Resolved Fluorescence Detection Research Articles

Fabrication, defect chemistry and microstructure of Mn-doped UO2

Mn-doped UO2 is under consideration for use as an accident tolerant nuclear fuel. We detail the synthesis of Mn-doped UO2 prepared via a wet co-precipitation method, which was refined to improve the yield of incorporated Mn. To verify the Mn-doped UO2 defect chemistry, X-ray absorption spectroscopy at the Mn K-edge was performed, in addition to X-ray diffraction, Raman spectroscopy and high-energy resolved fluorescence detection X-ray absorption near edge spectroscopy at the U M4-edge. It was established that Mn2+ directly substitutes for U4+ in the UO2 lattice, accompanied by oxygen vacancy (Ov) charge compensation. In contrast to other divalent-element doped UO2 materials, compelling evidence for U5+ in a charge compensating role was not found. This work furthers understanding of the structure and crystal chemistry of Mn-doped UO2, which could show potential advantages as a novel efficient advanced nuclear fuel.

Polarization Resolved Fluorescence Detection of Carbon Nanotube Corona Phases

Circular dichroism measures how chiral particles absorb right- and left- circularly polarized light differently to distinguish pairs of enantiomers. It can also elucidate differential photoabsorption of chiral fluorescent species via their emissions. However, this method has generally been restricted to bulk samples. Hence, these methods conceal information on the specific chiroptical response of a single particle. Furthermore, circular dichroism has been restricted in time- resolution as well as spectral range. In this work, we observed fluorescence detected circular dichroism (FDCD) of individually deposited single walled carbon nanotube (SWNT) corona. A custom-built setup compromising a linear polarizer (LP) and a quarter waveplate (QWP) was used to induce circularly polarized light (CPL). The light polarization state at the sample plane was analyzed using Stoke’s parameter. Experimental results showed that linear dichroism of the dichroic filter inside the microscope changes the polarization state of the incoming beam drastically, and thus inducing elliptically polarized light at the sample plane. A liquid crystal variable retarded (LCVR) was used to correct the polarization state of light and yield CPL at the sample plane. Singly-dispersed SWNT with different wrapping polymers were deposited onto a glass substrate. Alternating cycles of right- and left-handed circularly polarized light was delivered to excite the sample. The fluorescence intensities of each individual SWNT were tracked. The dissymmetry (g) parameters: g = 2(IL-IR)/(IL+IR) (where I L and I R are the measured fluorescence intensities with left and right circularly polarized light, respectively) were analyzed from the fluorescent signals to understand photoabsorption differences. A non-zero value of dissymmetry parameter provides information on how the corona modulates SWNT photoluminescence, an outcome not observable by bulk characterization methods. This observation can potentially enable the measurement of chiral species local interactions without a separations process.

Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay

Phosphorylation of the histone protein H2AX to form γ-H2AX foci directly represents DNA double-strand break formation. Traditional γ-H2AX detection involves counting individual foci within individual nuclei. The novelty of this work is the application of a time-resolved fluorescence assay using dissociation-enhanced lanthanide fluorescence immunoassay for quantitative measurements of γ-H2AX. For comparison, standard fluorescence detection was employed and analyzed either by bulk fluorescent measurements or by direct foci counting using BioTek Spot Count algorithm and Gen 5 software. Etoposide induced DNA damage in A549 carcinoma cells was compared across all test platforms. Time resolved fluorescence detection of europium as a chelated complex enabled quantitative measurement of γ-H2AX foci with nanomolar resolution. Comparative bulk fluorescent signals achieved only micromolar sensitivity. Lanthanide based immunodetection of γ-H2AX offers superior detection and a user-friendly workflow. These approaches have the potential to improve screening of compounds that either enhance DNA damage or protect against its deleterious effects.

Spectral Intensity Bioaerosol Sensor (SIBS): an instrument for spectrally resolved fluorescence detection of single particles in real time

Abstract. Primary biological aerosol particles (PBAPs) in the atmosphere are highly relevant for the Earth system, climate, and public health. The analysis of PBAPs, however, remains challenging due to their high diversity and large spatiotemporal variability. For real-time PBAP analysis, light-induced fluorescence (LIF) instruments have been developed and widely used in laboratory and ambient studies. The interpretation of fluorescence data from these instruments, however, is often limited by a lack of spectroscopic information. This study introduces an instrument – the Spectral Intensity Bioaerosol Sensor (SIBS; Droplet Measurement Technologies (DMT), Longmont, CO, USA) – that resolves fluorescence spectra for single particles and thus promises to expand the scope of fluorescent PBAP quantification and classification. The SIBS shares key design components with the latest versions of the Wideband Integrated Bioaerosol Sensor (WIBS) and the findings presented here are also relevant for the widely deployed WIBS-4A and WIBS-NEO as well as other LIF instruments. The key features of the SIBS and the findings of this study can be summarized as follows. Particle sizing yields reproducible linear responses for particles in the range of 300 nm to 20 µm. The lower sizing limit is significantly smaller than for earlier commercial LIF instruments (e.g., WIBS-4A and the Ultraviolet Aerodynamic Particle Sizer; UV-APS), expanding the analytical scope into the accumulation-mode size range. Fluorescence spectra are recorded for two excitation wavelengths (λex=285 and 370 nm) and a wide range of emission wavelengths (λmean=302–721 nm) with a resolution of 16 detection channels, which is higher than for most other commercially available LIF bioaerosol sensors. Fluorescence spectra obtained for 16 reference compounds confirm that the SIBS provides sufficient spectral resolution to distinguish major modes of molecular fluorescence. For example, the SIBS resolves the spectral difference between bacteriochlorophyll and chlorophyll a and b. A spectral correction of the instrument-specific detector response is essential to use the full fluorescence emission range. Asymmetry factor (AF) data were assessed and were found to provide only limited analytical information. In test measurements with ambient air, the SIBS worked reliably and yielded characteristically different spectra for single particles in the coarse mode with an overall fluorescent particle fraction of ∼4 % (3σ threshold), which is consistent with earlier studies in comparable environments.

X-Ray Absorption Spectroscopy Measurements of Cu-ProIAPP Complexes at Physiological Concentrations

The amyloidogenic islet amyloid polypeptide (IAPP) and the associated pro-peptide ProIAPP1–48 are involved in cell death in type 2 diabetes mellitus. It has been observed that interactions of this peptide with metal ions have an impact on the cytotoxicity of the peptides as well as on their deposition in the form of amyloid fibrils. In particular, Cu(II) seems to inhibit amyloid fibril formation, thus suggesting that Cu homeostasis imbalance may be involved in the pathogenesis of type 2 diabetes mellitus. We performed X-ray Absorption Spectroscopy (XAS) measurements of Cu(II)-ProIAPP complexes under near-physiological (10 μM), equimolar concentrations of Cu(II) and peptide. Such low concentrations were made accessible to XAS measurements owing to the use of the High Energy Resolved Fluorescence Detection XAS facility recently installed at the ESRF beamline BM16 (FAME-UHD). Our preliminary data show that XAS measurements at micromolar concentrations are feasible and confirm that ProIAPP1–48-Cu(II) binding at near-physiological conditions can be detected.

On-chip intermediate LED-IF-based detection for the control of electromigration in multichannel networks.

Monitoring analytes during the transfer step from the first to the second dimension in multidimensional electrophoretic separations is crucial to determine and control the optimal time point for sample transfer and thus to avoid band broadening or unwanted splitting of the sample band with consequent sample loss. A spatially resolved intermediate on-chip LED-induced fluorescence detection system was successfully implemented for a hybrid capillary-chip glass interface. The setup includes a high-power 455-nm LED prototype as an excitation light source and a linear light fiber array consisting of 23 light fibers with a diameter of 100μm for spatially resolved fluorescence detection in combination with a push-broom imager for hyperspectral detection. Using a basic FITC solution, the linear working range was determined to be 0.125 to 25μg/ml for a single light guide and the absolute detection limit was 0.04fmol at a signal-to-noise ratio of 4. With the setup presented here, labeled β-lactoglobulin focused via capillary isoelectric focusing was detectable on-chip with a sufficient intensity to monitor the analyte band transfer in the glass-chip interface demonstrating its applicability for full or intermediate on-chip detection.

Single-atom thermometer for ultracold gases

We use single or few Cs atoms as thermometer for an ultracold, thermal Rb cloud. Observing the thermometer atoms' thermalization with the cold gas using spatially resolved fluorescence detection, we find an interesting situation, where a fraction of thermometer atoms thermalizes with the cloud while the other fraction remains unaffected. We compare release-recapture measurements of the thermometer atoms to Monte-Carlo simulations while correcting for the non-thermalized fraction, and recover the cold cloud's temperature. The temperatures obtained are verified by independent time-of-flight measurements of the cold cloud's temperature. We also check the reliability of our simulations by first numerically modelling the unperturbed in-trap motion of single atoms in absence of the cold cloud, and second by performing release-recapture thermometry on the cold cloud itself. Our findings pave the way for local temperature probing of quantum systems in non-equilibrium situations.

Time-resolved FRET method for typing polymorphic alleles of the human leukocyteantigen system by using a single DNAprobe

By tens-of-picosecond resolved fluorescence detection we study Förster resonance energy transfer between a donor and a black-hole-quencher acceptor bound at the 5'- and 3'-positions of a probe-oligonucleotide matching an allelic region of a class II antigen of the human leukocyte antigen system, namely the region between codons 52 and 57 of the HLA DQB1 0201 allele. This dual-labelled probe is annealed with either the exactly complementary allelic sequence or with polymorphic sequences of the same region. We detect the longest-lived donor fluorescence in the case of the perfect hybridization achieved with the 0201 allele and definitely faster and distinct decays for the other allelic variants, some of which are single-nucleotide polymorphic variants.

Development, Characterization, and Application of a Versatile Single Particle Detection Apparatus for Time-Integrated and Time-Resolved Fluorescence Measurements—Part II: Experimental Evaluation

This paper describes the experimental realization and characterization of a versatile single particle detection apparatus. The system utilizes a novel particle beam inlet that can serve as either an on-line particle concentrator (i.e., all diameters confined in a narrow beam) or as a segregator (i.e., selected diameters confined in a narrow beam) and can be operated in a high-speed mode as well as in a low-speed mode, thus allowing different interaction times between the particles and the laser beam. An aerodynamic sizing technique has been incorporated into the system to provide rapid, real-time, and high-resolution sizing. Parameters such as transmission efficiency and size-segregation efficiency have been measured. The performance of the instrument has been demonstrated by on-line detection of spectrally resolved and time resolved fluorescence detection from airborne dye-doped particles and aerosolized endogenous fluorophores found in biological agents.

3.05] OCT of healthy skin, actinic skin and BCC lesions

Objective For the differentiation between healthy skin and actinic and BCC lesions two-dimensional images of the skin structure in situ might be of certain value. Optical coherence tomography (OCT) was used to assess whether the OCT images be of particular value or could act as a projection frame of further information obtained by various other methods, e.g. spatially resolved fluorescence detection (SRFD). Material and methods Images of healthy skin, actinic skin lesions with different degrees of dysplasia, and basal cell carcinoma (BCC) lesions were obtained from 10 patients referred for local photodynamic therapy (PDT). These were compared with regard to their appearance in OCT-images (SR-OCT Imaging System, Thorlabs). Results In healthy skin a good correlation between the images of histological samples and the two-dimensional OCT images was seen with regard to the discrimination of epithelial layer with its different zones, the basal membrane and adjacent dermal structures. In unhealthy skin, equivalents of typical histological features of epithelial dysplasia and changes in dermal layers could be found in OCT-images. Conclusion OCT may be used as investigative tool in the evaluation of actinic and BCC skin lesions. The comparison of images revealed a strong correlation with histological images for the upper layers of the dermis.

Feasibility of single nucleotide polymorphism genotyping with a single-probe by time-resolved Förster resonance energy transfer

By tens-of-picosecond resolved fluorescence detection we study Förster resonance energy transfer between a donor and a black-hole-quencher acceptor bound at the 5′- and 3′-positions of a synthetic DNA oligonucleotide. This dual-labelled oligonucleotide is annealed with either the complementary sequence or with sequences differing in one nucleotide at positions near either the ends or the centre of the oligonucleotide. We find donor fluorescence decay times whose values are definitely distinct and discuss the feasibility of single nucleotide polymorphism genotyping by this method.

Ps-LIF measurements of minor species concentration in a counterflow diffusion flame interacting with a vortex

Interactions of vortices and flame fronts may be considered as the basic structural elements of turbulent combustion. Additionally, they play an important role in flame instabilities as well as extinction and ignition processes. An ideal geometry to study these interactions is the counterflow diffusion burner with an additional actuator-driven nozzle for the generation of a vortex ring. This burner has already been well-characterized by other methods including CARS, LDA and PLIF. We present first quantitative measurements of minor species concentration in this flame using a short-pulse laser and time- and spatially resolved fluorescence detection with a streak camera. Quench-free OH concentrations are obtained by analysis of the time-resolved profiles. The high power density of the laser pulses allowed linewise detection of hydrogen using a three-photon excitation scheme. Simultaneously, shape and position of the vortex was monitored using two-dimensional detection of flame emissions. Spatially resolved concentration profiles of H and OH are presented for different interaction heights and times in the vortex. For steady flames, comparisons with model calculations are shown.

Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC)

A PC plug-in card for on-line time resolved fluorescence detection of single dye molecules based on a new time-correlated single photon counting (TCSPC) module is described. The module contains all electronic components constant fraction discriminators (CFDs), time-to-amplitude converter (TAC), analog-to-digital converter (ADC), multichannel analyzer (MCA timers) on board required for TCSPC. A fast TAC design in combination with a fast flash ADC and an error-correcting ADC/MCA principle results in a maximum count rate of 8 MHz (dead time 125 ns). A dual memory architecture allows for unlimited recording of decay curves with collection times down to 150 μs without time gaps between subsequent recordings. Applying a short-pulse diode laser emitting at 640 nm with a repetition rate of 60 MHz in combination with a confocal microscope, we studied bursts of fluorescence photons from individual dye labeled mononucleotide molecules (Cy5-dCTP) in a cone shaped microcapillary with an inner diameter of 0.5 μm at the end of the tip. The flow of the conjugates was controlled by electrokinetic forces. The presented technique permits the counting and identification of all labeled analyte molecules present in a given sample due to their characteristic velocities, burst sizes, and fluorescence decay times.

Fluorescence Investigation of Parametrically Excited Motional Wave Packets in Optical Lattices

We have used parametric excitation and time resolved fluorescence detection to investigate the coherent control of center-of-mass wave packets for atoms confined in an optical lattice. We have demonstrated the ability to manipulate the atomic wave packet motion using temporal variations of the lattice potential, and our results are in excellent agreement with a single atom model based on a solution of the Heisenberg equations of motion.

An intensely luminescent polymeric lanthanide chelator for multiple fluorescence labeling of biomolecules

A new bifunctional chelating agent has been synthesized and used to prepare a stable, intensely luminescent polymer, PLCS-Eu(III), with an extinction coefficient of 10 +6 (M-cm) −1, a very large stokes shift (Ex 337, Em 617nm), a 5nm emission bandwidth and a quantum yield of 0.6. The PLCS-Eu(III) complex was found stable towards EDTA and prolonged irradiation. It was coupled, as a coating, upon 3μm beads which in turn were coupled to oligonucleotides, for use in a nucleic acid sandwich assay. Its millisecond emission lifetime will allow it to be used for time resolved fluorescence detection. PLCS-Eu(III), a novel chelator, may be a useful new tool for array based studies of biopolymer interactions.

Capillary electrophoresis with wavelength-resolved fluorescence detection.

Single-channel fluorescence detectors provide extremely low limits of detection for analytes separated by capillary electrophoresis (CE), but little structural or diagnostic information. In wavelength-resolved fluorescence detection, complete fluorescence emission spectra are acquired for every analyte separated by CE. The fluorescence spectral information has been used for DNA sequencing and to identify the tyrosine and tryptophan content of peptides. In addition, a variety of diagnostic uses for the fluorescence information exist, including monitoring excitation source stability and the pH, organic content and impurities in the running buffer. The popularity of spectrally resolved fluorescence detection in CE is expected to increase just as diode-array detection has become relatively common for ultraviolet-visible absorbance.

Evidence for localized cell heating induced by infrared optical tweezers

The confinement of liposomes and Chinese hamster ovary (CHO) cells by infrared (IR) optical tweezers is shown to result in sample heating and temperature increases by several degrees centigrade, as measured by a noninvasive, spatially resolved fluorescence detection technique. For micron-sized spherical liposome vesicles having bilayer membranes composed of the phospholipid 1,2-diacyl-pentadecanoyl-glycero-phosphocholine (15-OPC), a temperature rise of approximately 1.45 +/- 0.15 degrees C/100 mW is observed when the vesicles are held stationary with a 1.064 microns optical tweezers having a power density of approximately 10(7) W/cm2 and a focused spot size of approximately 0.8 micron. The increase in sample temperature is found to scale linearly with applied optical power in the 40 to 250 mW range. Under the same trapping conditions, CHO cells exhibit an average temperature rise of nearly 1.15 +/- 0.25 degrees C/100 mW. The extent of cell heating induced by infrared tweezers confinement can be described by a heat conduction model that accounts for the absorption of infrared (IR) laser radiation in the aqueous cell core and membrane regions, respectively. The observed results are relevant to the assessment of the noninvasive nature of infrared trapping beams in micromanipulation applications and cell physiological studies.

Lifetime resolved fluorescence detection in chromatography. Global analysis of emission decay surfaces

Time resolved fluorescence detection adds another dimension to chromatography. Global analysis of the measured emission decay surfaces results in reliable lifetimes and concentration profiles for unknown and severely, up to completely, overlapping peak systems. The limits of the proposed algorithm are tested for three-component overlapping peak clusters with respect to chromatographic resolution, lifetime similarities, relative concentration (minor components), and data noise levels.

Lifetime resolved fluorescence detection in chromatography. Global analysis of emission decay surfaces

Time resolved fluorescence detection adds another dimension to chromatography. Global analysis of the measured emission decay surfaces results in reliable lifetimes and concentration profiles for unknown and severely, up to completely, overlapping peak systems. The limits of the proposed algorithm are tested for three-component overlapping peak clusters with respect to chromatographic resolution, lifetime similarities, relative concentration (minor components), and data noise levels.

Holographic pattern photobleaching apparatus for the measurement of lateral transport processes at interfaces—Design and performance

A new instrument was developed which allows the detection and characterization of lateral transport processes at the fluid–solid interface. It is based on holographic photobleaching combined with a laterally resolved fluorescence detection by a photon-counting camera. The geometry of the holographic pattern was chosen such that by ‘‘on-chip’’ integration of photoelectrons in the liquid-nitrogen-cooled camera the images are integrated along the symmetry axis of the pattern and subsequently stored on the chip. Time-dependent changes in the pattern caused by directed motion or by diffusion are then qualified by Fourier analysis. Lateral diffusion coefficients between 10−12 and 10−6 cm2/s and velocities between 10−10 and 10−4 m/s are measurable. In order to discriminate processes at the surface and in the volume, evanescent field illumination can be chosen. The penetration depth of the evanescent field can be varied from 96 to 800 nm. The performance of the instrument is demonstrated on selected examples.