The importance of temporal-fine structure to perceive time-compressed speech with and without the restoration of the syllabic rhythm.

Intelligibility of time-compressed (TC) speech decreases with increasing speech rate. However, intelligibility can be restored by 'repackaging' the TC speech by inserting silences between the syllables so that the original 'rhythm' is restored. Although restoration of the speech rhythm affects solely the temporal envelope, it is unclear to which extent repackaging also affects the perception of the temporal-fine structure (TFS). Here we investigate to which extent TFS contributes to the perception of TC and repackaged TC speech in quiet. Intelligibility of TC sentences with a speech rate of 15.6 syllables per second (sps) and the repackaged sentences, by adding 100 ms of silence between the syllables of the TC speech (i.e., a speech rate of 6.1 sps), was assessed for three TFS conditions: the original TFS and the TFS conveyed by an 8- and 16-channel noise vocoder. An overall positive effect on intelligibility of both the repackaging process and of the amount of TFS available to the listener was observed. Furthermore, the benefit associated with the repackaging TC speech depended on the amount of TFS available. The results show TFS contributes significantly to the perception of fast speech even when the overall rhythm/envelope of TC speech is restored.

Cyclopropanation-ring expansion of 3-chloroindoles with α-halodiazoacetates: novel synthesis of 4-quinolone-3-carboxylic acid and norfloxacin.

We present a short and efficient way of synthesizing two synthetically versatile 4-quinolone-3-carboxylate building blocks by cyclopropanation-ring expansion of 3-chloroindoles with α-halodiazoacetates as the key step. This novel transformation was applied towards the synthesis of the antibiotic drug norfloxacin.

Full-wafer in-situ fabrication and packaging of microfluidic flow cytometer with photo-patternable adhesive polymers.

Integration of microelectronics with microfluidics enables sophisticated lab-on-a-chip devices for sensing and actuation. In this paper, we investigate a novel method for in-situ microfluidics fabrication and packaging on wafer level. Two novel photo-patternable adhesive polymers were tested and compared, PA-S500H and DXL-009. The microfluidics fabrication method employs photo lithographical patterning of spin coated polymer films of PA or DXL and direct bonding of formed microfluidics to a top glass cover using die-to-wafer level bonding. These new adhesive materials remove the need for additional gluing layers. With this approach, we fabricated disposable microfluidic flow cytometers and evaluated the performance of those materials in the context of this application. DXL-009 exhibits lower autofluorescence compared to PA-S500H which improves detection sensitivity of fluorescently stained cells. Results obtained from the cytotoxicity test reveals that both materials are biocompatible. The functionality of these materials was demonstrated by detection of immunostained monocytes in microfluidic flow cytometers. The flexible, fully CMOS compatible fabrication process of these photo-patternable adhesive materials will simplify prototyping and mass manufacturing of sophisticated microfluidic devices with integrated microelectronics.

Computed ultrasound tomography in echo mode for imaging speed of sound using pulse-echo sonography: proof of principle.

The limitations of diagnostic echo ultrasound have motivated research into novel modalities that complement ultrasound in a multimodal device. One promising candidate is speed of sound imaging, which has been found to reveal structural changes in diseased tissue. Transmission ultrasound tomography shows speed of sound spatially resolved, but is limited to the acoustically transparent breast. We present a novel method by which speed-of-sound imaging is possible using classic pulse-echo equipment, facilitating new clinical applications and the combination with state-of-the art diagnostic ultrasound. Pulse-echo images are reconstructed while scanning the tissue under various angles using transmit beam steering. Differences in average sound speed along different transmit directions are reflected in the local echo phase, which allows a 2-D reconstruction of the sound speed. In the present proof-of-principle study, we describe a contrast resolution of 0.6% of average sound speed and a spatial resolution of 1 mm (laterally) × 3 mm (axially), suitable for diagnostic applications.

Effect of irradiation distance on image contrast in epi-optoacoustic imaging of human volunteers.

In combined clinical optoacoustic (OA) and ultrasound (US) imaging, epi-mode irradiation and detection integrated into one single probe offers flexible imaging of the human body. The imaging depth in epi-illumination is, however, strongly affected by clutter. As shown in previous phantom experiments, the location of irradiation plays an important role in clutter generation. We investigated the influence of the irradiation geometry on the local image contrast of clinical images, by varying the separation distance between the irradiated area and the acoustic imaging plane of a linear ultrasound transducer in an automated scanning setup. The results for different volunteers show that the image contrast can be enhanced on average by 25% and locally by more than a factor of two, when the irradiated area is slightly separated from the probe. Our findings have an important impact on the design of future optoacoustic probes for clinical application.

Lens-free imaging of magnetic particles in DNA assays.

We present a novel opto-magnetic system for the fast and sensitive detection of nucleic acids. The system is based on a lens-free imaging approach resulting in a compact and cheap optical readout of surface hybridized DNA fragments. In our system magnetic particles are attracted towards the detection surface thereby completing the labeling step in less than 1 min. An optimized surface functionalization combined with magnetic manipulation was used to remove all nonspecifically bound magnetic particles from the detection surface. A lens-free image of the specifically bound magnetic particles on the detection surface was recorded by a CMOS imager. This recorded interference pattern was reconstructed in software, to represent the particle image at the focal distance, using little computational power. As a result we were able to detect DNA concentrations down to 10 pM with single particle sensitivity. The possibility of integrated sample preparation by manipulation of magnetic particles, combined with the cheap and highly compact lens-free detection makes our system an ideal candidate for point-of-care diagnostic applications.

Vessel orientation-dependent sensitivity of optoacoustic imaging using a linear array transducer.

For clinical optoacoustic imaging, linear probes are preferably used because they allow versatile imaging of the human body with real-time display and free-hand probe guidance. The two-dimensional (2-D) optoacoustic image obtained with this type of probe is generally interpreted as a 2-D cross-section of the tissue just as is common in echo ultrasound. We demonstrate in three-dimensional simulations, phantom experiments, and in vivo mouse experiments that for vascular imaging this interpretation is often inaccurate. The cylindrical blood vessels emit anisotropic acoustic transients, which can be sensitively detected only if the direction of acoustic radiation coincides with the probe aperture. Our results reveal for this reason that the signal amplitude of different blood vessels may differ even if the vessels have the same diameter and initial pressure distribution but different orientation relative to the imaging plane. This has important implications for the image interpretation, for the probe guidance technique, and especially in cases when a quantitative reconstruction of the optical tissue properties is required.

Mechanisms of nanoparticle-mediated photomechanical cell damage.

Laser-assisted killing of gold nanoparticle targeted macrophages was investigated. Using pressure transient detection, flash photography and transmission electron microscopy (TEM) imaging, we studied the mechanism of single cell damage by vapor bubble formation around gold nanospheres induced by nanosecond laser pulses. The influence of the number of irradiating laser pulses and of particle size and concentration on the threshold for acute cell damage was determined. While the single pulse damage threshold is independent of the particle size, the threshold decreases with increasing particle size when using trains of pulses. The dependence of the cell damage threshold on the nanoparticle concentration during incubation reveals that particle accumulation and distribution inside the cell plays a key role in tissue imaging or cell damaging.

Real-time PCR to study the sequence specific magnetic purification of DNA.

The performance of various molecular techniques using complex biological samples greatly depends on the efficient separation and purification of DNA targets. In recent years, magnetic separation technology making use of small magnetic beads, has gained immense popularity. Most of these methods rely on the non-specific adsorption of DNA/RNA. However, as presented here, when functionalizing the beads with complementary DNA probes, the target of interest can selectively be isolated. Such sequence specific purification was evaluated for short DNA targets by means of simple fluorescent measurements, resulting in purification efficiencies around 80%. Besides standard fluorescent techniques, a real-time PCR (qPCR) method was applied for monitoring the purification of longer DNA targets. This qPCR method was specifically optimized for directly quantifying the purification efficiency of low concentrated DNA targets bound to magnetic beads. Additionally, parameters possibly affecting the magnetic isolation, including the length of the used capture probe or the hybridization location, were investigated. Using optimized conditions in combination with qPCR, purification efficiencies between 60% and 80% were observed and this over a large concentration window. These data also show the power of a direct qPCR approach to monitor the magnetic isolation of DNA at very low concentrations.

DNA sensors based on mixed self-assembled DNA/alkanethiol films.

When designing DNA biosensors, the immobilization of specific DNA probes is one of the most essential parts. Unfortunately, many of the existing strategies (e.g., adsorption, complexation, and entrapment) can only be used on standard microscope slides, while for more tailored surfaces alternative strategies are still required. In the case of gold surfaces, the self-assembly of mixed DNA/alkanethiol films is a very common approach to directly couple single-stranded DNA probes. The quality of these mixed films greatly depends on different parameters including the sensitivity and the detection limit. We have shown a positive relation between the length of the used carbon spacer and the general performance of the DNA biosensor. In this chapter an extended protocol for the controlled immobilization and subsequent hybridization of DNA is described.

Impact of spacers on the hybridization efficiency of mixed self-assembled DNA/alkanethiol films.

The immobilization of DNA strands is an essential step in the development of any DNA biosensor. Self-assembled mixed DNA/alkanethiol films are often used for coupling DNA probes covalently to the sensor surface. Although this strategy is well accepted, the effect of introducing a spacer molecule to increase the distance between the specific DNA sequence and the surface has rarely been assessed. The major goal of this work was to evaluate a number of such spacers and to assess their impact on for example the sensitivity and the reproducibility. Besides the commonly used mercaptohexyl (C(6)) spacer, a longer mercapto-undecyl (C(11)) spacer was selected. The combination of both spacers with tri(ethylene)glycol (TEG) and hexa(ethylene)glycol (HEG) was studied as well. The effect of the different spacers on the immobilization degree as well as on the consecutive hybridization was studied using surface plasmon resonance (SPR). When using the longer C(11) spacer the mixed DNA/alkanethiol films were found to be more densely packed. Further hybridization studies have indicated that C(11) modified probes improve the sensitivity, the corresponding detection limit as well as the reproducibility. In addition two different immobilization pathways, i.e. flow vs. diffusion controlled, were compared with respect to the hybridization efficiency. These data suggest that a flow-assisted approach is beneficial for DNA immobilization and hybridization events. In conclusion, this work demonstrates the considerable impact of spacers on the biosensor performance but also shows the importance of a flow-assisted immobilization approach.

Surface modification of gamma-Fe2O3@SiO2 magnetic nanoparticles for the controlled interaction with biomolecules.

Modifying the surface of magnetic nanoparticles (MNPs) to allow for controlled interaction with biomolecules enables their implementation in biomedical applications such as contrast agents for magnetic resonance imaging, labels in magnetic biosensing or media for magnetically assisted bioseparation. In this paper, self-assembly of trialkoxysilanes is used to chemically functionalize the surface of gamma-Fe2O3@SiO2 core-shell particles. First, the silane deposition procedure was optimized using infrared analysis in order to obtain maximum packing density of the silanes on the particles. The surface coverage was determined to be approximately 8 x 10(14) molecules/cm2. It was shown that the magnetic, crystalline, and morphological properties of the MNPs were not altered by deposition of a thin silane coating. The optimized procedure was transferred for the deposition of aldehyde and poly(ethylene glycol) (PEG) presenting silanes. The presence of both silanes on the particle surface was confirmed using XPS and FTIR. The interaction of proteins with silane-modified MNPs was monitored using a Bradford protein assay. Our results demonstrate that, by introducing aldehyde functions, the MNPs are capable of covalently binding human IgG while retaining their specific binding capacity. Maximum surface coverage occurs at 46 microg antibodies per mg particle, which corresponds to 35 antibodies bound to an average sized MNP (54 nm in diameter). The human IgG functionalized MNPs exhibit a high degree of specificity (approximately 90%) and retained a binding capacity of 32%. Using the same approach, streptavidin was coupled onto the MNPs and the biotin binding capacity was determined using biotinylated fluorescein. At maximum surface coverage, a biotin binding capacity of 1500 pmol/mg was obtained, corresponding to a streptavidin activity of 76%. On the other hand, by introducing PEG functions the non-specific adsorption of serum proteins could be significantly suppressed down to approximately 3 microg/mg. We conclude that self-assembly of silane films creates a generic platform for the controlled interactions of MNPs with biomolecules.