Microchip HPLC of peptides and proteins

Rapid microchip reversed-phase HPLC of peptides and proteins at pressure gradients of 12 bar/cm (180 psi/cm) has been performed using a microdevice that integrates subnanoliter on-chip injection and separation with a miniaturized fluorescence detector. Proteins and peptides were separated on a C18 side-chain porous polymer monolith defined by contact lithography, and injection was achieved via a pressure-switchable fluoropolymer valve defined using projection lithography. Preliminary separations of peptide standards and protein mixtures were performed in 40-200 s, and switching between samples with no detectible sample carryover has been performed. The injections and separations were reproducible; the relative standard deviation (RSD) for retention time was 0.03%, and peak area RSD was 3.8%. Sample volumes ranging from 220 to 800 pL could be linearly metered by controlling the pressure injection pulse duration with conventional timing and valving. The current prototype system shows the potential for rapid and autonomous HPLC separations with varying modalities and the potential for direct connection to mass spectrometers at nanospray flow rates.     

Wavelength references for interferometry in air

Cavity-mode wavelengths in air are determined by measuring a laser's frequency while it is locked to the mode in vacuum during a calibration step and subsequently correcting the mode wavelength for atmospheric pressure compression, temperature difference, and material aging. Using a Zerodur ring cavity, we demonstrate a repeatability of +/- 2 x 10(-8) (3sigma), with the wavelength accuracy limited to +/- 4 x 10(-8) by knowledge of the absolute helium gas temperature during the pressure calibration. Mirror cleaning perturbed the mode frequency by less than deltav/v approximately 3 x 10(-9), limited by temperature correction residuals.     

Magnetic-resonance-imaging-coupled broadband near-infrared tomography system for small animal brain studies

A novel magnetic-resonance-coupled broadband near-infrared (NIR) tomography system for small animal brain studies is described. Several features of the image formation approach are new in NIR tomography and represent major advances in the path to recovering high-resolution hemoglobin and oxygen saturation images of tissue. The NIR data were broadband and continuous wave and were used along with a second-derivative-based estimation of the path length from water absorption. The path length estimation from water was then used along with the attenuation spectrum to recover absorption and reduced scattering coefficient images at multiple wavelengths and then to recover images of total hemoglobin and oxygen saturation. Going beyond these basics of NIR tomography, software has been developed to allow inclusion of structures derived from MR imaging (MRI) for the external and internal tissue boundaries, thereby improving the accuracy and spatial resolution of the properties in each tissue type. The system has been validated in both tissue-simulating phantoms, with 10% accuracy observed, and in a rat cranium imaging experiment. The latter experiment used variation in inspired oxygen (FiO2) to vary the observed hemoglobin and oxygen saturation images. Quantitative agreement was observed between the changes in deoxyhemoglobin values derived from NIR and the changes predicted with blood-oxygen-level-dependent (BOLD) MRI. This system represents the initial stage in what will likely be a larger role for NIR tomography, coupled to MRI, and illustrates that the technological challenges of using continuous-wave broadband data and inclusion of a priori structural information can be met with careful phantom studies.     

Comparison of psychro-active arctic marine bacteria and common mesophillic bacteria using surface-enhanced Raman spectroscopy

Psychro-active bacteria, important constituents of polar ecosystems, have a unique ability to remain active at temperatures below 0 degrees C, yet it is not known to what extent the composition of their outer cell surfaces aids in their low-temperature viability. In this study, aqueous suspensions of five strains of Arctic psychro-active marine bacteria (PAMB) (mostly sea-ice isolates), were characterized by surface-enhanced Raman spectroscopy (SERS) and compared with SERS spectra from E. coli and P. aerigunosa. We find the SERS spectra of the five psychro-active bacterial strains are similar within experimental reproducibility. However, these spectra are significantly different from the spectra of P. aeruginosa and E. coli. We find that the relative intensities of many of the common peaks show the largest differences reported so far for bacterial samples. An indication of a peak was found in the PAMB spectra that has been identified as characteristic of unsaturated fatty acids and suggests that the outer membranes of the PAMB may contain unsaturated fatty acids. We find that using suspensions of silver colloid particles greatly intensifies the Raman peaks and quenches the fluorescence from bacterial samples. This technique is useful for examination of specific biochemical differences among bacteria.     

Multicomponent internal recalibration of an LC-FTICR-MS analysis employing a partially characterized complex peptide mixture: systematic and random errors

In high-throughput proteomics, a promising current approach is the use of liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) of tryptic peptides from complex mixtures of proteins. To apply this method, it is necessary to account for any systematic measurement error, and it is useful to have an estimate of the random error expected in the measured masses. Here, we analyze by LC-FTICR-MS a complex mixture of peptides derived from a sample previously characterized by LC-QTOF-MS. Application of a Bayesian probability model of the data and partial knowledge of the composition of the sample suffice to estimate both the systematic and random errors in measured masses.     

A scalable addressable positive-dielectrophoretic cell-sorting array

We present the first known implementation of a passive, scalable architecture for trapping, imaging, and sorting individual microparticles, including cells, using a positive dielectrophoretic (p-DEP) trapping array. Our array-based technology enables "active coverslips" where, when scaled, many individually held cells can be sorted based upon imaged spatial or temporally variant characteristics. Our design incorporates a unique "ring-dot" p-DEP trap geometry organized in a row/column array format. This trap design, implemented in a two-level metal process, provides strong and highly spatially localized holding fields enabling single-cell capture for all traps in the array. We release individual trapped microparticles during sorting using a passive transistor-independent approach where we electrically ground the row and column electrodes associated with specific traps in the array. The demand for chip-to-world electrical connections in our arrays scales proportionally with the square root of the number of traps in a given array, delivering a substantial improvement over prior designs. We demonstrate capture, holding, and release operations with both beads and cells in small arrays of this new architecture.     

Ion-exchanged glass waveguides with low birefringence for a broad range of waveguide widths

Optical communications networks require integrated photonic components with negligible polarization dependence, which typically means that the waveguides must feature very low birefringence. Recent studies have shown that waveguides with low birefringence can be obtained, e.g., by use of silica-on-silicon waveguides or buried ion-exchanged glass waveguides. However, many integrated photonic circuits consist of waveguides with varying widths. Therefore low birefringence is consequently required for waveguides having different widths. This is a difficult task for most waveguide fabrication technologies. We present experimental results on waveguide birefringence for buried silver-sodium ion-exchanged glass waveguides. We show that the waveguide birefringence of the order of 10(-6) for waveguide mask opening widths ranging from 2 to 10 microm can be obtained by postprocessing the sample through annealing at an elevated temperature. The measured values are in agreement with the values calculated with our modeling software for ion-exchanged glass waveguides. This unique feature of ion-exchanged waveguides may be of significant importance in a wide variety of integrated photonic circuits requiring polarization-independent operation.     

Effects of refractive index on near-infrared tomography of the breast

Near infrared (NIR) optical tomography is an imaging technique in which internal images of optical properties are reconstructed with the boundary measurements of light propagation through the medium. Recent advances in instrumentation and theory have led to the use of this method for the detection and characterization of tumors within the female breast tissue. Most image reconstruction approaches have used the diffusion approximation and have assumed that the refractive index of the breast is constant, with a bulk value of approximately 1.4. We have applied a previously reported modified diffusion approximation, in which the refractive index for different tissues can be modeled. The model was used to generate NIR data from a realistic breast geometry containing a localized anomaly. Using this simulated data, we have reconstructed optical images, both with and without correct knowledge of the refractive-index distribution to show that the modified diffusion approximation can accurately recover the anomaly given a priori knowledge of refractive index. But using a reconstruction algorithm without the use of correct a priori information regarding the refractive-index distribution is shown as recovering the anomaly but with a degraded quality, depending on the degree of refractive index mismatch. The results suggest that provided the refractive index of breast tissue is approximately 1.3-1.4, their exclusion will have minimal effect on the reconstructed images.     

Height effects in real and virtual environments

The study compared human perceptions of height, danger, and anxiety, as well as skin conductance and heart rate responses and postural instability effects, in real and virtual height environments. The 24 participants (12 men, 12 women), whose average age was 23.6 years, performed "lean-over-the-railing" and standing tasks on real and comparable virtual balconies, using a surround-screen virtual reality (SSVR) system. The results indicate that the virtual display of elevation provided realistic perceptual experience and induced some physiological responses and postural instability effects comparable to those found in a real environment. It appears that a simulation of elevated work environment in a SSVR system, although with reduced visual fidelity, is a valid tool for safety research. Potential applications of this study include the design of virtual environments that will help in safe evaluation of human performance at elevation, identification of risk factors leading to fall incidents, and assessment of new fall prevention strategies.     

Acoustic droplet vaporization for temporal and spatial control of tissue occlusion: a kidney study

Acoustic droplet vaporization (ADV) has been introduced with the potential application of tumor treatment via occlusion and subsequent necrosis. New Zealand White rabbits were anesthetized, and their left kidney was externalized. An imaging array and single-element transducer were positioned in a tank with direct access to the kidney's vasculature and renal artery. Filtered droplet emulsions (diameter <6 microm) were injected intra-arterially (IA) into the left heart during insonification of the renal artery, and the extent of blood flow reduction by ADV was compared to the untreated right kidney. Flow cytometry (using colored microspheres) of kidney tissue samples and reference blood from the femoral artery allowed the quantitative estimation of regional blood flow. A maximum regional blood flow reduction in the treated region of >90% and an average organ perfusion reduction of >70% was achieved using ADV. After treatment of the left kidney, the control kidney on the contralateral side showed a maximum decrease in regional blood flow of 18% relative to the pre-ADV baseline. Image-based hyper-echogenicity from ADV of IA injections was monitored for approximately 90 minutes, and cortex perfusion was reduced by >60% of its original value for more than 1 hour. This could be enough time for the onset of cell death and possible tumor treatment via ischemic necrosis. Moreover, currently used radiofrequency tissue ablation-based tumor treatment could benefit from ADV due to the decreased heat loss via vascular cooling.