Probe Volume Research Articles

Solid and liquid spectroscopic analysis (SALSA)–a soft x-ray spectroscopy endstation with a novel flow-through liquid cell

We present a novel synchrotron endstation with a flow-through liquid cell designed to study the electronic structure of liquids using soft x-ray spectroscopies. In this cell, the liquid under study is separated from the vacuum by a thin window membrane, such that the sample liquid can be investigated at ambient pressure. The temperature of the probing volume can be varied in a broad range and with a fast temperature response. The optimized design of the cell significantly reduces the amount of required sample liquid and allows the use of different window membrane types necessary to cover a broad energy range. The liquid cell is integrated into the solid and liquid spectroscopic analysis (SALSA) endstation that includes a high-resolution, high-transmission x-ray spectrometer and a state-of-the-art electron analyzer. The modular design of SALSA also allows the measurement of solid-state samples. The capabilities of the liquid cell and the x-ray spectrometer are demonstrated using a resonant inelastic x-ray scattering map of a 25 wt % NaOD solution.

Chemical U-Th-Pb dating of monazite by 3D-Micro X-ray fluorescence analysis with synchrotron radiation

A confocal set-up for three-dimensional (3D) micro X-ray fluorescence (micro-XRF) was used at the mySpot beamline at BESSY II, which allows compositional depth profiling for various applications. We present results obtained with a confocal 3D micro-XRF set-up for chemical age dating using the U, Th and Pb concentrations of monazite within rock thin sections. The probing volume was determined to be approximately 21 × 21 × 24 μm 3 for W- L α using an excitation energy of 19 keV. The relative detection limits particularly for Pb are below 10 ppm (for counting times of 1000 s). Therefore, this 3D micro-XRF set-up is suitable for dating of minerals with low Pb concentrations as long as all Pb is radiogenic, allowing spatial resolution comparable to ion microprobe or laser ablation techniques. The set-up was tested on monazites that are well characterized by isotopic techniques and have a wide range of ages, varying from 20 Ma to 1.82 Ga. Reference materials (GM3, F6, 3345) can be reproduced within error. The spread in the ages of all points determined by 3D micro-XRF is within 8 % of the isotopic reference value. The average 3D micro-XRF dates reproduce the reference ages with discrepancies between 10 and 13 Ma which translate to deviations of 1–4 %. Younger monazite samples from the European Alps (Baceno2, Bi9801) show slightly older 3D micro-XRF dates than those determined by isotopic techniques and table-top micro-XRF on whole grains. We interpret this to be related to age heterogeneities smaller than the spatial resolution and/or contributions by common Pb, which is a limitation for this technique. The method was then applied to monazites of unknown age from sapphirine-bearing granulites originating from the Gruf Complex (Alps, N-Italy). The 3D micro-XRF mean date was determined as 33 ± 4.4 Ma (2 σ) and was verified by ID-TIMS techniques. This age can be reliably interpreted as the age of the high-temperature (H T ) event because the monazites are included in and intergrown with H T minerals (sapphirine, high-Al orthopyroxene).

Latest developments and opportunities for 3D analysis of biological samples by confocal μ-XRF

X-ray fluorescence analysis performed with a primary radiation focused in the micrometer range is known as micro-X-ray fluorescence (μ-XRF). It is characterized by a penetration depth higher than other micro-analytical methods, reaching hundreds of micrometers in biological samples. This characteristic of the X-ray beam can be employed in 3D analysis. An innovative method to perform 3D analysis by μ-XRF is the so-called confocal setup. The confocal setup consists of X-ray lenses in the excitation as well as in the detection channel. In this configuration, a micro-volume defined by the overlap of the foci of both X-ray lenses is analyzed. Scanning this micro-volume through the sample can be used to perform a study in three dimensions. At present, X-ray lenses used in confocal μ-XRF experiments are mainly glass capillaries and polycapillaries. Glass capillaries are used in the excitation channel with sources of high photon flux like synchrotron radiation. Half polycapillaries or conical polycapillary concentrators are used almost exclusively in the detection channel. Spatial resolution of the confocal μ-XRF depends on the dimensions of the foci of both X-ray lenses. The overlap of these foci forms an ellipsoid which is the probing volume of the confocal setup. The axis length of the probing volume reported in confocal μ-XRF experiments are of order of few tens of micrometer. In our confocal setup, we used a commercial glass monocapillary in the excitation channel and a monolithic half polycapillary in the detection channel. The polycapillary was home-made by means of drawing of multibundles of glass capillaries in a heating furnace. The experiment was carried out at the beamline D09B-XRF of the Synchrotron Light National Laboratory (Laboratório Nacional de Luz Síncrotron, LNLS) using white beam. A model for the theoretical description of X-ray fluorescence intensity registered by confocal μ-XRF was introduced by Malzer and Kanngieβer [2005. A model for the confocal volume of 3D micro-X-ray fluorescence spectrometer. Spectrochim. Acta B 60, 1334–1341]. These authors showed that the measured scanning of fluorescent intensity is the spatial convolution of the sensitivity and the X-ray fluorescence emission rate detected. In a previous work we demonstrated that the convolution theorem can simplify the calibration and quantification process in confocal μ-XRF. In the present work, we applied these ideas to analyze samples of aquatic plants and a sample of tooth by confocal μ-XRF. We showed that confocal μ-XRF can be successfully applied to help the study of the effects of water bioremediation on aquatic plants. We also showed that confocal μ-XRF can be used to make topological studies of teeth.

A High-Precision, Low-Cost Hybrid System for Biomedical Multi-Marker Diagnostic Applications

Summary Point-of-care biosensing for multiple marker detection requires multiple-site detection, ease of use, portability, and low cost. State-of-the-art measuring systems are based on optical detection, but such techniques demand bulky, expensive, and often delicate instrumentation. Systems based on electronic detection are good candidates for the next generation technology for biosensors. Integration of electrodes and electronics onto the same silicon die for biosensing and molecular diagnostic applications has advantages in terms of high parallelism, small probe volumes and low noise. On the other hand, VLSI solutions have high non-recurring-engineering cost and are not suited for small volume production. Hybrid electronic circuits connected to a passive disposable support featuring a limited number of electrodes represent a more cost-effective solution for moderate-parallelism (less than 100 sites) applications, such as protein biomarker monitoring. The main challenge in this case is lower accuracy and higher noise levels due to connectors and limited device matching. In this work we demonstrate a flexible system based on hybrid electronics for capacitive biosensing that achieves signal-to-noise ratios compatible with micro-fabricated arrays of passive electrodes. Biosensor operating principle Our biosensing approach relies on the principle of chronoamperometry, i.e., the measurement of the time constant associated with the electrical transient which follows the application of a voltage step to a pair of bio-functionalized electrodes immersed in a solution. The system presented in this work is based on a printed circuit board and allows addressing and excitation of multiple electrode couples (in this prototype implementation up to 16), as well as the parallel measurement of the transient characteristics. The key design specification is detection of capacitive impedance variations between 4% and 20%. Results The measurement circuit provides a square wave through a follower stage and switches to excite several sensing sites of the biosensor independently. The response RC decay signal is amplified and sampled by an NI-DAQ acquisition device (sampling rate: 400 kHz). The load resistor and the amplification factor can be selected to tune the decay constant in a certain range (around 100 μsec). The addressing devices on the PCB allow an independent measurement of each site. The switches and the follower stage can be considered transparent to the excitation signal, and the noise is independent of the excitation voltage between −100 mV and 100 mV. Signal distortion caused by the limited bandwidth of the amplifier is negligible for transients longer than 1 μs decay time. Parasitic elements influence the response transient when the measured capacitance is lower than 100 pF. This lower capacitance limit corresponds to an electrode size of 100 μm × 100 μm, which is fully adequate to moderate density arrays. As an example of the use of the system, 1 mm2 gold electrodes with a self-assembled monolayer of ethylene glycols were measured. The response transient was well represented by an exponential, and the capacitance value was 11.6 nF and was determined with a relative standard deviation of 1.5%, which fully meets the design specification.

Development of Laser Techniques for Acoustic Boundary Layer Measurements.<BR>Part I: LDV Signal Processing for High Acoustic Displacements

A statistical model of the Laser Doppler signal in the case of pure acoustics is proposed. It appears that two different cases should be considered depending on the ratio of acoustic displacement amplitude to probe volume diameter. The processing of Laser Doppler Velocimetry signal in the case of high particle displacements (with oscillations across the measuring volume) is then considered. A specific signal post-processing strategy is proposed to determine the acoustic frequency, the acoustic velocity amplitude and its phase. First, the acoustic frequency is estimated by means of synchronous analysis weighted by arrival times. Then, the signal is uniformly re-sampled and the phase of the acoustic velocity is calculated. Lastly, a least-square method weighted by local probability density function is used to determine the acoustic velocity amplitude. This method permits an accurate estimation of the three acoustic parameters (frequency, velocity amplitude and phase) even in the adverse conditions induced by the proximity of a wall and is applied to oscillating viscous boundary layer measurements.

Bragg cell laser intensity modulation: effect on laser Doppler velocimetry measurements

In most laser Doppler velocimetry (LDV) systems, the frequency of one of the two laser beams that intersect to create the probe volume is shifted with an acousto-optic element. It is shown here that Bragg shifting can impose a problematic fluctuation in intensity on the frequency-shifted beam, producing spurious velocity measurements. This fluctuation occurs at twice the Bragg cell frequency, and its relative amplitude to the time average intensity is a function of the ratio of the laser beam diameter to the Bragg cell acoustic wavelength. A physical model and a configuration procedure to minimize adverse effects of the intensity modulations are presented.

Light Tolerance of R-Phycoerythrin and a Tandem Conjugate Observed by Single Molecule Recrossing Events

The recrossing of single molecules in a probe volume was used to investigate light harvesting and energy transfer between R-phycoerythrin (R-PE) and a tandem conjugate dye. The normalized recrossing ratio, Nr/Nt, was defined as the number of molecules that reenter the probe volume (Nr) to the total number of molecules detected (Nt). The energy transfer process in phycobiliproteins was studied as a function of excitation irradiation and irradiation time. This was achieved by investigating the average baseline-subtracted fluorescence intensity, normalized molecular recrossing ratio (Nr/Nt), and the number of molecules detected per second. The photon saturation irradiance of the R-PE and the tandem conjugate were compared with each other, showing that energy transfer to the tandem dye significantly improves photostability and light tolerance of the phycobiliprotein. The Nr/Nt ratio was used to study the photophysical properties of R-phycoerythrin and the tandem conjugate Streptavidin R-Phycoerythrin-AlexaFluor-647 (PE-647). Normalized molecular recrossings showed that energy transfer to a tandem conjugate could reduce the formation of triplet states in R-phycoerythrin and extend the light tolerance of certain phycobiliproteins.

Errors analysis in the evaluation of particle concentration by PDA on a turbulent two-phase jet: application for cross section and transit time methods

Phase-Doppler anemometry (PDA) is a powerful tool for two-phase flow measurements and testing. Particle concentration and mass flux can also be evaluated using the raw particle data supplied by this technique. The calculation starts from each particle velocity, diameter, transit time data, and the total measurement time. There are two main evaluation strategies. The first one uses the probe volume effective cross section, and it is usually simplified assuming that particles follow quasi one-directional trajectories. In the text, it will be called the cross section method. The second one includes a set of methods which will be denoted as “Generalized Integral Methods” (GIM). Concentration algorithms such as the transit time method (TTM) and the integral volume method (IVM) are particular cases of the GIM. In any case, a previous calibration of the measurement volume geometry is necessary to apply the referred concentration evaluation methods. In this study, concentrations and mass fluxes both evaluated by the cross-section method and the TTM are compared. Experimental data are obtained from a particle-laden jet generated by a convergent nozzle. Errors due to trajectory dispersion, burst splitting, and multi-particle signals are discussed.

Investigation on the influence of soot size on prompt LII signals in flames

Theoretical papers predict that prompt LII signals are weakly dependent on the soot size due to the fact that larger particles reach higher temperatures during the heating process by nanosecond laser pulses. This question is of crucial importance for establishing LII as a practical technique for soot volume fraction measurements. In this work two-color prompt LII measurements have been performed in several locations of diffusion and rich premixed ethylene-air flames. The experimental apparatus was carefully designed with a probe volume of uniform light distribution and sharp edges, a 4 ns integration time around the signal pulse peak and narrow spectral bandwidth. Measurements did not confirm the theoretical predictions concerning an increase of temperature for larger particles. On the contrary, larger particles in richer premixed flames exhibit a lower 400/700 signal ratio. This can probably be attributed to small differences in the refractive index of soot.

Development of an Ultrasensitive Dual-Channel Flow Cytometer for the Individual Analysis of Nanosized Particles and Biomolecules

A compact, high-sensitivity, dual-channel flow cytometer (HSDCFCM) was developed for the individual analysis of nanosized particles and biomolecules. A hydrodynamic focusing technique was applied to confine the sample stream and enable small probe volume. Fluorescence bursts from single R-phycoerythrin (R-PE) molecules passing through the laser beam were well resolved from the background with signal-to-noise ratio of 17. Excellent size discrimination was demonstrated with a mixture of three sizes of polystyrene nanoparticles. Simultaneous measurement of fluorescence and light scattering signals from individual nanoparticles was demonstrated with the 100 nm fluorescent latex beads. Doxorubicin-loaded ZrO(2) nanoparticles and fluorescently stained Escherichia coli ER2738 cells were analyzed successfully with dual-channel detection. Particle counting is demonstrated with the 210 nm fluorescent latex beads, and excellent correlation (R(2) > 0.998) between the manufacturer-reported concentrations and those measured by HSDCFCM enumeration was obtained. The measured sample detection efficiency was approximately 90% on average for particle concentrations ranging from 1.62 x 10(5) to 3.93 x 10(7) particles/mL. Sample mixtures with varying proportions of fluorescently labeled and unlabeled nanoparticles were also analyzed with good ratio correspondence. By providing rapid, quantitative, and multiparameter characterization of nanoparticles, it is believed that the HSDCFCM will find many applications in the fields of bionanotechnology, bioanalytical chemistry, and biomedicine.

Finite Instrumental Resolution Scales in Plasma Modeling

AbstractThe classical statistical approach is based on the assumption that coordinates of all particles are distinguishable what results in one‐particle kinetic models. By considering finite instrumental resolution scales we have to suppose that particles' coordinates are indistinguishable within a probing volume. This approach produces models based on multiparticle probability functions that are derived by integration of a probability function of total particles' system. The system is formed by separating within plasma (gas) flow a volume that is in dynamic equilibrium with surroundings. The probability function of the separated system satisfies Liouville theorem if the hypothesis of detailed dynamic equilibrium is assumed. The produced multiparticle models describe plasma flow in terms of fluctuations of macroscopic parameters. The discussed approach contributes to solution of Gibbs' paradox. It is tested by wellknown classical problems and applied to modeling of solar wind plasma expansion. The derived macroscopic parameters (density and speed) of plasma flow coincide under some assumptions with results of the two‐particle kinetic model and are consistent with observational data (in contrast, the classical one‐particle models do not provide the observed solar wind acceleration under the same assumptions). The results can be interpreted in favor of the suggested multiparticle approach (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Stress Perturbation Method for the Assessment of Cathodoluminescence Probe Response Functions

A method to determine the in-plane cathodoluminescence (CL) probe response function (PRF) (i.e., the function characterizing the in-plane luminescence intensity distribution within the electron probe volume) is proposed, which is based on "perturbing" the spectral position of a selected luminescence band using a highly graded stress field. The method is applied to the stress field developed ahead of the tip of an equilibrium crack in three different cases of CL bands, which arise from different structural phenomena: (i) the F(+) (oxygen excess) defect band in a nominally stoichiometric sapphire (alpha-Al(2)O(3)) single crystal; (ii) the chromophoric R-line in ruby lattice (alpha-Al(2-x)Cr(x)O(3)); and (iii) the near band-gap line in n-type GaN semiconductor crystal. A computer-aided data restoration procedure was applied to rationalize data retrieved from crack-tip line scans performed at different acceleration voltages. For the excitonic band-gap in GaN and for F(+) emission in sapphire the CL probe in the electron focal plane was found to be comparable, but not necessarily coincident, in size to the electron probe. On the other hand, the occurrence of self-absorption in the case of R-line photons in ruby resulted in a significantly broadened CL probe with respect to the average scattering length of electrons.

Quantitative analysis in confocal micro-PIXE—general concept and layered materials

In a typical micro-PIXE set-up the insertion of an X-ray optic such as a polycapillary half lens, in front of the X-ray detector in combination with the focused charged particle micro-beam creates a confocal geometry and thus a probing-volume. By moving the sample across the probing-volume, spatially resolved characteristic X-ray intensities are detected. The aim of this work is to present a theoretical model that describes the micro-PIXE intensities in confocal geometry in the case of either layered-structured materials or materials composed of major–minor elements exhibiting concentration gradients within depth. The influence of various experimental parameters such as the beam energy, spatial resolution of the X-ray lens, micro-beam dimensions, irradiation and detection angles, as well as the structure (thickness, composition) of the individual layers that compose the analyzed sample, is investigated by means of simulations of the PIXE yield at various positions of the analyzed sample within the probing volume. The advantages and limitations of the micro-PIXE analysis in confocal geometry are presented through examples and discussed, in particular with respect to the differential PIXE analysis.

Determination of probe volume dimensions in coherent measurement techniques

When investigating combustion phenomena with pump-probe techniques, the spatial resolution is given by the overlapping region of the laser beams and thus defines the probe volume size. The size of this probe volume becomes important when the length scales of interest are on the same order or smaller. We present a new approach to measure the probe volume in three dimensions, which can be used to determine the probe volume length, diameter, and shape. The optical arrangement and data evaluation are demonstrated for a dual-pump dual-broadband coherent anti-Stokes Raman scattering setup that is used for combustion diagnostics. This new approach offers a simple, quick alternative with more capabilities than formerly used probe volume measurement methods.

Molecular mobility of nitroxide spin probes in glassy polymers. Quasi‐libration model

AbstractESR spectra of three spin probes with different molecular volumes: 2,2,6,6‐tetramethyl‐4‐oxopiperidine‐1‐oxyl, di‐p‐anisylnitroxide, and nitroxide derivative of fullerene in glassy polystyrene, polyvinyl trimethylsilane, and Teflon AF‐2400 were calculated numerically within the model of quasi‐libration motions. Temperature ranges, where the model is capable to reproduce spectra within experimental errors, were defined. It was found that simulation of X‐band ESR spectra allows to determine quasi‐libration amplitudes around molecular axes X and Y with accuracy ∼ 3° and around Z axis with accuracy ∼ 15–20°. A shape of distribution of quasi‐libration amplitudes was also determined qualitatively by ESR spectra simulations. It was established that the average amplitude of quasi‐libration motion depends on the free volume of each polymer and geometrical molecular volume of a spin probe. Quasi‐libration amplitudes increase as the temperature increases, and reach the value of 40 degrees. We found that upon further temperature increase, quasi‐libration model becomes inapplicable for quantitative numerical spectra simulation. © 2008 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 47: 107–120, 2009

Multi-scalar measurements in a premixed swirl burner using 1D Raman/Rayleigh scattering

Instantaneous measurements of temperature, equivalence ratio, and major species were performed along a one-dimensional probe volume using simultaneous Raman/Rayleigh scattering in an unconfined turbulent lean-premixed swirling methane/air flame. Temperature was determined from Rayleigh scattering and the major species, CO 2, O 2, N 2, CH 4, H 2O, and H 2 from Raman scattering. Effective Rayleigh cross-sections were corrected using the local chemical composition obtained from Raman scattering. These experiments were conducted to investigate the compositional structure of a lean-premixed swirling flame in detail and to complement previous measurements of the underlying flow field. The flame was classified within a revised regime diagram at the cross-over between corrugated flames and thin reaction zones. Instantaneous temperature profiles varied significantly showing shapes ranging from laminar-like flamelets to mixing between reacted fluid elements and secondary air. Different thermo-kinetic states could be assigned to the inner and outer recirculation zones and to the inner and outer mixing layers. Linked to published velocity data of this flame, the present multi-scalar data are useful for validation of numerical simulations.

Nano-Biophotonics: new tools for chemical nano-analytics

The nondestructive chemical analysis of biological processes in the crowded intracellular environment, at cellular membranes, and between cells with a spatial resolution well beyond the diffraction limit is made possible through Nano-Biophotonics. A number of sophisticated schemes employing nanoparticles, nano-apertures, or shaping of the probe volume in the far field have significantly extended our knowledge about lipid rafts, macromolecular complexes, such as chromatin, vesicles, and cellular organelles, and their interactions and trafficking within the cell. Here, I review some of the most recent developments in Nano-Biophotonics that already are or soon will become relevant to the analysis of intracellular processes. The pros and cons of the various techniques will be discussed and an outlook of their prospects for the near future will be provided.

Measurement of liquid particle concentrations in a free jet flow

An experimental model was developed to quantify the concentration of liquid particles in a two-phase jet induced by a coaxial injector. The particles tested here, having 40 μm as mean diameter, are generated and transported by an air flow. The concentrations of particles are calculated through measurements of their characterizing parameters such as their velocities, their sizes and their crossing frequencies in the probe volume of a laser-Doppler apparatus. For a water column flowing with a velocity of 1.4 m/s and sprayed by an air stream with initial injection velocity of 80 m/s, the concentration of the liquid particles measured in six stations downstream the injection orifice varies from 70 g m −3 to 24 g m −3. The obtained concentrations are in good agreement with those obtained, directly by the use of the isokinetic sampling method.

Monitoring Protein Conformation along the Pathway of Chaperonin-Assisted Folding

The GroEL/GroES chaperonin system mediates protein folding in the bacterial cytosol. Newly synthesized proteins reach GroEL via transfer from upstream chaperones such as DnaK/DnaJ (Hsp70). Here we employed single molecule and ensemble FRET to monitor the conformational transitions of a model substrate as it proceeds along this chaperone pathway. We find that DnaK/DnaJ stabilizes the protein in collapsed states that fold exceedingly slowly. Transfer to GroEL results in unfolding, with a fraction of molecules reaching locally highly expanded conformations. ATP-induced domain movements in GroEL cause transient further unfolding and rapid mobilization of protein segments with moderate hydrophobicity, allowing partial compaction on the GroEL surface. The more hydrophobic regions are released upon subsequent protein encapsulation in the central GroEL cavity by GroES, completing compaction and allowing rapid folding. Segmental chain release and compaction may be important in avoiding misfolding by proteins that fail to fold efficiently through spontaneous hydrophobic collapse.

Sizing subcellular organelles and nanoparticles confined within aqueous droplets.

This article describes two complementary techniques, single-particle tracking and correlation spectroscopy, for accurately sizing nanoparticles confined within picoliter volume aqueous droplets. Single-particle tracking works well with bright particles that can be continuously illuminated and imaged, and we demonstrated this approach for sizing single fluorescent beads. Fluorescence correlation spectroscopy detects small intensity bursts from particles or molecules diffusing through the confocal probe volume, which works well with dim and rapidly diffusing particles or molecules; we demonstrated FCS for sizing synaptic vesicles confined in aqueous droplets. In combination with recent advances in droplet manipulations and analysis, we anticipate this capability to size single nanoparticles and molecules in free solution will complement existing tools for probing cellular systems, subcellular organelles, and nanoparticles.