements of skin and brain tissues are presented and show that fluorescence and reflection could be used to diagnose and demarcat cancer boundaries.

Depth dependence of vascular fluorescence imaging

In vivo surface imaging of fluorescently labeled vasculature has become a widely used tool for functional brain imaging studies. Techniques such as phosphorescence quenching for oxygen tension measurements and indocyanine green fluorescence for vessel perfusion monitoring rely on surface measurements of vascular fluorescence. However, the depth dependence of the measured fluorescence signals has not been modeled in great detail. In this paper, we investigate the depth dependence of the measured signals using a three-dimensional Monte Carlo model combined with high resolution vascular anatomy. We found that a bulk-vascularization assumption to modeling the depth dependence of the signal does not provide an accurate picture of penetration depth of the collected fluorescence signal in most cases. Instead the physical distribution of microvasculature, the degree of absorption difference between extravascular and intravascular space, and the overall difference in absorption at the excitation and emission wavelengths must be taken into account to determine the depth penetration of the fluorescence signal. Additionally, we found that using targeted illumination can provide for superior surface vessel sensitivity over wide-field illumination, with small area detection offering an even greater amount of sensitivity to surface vasculature. Depth sensitivity can be enhanced by either increasing the detector area or increasing the illumination area. Finally, we see that excitation wavelength and vessel size can affect intra-vessel sampling distribution, as well as the amount of signal that originates from inside the vessel under targeted illumination conditions.

Magnetic microparticle-based multiplexed DNA detection with biobarcoded quantum dot probes

We have developed a new analytical method to detect multiple DNA simultaneously based on the biobarcoded CdSe/ZnS quantum dot (QD) and magnetic microparticle (MMP). It was demonstrated by using oligonucleotide sequences of 64 bases associated with human papillomavirus 16 and 18 L1 genes (HPV-16 and HPV-18) as model systems. This analytical system involves three types of probes, a MMP probe and two streptavidin-modified QD probes. The MMPs are functionalized with HPV-16 and HPV-18 captures DNA to form MMP probes. The QDs are conjugated with HPV-16 or HPV-18 probe DNA along with FAM- or Rox-labeled random DNA to form HPV-16 and HPV-18 QD probes, respectively. A one-step hybridization reaction was performed by mixing the MMP probes, HPV-16 and HPV-18 target DNA (T-16 and T-18), HPV-16 and HPV-18 QD probes. Afterwards, the hybrid-conjugated microparticles were separated by a magnet and heated to remove the MMPs. Finally, the detections of T-16 and T-18 were done by measuring fluorescence signals of FAM and Rox, respectively. Under the optimum conditions, the fluorescence intensity exhibited a good linear dependence on target DNA concentration in the range from 8 × 10 −11 to 8 × 10 −9 M. The detection limit of T-16 is up to 7 × 10 −11 M (3 σ), and that of T-18 is 6 × 10 −11 M. Compared with other biobarcode assay methods, the proposed method that QDs were used as the solid support has some advantages including shorter preparation time of QD probes, faster binding kinetics and shorter analytical time. Besides, it is simple and accurate.

Signal/Noise Analysis of FRET-Based Sensors

Molecular sensors based on intramolecular Förster resonance energy transfer (FRET) have become versatile tools to monitor regulatory molecules in living tissue. However, their use is often compromised by low signal strength and excessive noise. We analyzed signal/noise (SNR) aspects of spectral FRET analysis methods, with the following conclusions: The most commonly used method (measurement of the emission ratio after a single short wavelength excitation) is optimal in terms of signal/noise, if only relative changes of this uncalibrated ratio are of interest. In the case that quantitative data on FRET efficiencies are required, these can be calculated from the emission ratio and some calibration parameters, but at reduced SNR. Lux-FRET, a recently described method for spectral analysis of FRET data, allows one to do so in three different ways, each based on a ratio of two out of three measured fluorescence signals (the donor and acceptor signal during a short-wavelength excitation and the acceptor signal during long wavelength excitation). Lux-FRET also allows for calculation of the total abundance of donor and acceptor fluorophores. The SNR for all these quantities is lower than that of the plain emission ratio due to unfavorable error propagation. However, if ligand concentration is calculated either from lux-FRET values or else, after its calibration, from the emission ratio, SNR for both analysis modes is very similar. Likewise, SNR values are similar, if the noise of these quantities is related to the expected dynamic range. We demonstrate these relationships based on data from an Epac-based cAMP sensor and discuss how the SNR changes with the FRET efficiency and the number of photons collected.

Assessment of Fluorescence Resonance Energy Transfer for Two-Color DNA Microarray Platforms

Two-color DNA microarray platforms are widely used for determining differential amounts of target sequences in parallel between sample pairs. However, the fluorescence (or Forster) resonance energy transfer (FRET) between two fluorophores can potentially result in the distortions of the measured fluorescence signals. Here we assessed the influence of FRET on the two-color DNA microarray platform and developed a reliable and convenient method for the correction of FRET distortion. Compared to current methods of normalization based on the statistical analysis and the hypothesis that only a small part of target sequences are differentially presented between sample pairs, our FRET correction method can recover the undistorted signals by the compensation of fluorescence emission, without considering the number of target sequences differentially presented. The correction method was validated with samples at different target ratios and with microarrays spotted in different probe concentrations. We also applied the FRET correction method to gene expression profiling arrays, and the results show that FRET was present when the content of target sequence was beyond a threshold amount and that the process incorporating our FRET correction method can improve the reliability of the gene expression profiling microarray platform in comparison with the current process without FRET correction.

A new spectrometer for grazing incidence X-ray fluorescence for the characterization of Arsenic implants and Hf based high-k layers

Grazing Incidence X-ray Fluorescence Analysis (GIXRF) is a powerful technique for depth-profiling and characterization of thin layers in depths up to a few hundred nanometers. By measurement of fluorescence signals at various incidence angles Grazing Incidence X-ray Fluorescence Analysis provides information on depth distribution and total dose of the elements in the layers. The technique is very sensitive even in depths of a few nanometers. As Grazing Incidence X-ray Fluorescence Analysis does not provide unambigous depth profile information and needs a realistic input depth profile for fitting, in the context of the EC funded European Integrated Activity of Excellence and Networking for Nano and Micro-Electronics Analysis (ANNA) Grazing Incidence X-ray Fluorescence Analysis is used as a complementary technique to Secondary Ion Mass Spectrometry (SIMS) for the characterization of Ultra Shallow Junctions (USJ). A measuring chamber was designed, constructed and tested to meet the requirements of Grazing Incidence X-ray Fluorescence Analysis. A measurement protocol was developed and tested. Some results for As implants as well as Hf based high k layers on Silicon are shown. For the determination of the bulk As content of the wafers, Instrumental Neutron Activation Analysis has also been applied for comparison.

Measurement of the hydroxyl radical formation from H 2O 2, NO 3−, and Fe(III) using a continuous flow injection analysis

Production of hydroxyl radical ( OH) is of significant concern in engineered and natural environment. A simple in situ method was developed to measure OH formation in UV/H 2O 2, UV/Fe(III), and UV/NO 3 − systems using trapping of OH by benzoic acid (BA) and measuring fluorescence signals from hydroxylated products of BA. Method development included characterization of OH trapping mechanism and measurement of quantum yields ( Φ OH ) for OH. The distribution of OHBA isomers was in the order of o-OHBA > p-OHBA > m-OHBA, although it changed with the H 2O 2 concentration and light intensity. This supports that OH attacks dominantly on the benzene rings. The quantum yields for OH formation in the UV/H 2O 2 process were 1.02 and 0.59 at 254 and 313 nm, which were in good agreement with the literature values, confirming that the method is suitable for the measurement of OH production from UV/H 2O 2 processes. Using the continuous flow method developed, quantum yields for OH in UV/H 2O 2, UV/Fe(III), and UV/NO 3 − systems were measured varying the initial concentration of OH precursors. The Φ OH values increased with increasing concentrations of H 2O 2, Fe(III), and NO 3 − and approached constant values as the concentration increased. The Φ OH values were 0.009 for H 2O 2 at 365 nm, showing that OH production is not negligible at such high wavelength. The Φ OH values during the photolysis of Fe(OH) 2+ (pH 3.0) and Fe(OH) 2 + (pH 6.0) at 254 nm were 0.34 and 0.037, respectively. The Φ OH values for NO 3 − approached a constant value of 0.045 at 254 nm at the initial concentration of 10 mM.

Concentration dependence of fluorescence signal in a microfluidic fluorescence detector

Fluorescence detection is one of the most widely used techniques for measuring analyte concentration in biological applications. Although used frequently with bulk systems, there are important considerations in using this approach in thin (<100 μm) microfluidic lab-on-a-chip (LOC) systems in relating the measured fluorescence signal to analyte concentration. In this work, a general relationship between the photoresponse of an on-chip organic photodetector (OPD) and concentration of a fluorescent dye Rhodamine 6G in a microfluidic chip is presented. The developed theoretical model for photoresponse matches well to the extreme sub-linearity observed in measurements in a dilution series from 10 nM to 1 mM. The influence of the system factors, such as noise, detector sensitivity, and optical power were also analyzed to indicate design changes to improve the system performance and limits of detection even further.

Feasibility of Monte CarlO simulations in Quantitative Tissue Imaging

Purpose The feasibility of Monte Carlo simulations as a tool to facilitate quantitative image analysis is investigated by means of simulating light transport in skin phantoms. Methods A Monte Carlo tool is used to compare if simulated fluorescent signals show agreement with measured data. The lipophilic fluorescent probe Nile Red and dedicated skin phantoms are also used in simulations to investigate the influence of the optical properties of the skin on the signal. Results It is shown that the simulated and measured fluorescence signals show linear behavior up to a certain concentration of Nile Red. The simulations of the skin phantoms show the varying influence of single skin layers on the fluorescence signal. A calibration factor for quantitative analysis can be determined for the different skin layers. Conclusion Characterizing the influence of different media on imaging signals is a primary task in developing quantitative analysis methods. Monte Carlo simulations are a useful tool to investigate imaging properties of biological specimen where quantifying signals is important. However, detailed models must be provided.

Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence

A time-resolved spectroscopic imaging system is built to study the fluorescence characteristics of nicotinamide adenine dinucleotide (NADH), an important metabolic coenzyme and endogenous fluorophore in cells. The system provides a unique approach to measure fluorescence signals in different cellular organelles and cytoplasm. The ratios of free over protein-bound NADH signals in cytosol and nucleus are slightly higher than those in mitochondria. The mitochondrial fluorescence contributes about 70% of overall cellular fluorescence and is not a completely dominant signal. Furthermore, NADH signals in mitochondria, cytosol, and the nucleus respond to the changes of cellular activity differently, suggesting that cytosolic and nuclear fluorescence may complicate the well-known relationship between mitochondrial fluorescence and cellular metabolism.

Biosensors based on surface plasmon-enhanced fluorescence spectroscopy (Review)

The implementation of surface plasmon-enhanced fluorescence spectroscopy (SPFS) to surface plasmon resonance (SPR) biosensors enables increasing their sensitivity by several orders of magnitude. In SPR-based biosensors, surface plasmons probe the binding of target molecules contained in a liquid sample by their affinity partners attached to a metallic sensor surface. SPR biosensors relying on the detection of refractive index changes allow for direct observation of the binding of large and medium size molecules that produces sufficiently large refractive index changes. In SPR biosensors exploiting SPFS, the capture of fluorophore-labeled molecules to the sensor surface is observed by the detection of fluorescence light emitted from the surface. This technique takes advantage of the enhanced intensity of electromagnetic field accompanied with the resonant excitation of surface plasmons. The interaction with surface plasmons can greatly increase the measured fluorescence signal through enhancing the excitation rate of fluorophores and by more efficient collecting of fluorescence light. SPFS-based biosensors were shown to enable the analysis of samples with extremely low analyte concentrations and the detection of small molecules. In this review, we describe the fundamental principles, implementations, and current state of the art applications of SPFS biosensors. This review focuses on SPFS-based biosensors employing the excitation of surface plasmons on continuous metal-dielectric interfaces.

Composition determination for complex and transmitting samples in x‐ray quantitative analysis

AbstractIn this work, quantitative analysis of x‐ray fluorescence measurements of transmitting samples with complex chemical composition is considered. A method is presented for analytical solution of sample composition including matrix effects, independent of sample thickness and requiring no standards. The method uses fundamental parameters and measured fluorescence signal intensities, and is applicable to transmission geometry measurements for which standard analyses are not applicable. Limitations of the analysis presented here are discussed. Copyright © 2008 John Wiley &amp; Sons, Ltd.

A novel homogeneous bioluminescence resonance energy transfer element for biomolecular detection with CCD camera or CMOS device

A novel optical signal element based on homogeneous bioluminescence resonance energy transfer (BRET) was developed for biomolecular detection. A fluorescent dye and alkaline phosphatase (AP) conjugate was used as a reporter and light-generation element for imaging detection platforms that use a CCD camera or CMOS chip-based devices. In the presence of a luminescence substrate, the energy from the first light emission of a bioluminescence enzymatic reaction was transferred to fluorescent dyes which were conjugated to an enzyme. This resulted in a second light emission with a shorter wavelength. The second light was localized at the position of target molecules without the diffusion problems present in current technology. To optimize energy transfer efficiency, the ratio of enzyme to fluorophore in the conjugates, the fluorescent dyes used in the conjugates and the luminescence substrates used for BRET were investigated. BRET was demonstrated by using both a CCD camera and a CMOS imaging device. Image spatial resolution was greatly improved compared with conventional chemiluminescence detection. This new signal element opens a door for the direct measurement of fluorescent signals on an imaging chip without an external light source and portable instrumentation normally required for the fluorescent detection of biomolecules.

Fluorescence intensity is a poor predictor of saturation effects in two‐photon microscopy: Artifacts in fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) has become an increasingly important measurement tool for biological and biomedical investigations, with the capability to assay molecular dynamics and interactions both in vitro and within living cells. Information recovery in FCS requires an accurate characterization and calibration of the observation volume. A number of recent reports have demonstrated that the calibration of the observation volume is excitation power dependent, a complication that arises due to excitation saturation. While quantitative models are now available to account for these volume variations, many researchers attempt to avoid saturation issues by working with low nonsaturating excitation intensities. For two-photon excited fluorescence, this is typically thought to be achievable by working with excitation powers for which the total measured fluorescence signal maintains its quadratic dependence on excitation intensity. We demonstrate that observing only the power dependence of the fluorescence intensity will tend to underestimate the importance of saturation, and explain these findings in terms of basic physical models.

3-Pentanone LIF at elevated temperatures and pressures: measurements and modeling

The objective of this study is to investigate 3-pentanone fluorescence experiments in a constant volume vessel at high temperature and high pressure to underline the influent parameters in conditions close to those encountered in internal combustion engines. To obtain quantitative analysis, measured fluorescence signals must be corrected by considering the influence of preponderant parameters such as temperature, pressure and gas composition. Quantitative dependences of fluorescence on thermodynamic parameters are measured and compared with the predictions of a photophysical model, which combines the effects of temperature, pressure, excitation wavelength on fluorescence quantum yield. The increase of 3-pentanone fluorescence with pressure is due to the vibrational relaxation of energy levels. The fluorescence decreases with increasing temperature, except at low temperature where the fluorescence increase is due to an activation of intersystem crossing between triplet toward singlet levels. The influences of thermodynamic parameters are based on an increase of the non-radiative decay rate with the vibrational energy level of excited electronic state and the important collisions to remove the excess vibrational energy. Experimental and calculated results show a satisfactory agreement.

Novel Lipophilic Tracking Dyes for Monitoring Cell Proliferation

The advent of contemporary digital instrumentation has enhanced both the potential and the complexity of flow cytometric experiments, allowing for the detailed dissection of immune cell subsets and their functions. The use of cell tracking labels such as PKH26 and CFSE has been important in observing such cellular functions, but their visible emission characteristics have limited the design of such analyses. As the demand for multiparametric flow cytometry intensifies, it will become increasingly important to utilize a broader range of cell tracking reagents to optimize the measurement of fluorescence signals and to provide flexibility in the use of commercially available fluorochrome - antibody combinations. We report on the evaluation of three lipophilic membrane dyes, CellVue® Lavender, CellVue® Plum and CellVue® NIR780; with fluorescence emissions in the violet, far-red and near infrared wavelength regions, respectively. These reagents are similar to established tracking dyes such as PKH26 and CFSE in terms of staining procedure, membrane stability, optimal concentration, and lack of effect on cellular proliferation. The CellVue dyes however, exhibit different spectral characteristics than existing tracking compounds, and capitalize upon the increased number of lasers incorporated into commercially available instrumentation; thus permitting measurement of labeled populations in underexploited regions of the spectrum.

Role for Macrophage Metalloelastase in Glomerular Basement Membrane Damage Associated with Alport Syndrome

Alport syndrome is a glomerular basement membrane (GBM) disease caused by mutations in type IV collagen genes. A unique irregular thickening and thinning of the GBM characterizes the progressive glomerular pathology. The metabolic imbalances responsible for these GBM irregularities are not known. Here we show that macrophage metalloelastase (MMP-12) expression is >40-fold induced in glomeruli from Alport mice and is markedly induced in glomeruli of both humans and dogs with Alport syndrome. Treatment of Alport mice with MMI270 (CGS27023A), a broad spectrum MMP inhibitor that blocks MMP-12 activity, results in largely restored GBM ultrastructure and function. Treatment with BAY-129566, a broad spectrum MMP inhibitor that does not inhibit MMP-12, had no effect. We show that inhibition of CC chemokine receptor 2 (CCR2) receptor signaling with propagermanium blocks induction of MMP-12 mRNA and prevents GBM damage. CCR2 receptor is expressed in glomerular podocytes of Alport mice, suggesting MCP-1 activation of CCR2 on podocytes may underlie induction of MMP-12. These data indicate that the irregular GBM that characterizes Alport syndrome may be mediated, in part, by focal degradation of the GBM due to MMP dysregulation, in particular, MMP-12. Thus, MMP-12/CCR2 inhibitors may provide a novel and effective therapeutic stra-tegy for Alport glomerular disease.