Angular distribution of light scattered by single biological cells and oriented particle agglomerates

We used a flow cytometer together with an intensified CCD camera to record spatially resolved light scattering from micrometer-sized single particles and single oriented particle agglomerates. Experimental differential cross sections of an oriented dumbbell made from two identical polystyrene spheres were compared with theoretical values calculated within the discrete dipole approximation, and good agreement was achieved. Furthermore, characteristic two-dimensional patterns of the scattered-light intensity were recorded for single blood cells, yielding information on the cells' shape and volume. Besides flow cytometry, we observed and analyzed differential light scatter of particle clusters of known size, shape, and orientation located within an optical trap.     

Dual-modality single particle orientation and rotational tracking of intracellular transport of nanocargos

The single particle orientation and rotational tracking (SPORT) technique was introduced recently to follow the rotational motion of plasmonic gold nanorod under a differential interference contrast (DIC) microscope. In biological studies, however, cellular activities usually involve a multiplicity of molecules; thus, tracking the motion of a single molecule/object is insufficient. Fluorescence-based techniques have long been used to follow the spatial and temporal distributions of biomolecules of interest thanks to the availability of multiplexing fluorescent probes. To know the type and number of molecules and the timing of their involvement in a biological process under investigation by SPORT, we constructed a dual-modality DIC/fluorescence microscope to simultaneously image fluorescently tagged biomolecules and plasmonic nanoprobes in living cells. With the dual-modality SPORT technique, the microtubule-based intracellular transport can be unambiguously identified while the dynamic orientation of nanometer-sized cargos can be monitored at video rate. Furthermore, the active transport on the microtubule can be easily separated from the diffusion before the nanocargo docks on the microtubule or after it undocks from the microtubule. The potential of dual-modality SPORT is demonstrated for shedding new light on unresolved questions in intracellular transport.     

Second order standard addition method and fluorescence spectroscopy in the quantification of ibuprofen enantiomers in biological fluids

The second order standard addition method and spectrofluorimetry were used for determination of ibuprofen enantiomers in human plasma and urine. The methodology was based on chiral recognition of ibuprofen by formation of an inclusion complex with a chiral auxiliary, β-cyclodextrin, in the presence of 1-butanol. The strategy combines the use of PARAFAC, for extraction of the pure analyte signal, with the standard addition method, for determinations in presence of a matrix effect. A specific PARAFAC model was built for each sample and the scores were related to (S)-ibuprofen concentration using a linear regression in the standard addition method. Feasible results were obtained for determinations in the molar fraction range from 50 to 80% of (S)-ibuprofen, providing absolute errors lowers than 4.0% for plasma and urine.     

Quantum dot photon statistics measured by three-dimensional particle tracking

We present an instrument for performing correlation spectroscopy on single fluorescent particles while tracking their Brownian motion in three dimensions using real-time feedback. By tracking CdSe/ZnS quantum dots in water (diffusion coefficient approximately 20 microm2/s), we make the first measurements of photon antibunching (at approximately 10 ns) on single fluorophores free in solution and find fluorescence lifetime heterogeneity within a quantum dot sample. In addition, we show that 2-mercaptoethanol suppresses short time-scale intermittency (1 ms to 1 s) in quantum dot fluorescence by reducing time spent in the off-state.     

Fine particle formation using supercritical fluids

The use of supercritical fluids as solvents for the formation of micro- and nano-particles has shown tremendous over the past few years due to increased demands from industry for materials with high performance specifications and high or unique functionality. Supercritical fluids allow synthesis of many types of particles since the solvent's chemical and physical properties can be varied with temperature or pressure, both of which can affect the degree of supersaturation and nucleation. In this review, we describe methods for the formation of fine particles using supercritical fluids CO₂ and water. Basic principles and specific features of each solvent are described, and the advantages and limitations are discussed.     

Direct determination of benzodiazepines in biological fluids by restricted-access solid-phase microextraction

A biocompatible solid-phase microextraction (SPME) fiber was prepared using an alkyl-diol-silica (ADS) restricted-access material as the SPME coating. The ADS-SPME fiber was able to simultaneously fractionate the protein component from a biological sample, while directly extracting several benzodiazepines, overcoming the present disadvantages of direct sampling in biological matrixes by SPME. The fiber was interfaced with an HPLC-UV system, and an isocratic mobile phase was used to desorb, separate, and quantify the extracted compounds. The calculated clonazepam, oxazepam, temazepam, nordazepam, and diazepam detection limits were 600, 750, 333, 100, and 46 ng/mL in urine, respectively. The method was confirmed to be linear over the range of 500-50000 ng/mL with an average linear coefficient (R2) value of 0.9918. The injection repeatability and intraassay precision of the method were evaluated over 10 injections, resulting in a RSD of approximately 6%. The ADS-SPME fiber was robust and simple to use, providing many direct extractions and subsequent determination of benzodiazepines in biological fluids.     

Calibration curves for biological dosimetry by fluorescence in situ hybridisation

Dose-response curves were measured for the induction of chromosomal aberrations in peripheral blood lymphocytes after acute exposure in vitro to 60Co gamma rays. Blood was obtained from four different healthy donors, and chromosomes were either observed at metaphase, following colcemid accumulation, or prematurely condensed by calyculin A. Cells were analysed in three different Italian laboratories. Chromosomes 1, 2, and 4 were painted, and simple-type interchanges between painted and non-painted chromosomes were scored in cells exposed in the dose range 0.1-3.0 Gy. The chemical-induced premature chromosome condensation method was also used combined with chromosome painting (chromosome 4 only) to determine calibration curves for high dose exposures (up to 20 Gy X rays). Calibration curves described in this paper will be used in our laboratories for biological dosimetry by fluorescence in situ hybridisation.     

Novel aqueous magnetic fluids stabilized by albumin for biological application

This paper demonstrates bovine serum albumin (A1) and ovalbumin (A2) can be used as stabilizer to prepare aqueous magnetic fluids for gene therapy.The content of magnetite depends on the reaction temperature and time. The reaction is performed at 70 °C for 30 min, which is appropriate to obtain stable aqueous magnetic fluids. The super paramagnetic magnetic fluids composed of Fe₃O₄ cores, which have average diameter about 8 nm. A high positive ζ-potential of +46.0 mV and suitable hydrodynamic diameters were realized.     

Fluorescent nanocrystals as colloidal probes in complex fluids measured by fluorescence correlation spectroscopy

The diffusion properties of fluorescent colloidal CdSe and CdSe/ZnS nanocrystals (QDs) with different hydrophilic coatings were characterized in complex fluids such as actin solutions using fluorescence correlation spectroscopy (FCS). The hydrodynamic radii of the QDs were determined both in organic solvents and water. Attention was given to the potential artifacts arising from the fluorescence properties of the QDs. With increasing excitation intensities, the apparent particle concentration and diffusion times are overestimated if using a simple diffusion model. This can be explained by a numerical simulation. The diffusion behavior of QDs in actin networks of different concentrations was determined to demonstrate the potential use of nanocrystals as probes in soft biological matter. The decreasing diffusion coefficient of the nanocrystals with increasing actin concentration results in an intrinsic polymer viscosity of 0.12+/-0.02 ml mg(-1), in accordance with literature values.     

Ion-counting nanodosemeter with particle tracking capabilities

An ion-counting nanodosemeter (ND) yielding the distribution of radiation-induced ions in a low-pressure gas within a millimetric, wall-less sensitive volume (SV) was equipped with a silicon microstrip telescope that tracks the primary particles, allowing correlation of nanodosimetric data with particle position relative to the SV. The performance of this tracking ND was tested with a broad 250 MeV proton beam at Loma Linda University Medical Center. The high-resolution tracking capability made it possible to map the ion registration efficiency distribution within the SV, for which only calculated data were available before. It was shown that tracking information combined with nanodosimetric data can map the ionisation pattern of track segments within 150 nm-equivalent long SVs with a longitudinal resolution of approximately 5 tissue-equivalent nanometers. Data acquired in this work were compared with results of Monte Carlo track structure simulations. The good agreement between 'tracking nanodosimetry' data acquired with the new system and simulated data supports the application of ion-counting nanodosimetry in experimental track-structure studies.     

Three-dimensional particle tracking via bifocal imaging

We introduce a bifocal imaging method that enables three-dimensional (3D) tracking of both fluorescent and nonfluorescent particles. We accomplish this by simultaneously imaging a focused plane, for in-plane position (x,y), and a defocused plane, for out-of-plane position (z), of a molecule using a single CCD camera. We applied our method to several systems including in vivo melanosome tracking and phagocytosed fluorescent bead tracking. We have achieved 2-5 nm accuracy with a 2-50 ms time resolution.     

Pulse shapes in single photon,single particle calorimeters

Single photon or single particle calorimeters with energy resolutions of order 1 eV fwhm have recently been proposed as detectors for X-ray astronomy and many other applications. Such devices consist, essentially, of an absorber (linked to a heat bath at ∼ 0.1 K) and one or more thermistors The temperature rise induced in the former element by the absorption of a single photon or particle produces voltage waveforms at the ouput of the latter from which energy information may be abstracted. In this paper, we consider the practical limits to the energy resolution which arise from the “thermal nonuniformity” of the absorber mass. The variation in thermistor pulse shape with position of photon absorption is estimated from solutions of the heat conduction equation, for a number of linear calorimeter designs. An assessment is made of the position-sensing properties of a calorimeter with two line-end thermistors.     

Novel approach for measurement of restitution coefficient by magnetic particle tracking

Inter particular contacts between particles are of great interest, as the intensity and quantity of contacts impact the behavior of a wide variety of industrial processes. The coefficient of restitution is commonly used to describe the collision of a particle either with other particles or with walls and components of plant equipment. The coefficient considers the amount of energy that is dissipated in the collision event. Usually the restitution coefficient is examined by the use of high speed cameras and image velocimetry. In this study a new method based on magnetic particle tracking (MPT) was developed to measure the restitution coefficient of magnetic particles. By performing simultaneous image velocimetry and MPT measurements, the novel approach is validated. The comparison shows a high coincidence of the results from both measurement methods, both for normal direction impacts as well as impacts on oblique surfaces. Furthermore, the rotation of particles was investigated (by rolling down a tilted plate), which has also shown the applicability of the MPT measurement method.     

Space mapping-focused control techniques for particle dispersions in fluids

We present and investigate techniques for optimizing particle dispersions in all kinds of Reynolds number flows. In particular, we show the range of application, power and efficiency of space mapping approaches that are based on a hierarchy of models ranging from a complex (fine, accurate, costly) to simple (coarse, rough, cheap) model. Space mapping turns out to be a reasonable approximation to optimal control and a competitive alternative to instantaneous control regarding speed and memory demands when dealing with complex, non-stationary problems. Moreover it allows the easy and efficient treatment of stochastic design problems. To control random particle dynamics in a turbulent flow, we suggest a Monte-Carlo aggressive space mapping algorithm which yields very convincing numerical results.     

Alternating-color quantum dot nanocomposites for particle tracking

Because of their extraordinary brightness and photostability, quantum dots (QDs) have tremendous potential for long-term, particle tracking in heterogeneous systems (e.g., living cells, microfluidic flow). However, one of their major limitations is blinking, an intermittent loss of fluorescence, characteristic of individual and small clusters of QDs, that interrupts particle tracking. Recently, several research groups have reported "nonblinking QDs". However, blinking is the primary method used to confirm nanoparticle aggregation status in situ, and single or small clusters of nanoparticles with continuous fluorescence emission are difficult to discern from large aggregates. Here, we describe a new class of quantum dot-based composite nanoparticles that solve these two seemingly irreconcilable problems by exhibiting near-continuous, alternating-color fluorescence, which permits aggregation status discrimination by observable color changes even during motion across the focal plane. These materials will greatly enhance particle tracking in cell biology, biophysics, and fluid mechanics.