Dark-field Laser Research Articles

Improving sensory quality and properties of stirred high-protein yogurts with a rotor/stator device

This study investigated the impact of post-fermentation processing and fat content on the quality of stirred high-protein yogurts (6 wt% protein) during 13 weeks of storage. A rotor/stator device was used to apply two post-fermentation treatments (119W and 296W, 290 L-1h) to yogurts containing 0 or 3 wt % fat, while a control yogurt did not undergo post-fermentation processing. Sensory quality was analyzed by Quantitative descriptive analysis (QDA) and Quality control (QC). QC results showed significant effects of post-fermentation processing on appearance, consistency, and odor/flavor. Post-fermentation processing significantly reduced whey separation, stickiness, thickness, sticky mouthfeel and mealiness, and increased shininess and smooth mouthfeel according to QDA results. Confocal laser scanning microscopy and darkfield microscopy revealed larger protein aggregates in the control samples compared to the post-processed samples. The post-processed yogurts maintained acceptable quality after 13 weeks of storage, contrasting with the control samples that had unacceptable quality throughout the storage period.

Comparative cytotoxicity of kaolinite, halloysite, multiwalled carbon nanotubes and graphene oxide

This study aimed at comparative examining of the interactions between conventionally used clay and carbon nanomaterials and human lung adenocarcinoma cells (A549 cells). The following platy and tubular nanomaterials were tested: carbon nanoparticles, i.e. multi-walled carbon nanotubes (MWCNTs) and graphene oxide nanosheets (GO) as well as nanoclays, i.e. halloysite nanotubes (HNTs) and kaolinite nanosheets (Kaol). Nanoparticle physicochemical properties and their internalization into cells were examined using dynamic light scattering as well as atomic force, 3D laser scanning confocal and darkfield hyperspectral microscopies. Biological aspects of the nanomaterial-cell interaction included assessment of cellular toxicity, DNA damage, metabolic activity, and physical parameters of the cells. Regardless of a shape, carbon nanomaterials demonstrated cell surface adsorption, but negligible penetration into cells compared to nanoclays. However, carbon nanomaterials were found to be the most toxic for cells as probed by the MTS assay. They also turned out to be the most genotoxic for cells compared to nanoclays as revealed by the DNA-Comet assay. GO significantly increased the fraction of apoptotic cells and was the most cytotoxic and genotoxic nanomaterial. Comparison of flow cytometry and MTS data indicated that a cytotoxic effect of MWCNTs was not associated with increased cell death, but was rather due to a decrease in cell metabolic activity and/or proliferation. Finally, no significant effect of the shape of the tested nanomaterials on their internalization and cytotoxicity was revealed.

Dark Field and Coherent Anti-Stokes Raman (DF-CARS) Imaging of Cell Uptake of Core-Shell, Magnetic-Plasmonic Nanoparticles.

Magnetic-plasmonic, Fe3O4-Au, core-shell nanoparticles are popular in many applications, most notably in therapeutics and diagnostics, and thus, the imaging of these nanostructures in biological samples is of high importance. These nanostructures are typically imaged in biological material by dark field scatter imaging, which requires an even distribution of nanostructures in the sample and, therefore, high nanoparticle doses, potentially leading to toxicology issues. Herein, we explore the nonlinear optical properties of magnetic nanoparticles coated with various thicknesses of gold using the open aperture z-scan technique to determine the nonlinear optical properties and moreover, predict the efficacy of the nanostructures in nonlinear imaging. We find that the magnetic nanoparticles coated with gold nanoseeds and thinner gold shells (ca. 4 nm) show the largest nonlinear absorption coefficient β and imaginary part of the third-order susceptibility Im χ(3), suggesting that these nanostructures would be suitable contrast agents. Next, we combine laser dark field microscopy and epi-detected coherent anti-Stokes Raman (CARS) microscopy to image the uptake of magnetic-plasmonic nanoparticles in human pancreatic cancer cells. We show the epi-detected CARS technique is suitable for imaging of the magnetic-plasmonic nanoparticles without requiring a dense distribution of nanoparticles. This technique achieves superior nanoparticle contrasting over both epi-detected backscatter imaging and transmission dark field imaging, while also attaining label-free chemical contrasting of the cell. Lastly, we show the high biocompatibility of the Fe3O4 nanoparticles with ca. 4-nm thick Au shell at concentrations of 10–100 µg/mL.

Limiting (zero-load) speed of the rotary motor of Escherichia coli is independent of the number of torque-generating units

Rotation of the bacterial flagellar motor is driven by multiple torque-generating units (stator elements). The torque-generating dynamics can be understood in terms of the "duty ratio" of the stator elements, that is, the fraction of time a stator element engages with the rotor during its mechanochemical cycle. The dependence of the limiting speed (zero-load speed) of the motor on the number of stator elements is the determining test of the duty ratio, which has been controversial experimentally and theoretically over the past decade. Here, we developed a method combining laser dark-field microscopy and optical trapping to resolve this controversy. We found that the zero-load speed is independent of the number of stator elements for the bacterial flagellar motor in Escherichia coli, demonstrating that these elements have a duty ratio close to 1.

Line-scanning laser scattering system for fast defect inspection of a large aperture surface.

Inspection of defects with micrometer level on large aperture surfaces with hundreds of millimeters is one of the challenges in surface quality evaluation. Various microscopic imaging methods have been applied to inspecting the surface defects, while they are time-consuming for the small field of view and the sub-aperture stitching. To tackle this problem, a high-speed line scanning system based on the dark-field laser scattering method is proposed. The laser beam is scanned by the rotating polygon mirror to a laser line for high throughput and then the telecentric F-theta lens converges each incoming laser beam to a focused spot that creates a high intensity to enhance the signal-to-noise ratio. The scattered light from surface defect is collected by the designed integrating sphere for low background noise and the scattering signal is detected for each focused spot at a proper acquisition rate by a photomultiplier (PMT) detector with extremely short response time. In the meanwhile, the tested surface is moving perpendicular to the laser line to realize high-speed large area inspection. The defect inspection system is confirmed experimentally with laser line length of 60mm, minimum detectable size less than 0.5μm, and figure of merit of 9.6 cm2 s-1 μm-1. The work put forward an effective method for automatic discovery of surface defects such as scratches, digs, and contaminants on large aperture surfaces.

Towards high-cooperativity strong coupling of a quantum dot in a tunable microcavity

We investigate the strong coupling regime of a self-assembled quantum dot in a tunable microcavity with dark-field laser spectroscopy. The high quality of the spectra allows the lineshapes to be analyzed revealing subtle quantum interferences. Agreement with a model calculation is achieved only by including exciton dephasing which reduces the cooperativity from a bare value of 9.0 to the time-averaged value 5.5. In the pursuit of high cooperativity, besides a high-Q and low mode-volume cavity, we demonstrate that equal efforts need to be taken towards lifetime-limited emitter linewidths.

On-chip immunoelectrophoresis of extracellular vesicles released from human breast cancer cells.

Extracellular vesicles (EVs) including exosomes and microvesicles have attracted considerable attention in the fields of cell biology and medicine. For a better understanding of EVs and further exploration of their applications, the development of analytical methods for biological nanovesicles has been required. In particular, considering the heterogeneity of EVs, methods capable of measuring individual vesicles are desired. Here, we report that on-chip immunoelectrophoresis can provide a useful method for the differential protein expression profiling of individual EVs. Electrophoresis experiments were performed on EVs collected from the culture supernatant of MDA-MB-231 human breast cancer cells using a measurement platform comprising a microcapillary electrophoresis chip and a laser dark-field microimaging system. The zeta potential distribution of EVs that reacted with an anti-human CD63 (exosome and microvesicle marker) antibody showed a marked positive shift as compared with that for the normal immunoglobulin G (IgG) isotype control. Thus, on-chip immunoelectrophoresis could sensitively detect the over-expression of CD63 glycoproteins on EVs. Moreover, to explore the applicability of on-chip immunoelectrophoresis to cancer diagnosis, EVs collected from the blood of a mouse tumor model were analyzed by this method. By comparing the zeta potential distributions of EVs after their immunochemical reaction with normal IgG, and the anti-human CD63 and anti-human CD44 (cancer stem cell marker) antibodies, EVs of tumor origin circulating in blood were differentially detected in the real sample. The result indicates that the present method is potentially applicable to liquid biopsy, a promising approach to the low-invasive diagnosis of cancer.

Measurement of Individual Nanobioparticles on Microfluidic Chips by Laser Dark-field Imaging

Measurement of Individual Nanobioparticles on Microfluidic Chips by Laser Dark-field Imaging

Use of Darkfield Laser Technology in Monitoring Particles Generated by Process Tools in Cu Pillar Bumping & Wafer Edge Inspection

In recent years, the semiconductor industry has witnessed an increasingly fast adoption rate of Cu pillar bumps for a variety of devices including memory, logic, processors, and FPGA. Cu pillar bumps provide several advantages over their counterparts, among which are tighter pitch, large number of I/Os as well as better heat dissipation. Due to the constant drive for miniaturization, the size and pitch of these bumps are getting smaller and smaller. As the sizes shrink, the need to minimize size and number of particles generated by the process tools increases. Unwanted particles generated by tools such as sputtering or etch can create killer defects driving down yields. The purpose of this paper is to examine how a darkfield laser technique can be used to monitor process tools for the amount of particles they generate. Determination of excessive particle density could trigger a chamber clean. After the chamber is cleaned, particle density needs to be re-characterized before releasing tool into production. Figure 1 shows the working principal of this sensor and figure two shows an example of maps generated by the inspection tool. Particle and haze data generated on a monitor wafer allows engineers to maintain process tools at optimal cleaning levels.

Stepping Dynamics of the Bacterial Flagellar Motor

The bacterial flagellar motor (BFM) is a rotary molecular motor which is self-assembled and embedded within the cellular envelope of many species of swimming bacteria. Powered by a flux of ions across the cytoplasmic membrane, the BFM rotates the helical flagellar filaments that propel the cell. It is one of the best characterized large biomolecular complexes. In 2005, a sodium-driven chimeric flagellar motor in E. coli at low energization and low expression of torque-generating units was observed to take 26 steps per revolution1. However, achieving sufficient spatial and temporal resolution to resolve kinetics and discrete stepping behavior under normal energization conditions and with multiple torque-generating units remains a challenge. We have used a novel objective-type laser darkfield microscope to image small gold nanoparticles attached to the hooks of rotating BFMs with sub-nanometer and microsecond resolution. We present preliminary results for the stepping behaviour of the BFM rotating at up to hundreds of revolutions per second and with a varying number of torque-generating units.1. Y. Sowa, A. D. Rowe, M. C. Leake, T. Yakushi, M. Homma, A. Ishijima, and R. M. Berry. Direct observation of steps in rotation of the bacterial flagellar motor. Nature, 437(7060): 916-9, 2005.

Electrokinetic Evaluation of Individual Exosomes by On-Chip Microcapillary Electrophoresis with Laser Dark-Field Microscopy

Cell-secreted nanovesicles called exosomes are expected as a promising candidate biomarker of various diseases. Toward the future application of exosomes as a disease biomarker for low-invasive diagnostics, challenges remain in the development of sensitive and precise analysis methods for exosomes. In this study, we performed the electrokinetic evaluation of individual exosomes by the combined use of on-chip microcapillary electrophoresis and laser dark-field microscopy. We extracted exosomes from six types of human cell cultured in a serum-free medium by differential ultracentrifugation and their zeta potential (electrophoretic mobility) were evaluated. We demonstrated that the proposed electrophoresis apparatus is particularly suitable for the tracking analysis of the electrophoretic migration of individual exosomes and enables the accurate evaluation of the zeta potential distribution of exosomes, for the first time. From the experimental results, we found that there is a strong correlation between the average zeta potentials of exosomes and their cells of origin.

Differential interferometric particle tracking on the subnanometer- and submillisecond-scale

We describe an interferometric method to measure the movement of a subwavelength probe particle relative to an immobilized reference particle with high spatial (Δx = 0.9nm) and temporal (Δt = 200μs) resolution. The differential method eliminates microscope stage drift. An upright microscope is equipped with laser dark field illumination (λ(0) = 532nm, P(0) = 30mW) and a compact modified Mach-Zehnder interferometer is mounted on the camera exit of the microscope, where the beams of scattered light of both particles are combined. The resulting interferograms provide in two channels subnanometer information about the motion of the probe particle relative to the reference particle. The interferograms are probed with two avalanche photodiodes. We applied this method to measuring the movement of kinesin along microtubules and were able to resolve the generic 8-nm steps at high ATP concentrations without external forces.

背景別しきい値設定によるDie-to-Golden Die比較と画像内自己参照比較に基づく高感度複合欠陥検査

Laser dark field inspection tools for LSI wafer patterns are very fast and often used in LSI fabrications. However with non-repetitive LSI patterns, the sensitivity of the conventional die-to-die inspection method is not sufficient due to the increase in noise that occurs in connection with highly coherent laser illumination and the variation of the thin film thickness of the LSI patterns. In this paper two methods are combined for highly sensitive image comparison : self-reference comparison and die-to-golden die comparison. The self-reference comparison searches for parts of similar patterns in each die and the die-to-golden die comparison generates images with similar patterns on the basis of adjacent dies. The combined methods adaptively select the parts to be referenced pixel by pixel. Experimental results showed they could successfully detect a low S/N defect signal of 0.12 in the non-repetitive patterns.

Blood oxygen flux estimation with a combined photoacoustic and high-frequency ultrasound microscopy system: a phantom study

The metabolic rate of oxygen consumption, an important indicator of tissue metabolism, can be expressed as the change of net blood oxygen flux into and out of a tissue region per 100 g of tissue. In this work, we propose a photoacoustic and Doppler ultrasound method for imaging local blood oxygen flux of a single vessel. An imaging system for combined photoacoustic and high-frequency ultrasound microscopy is presented. This system uses a swept-scan 25-MHz ultrasound transducer with confocal dark-field laser illumination optics. A pulse-sequencer enables ultrasonic and laser pulses to be interlaced so that photoacoustic and Doppler ultrasound images are co-registered. Since the mean flow speed can be measured by color Doppler ultrasound, the vessel cross-sectional area can be measured by power Doppler or structural photoacoustic imaging, and multi-wavelength photoacoustic methods can be used to estimate oxygen saturation (sO(2)) and total concentration of haemoglobin (C(Hb)), all of the parameters necessary for oxygen flux estimation can be provided. The accuracy of the flow speed and sO(2) estimation has been investigated. In vitro sheep blood phantom experiments have been performed at different sO(2) levels and mean flow speeds. Blood oxygen flux has been estimated, and the uncertainty of the measurement has been quantified.

Examination of Bow-Shock Formation in Supersonic Radiatively Cooled Plasma Flows

Radiative high-Z plasma bow shocks driven by a 200-kA current are investigated by using high-resolution dark-field laser Schlieren imaging and a Mach-Zehnder interferometer. Results demonstrate stationary high-density compressible bow shocks and provide data on the plasma Mach number and electron density.

Single Molecule Rotation of F1-ATPase from S. Cerevisiae

Single molecule studies of the thermophilic Bacillus PS3 F1-ATPase have revealed kinetic and structural information that cannot be discerned using other methods, including the presence of 40 and 80 degree physical substeps (Yasuda et al. 2001) and the order and kinetics of chemical substeps. We use single molecule techniques to observe the effects of Mitochondrial Genome Integrity mutations on enzyme kinetics and torque production in F1 from the yeast S. cerevisiae.Mitochondrial Genome Integrity (mgi) mutations allow yeast to survive the loss of mitochondrial DNA. A number of these mutations occur in the genes encoding the F1 portion of the ATP Synthase, and have been shown to uncouple ATP Synthase (Wang et al. 2007). The mutations cluster around the collar region of F1 where the alpha, beta and gamma subunits interact and are thus likely to affect F1 rotation.Using a high speed camera and a novel method for laser darkfield microscopy, we captured the rotation of wild-type and mgi forms of yeast F1-ATPase. We show for the first time that at saturating ATP, wild-type yeast F1 rotates approximately four times faster than the thermophilic F1. Kinetic and substepping behaviour in wildtype yeast appears to be similar to that observed in bacterial F1, but some of the mgi forms show behaviour that is different to both wildtype and previously reported forms of F1. We will use the results from these single molecule experiments in conjunction with structural studies to elucidate the mechanisms underlying rotation in wildtype and mgi forms of F1.This work was supported by NIH R01GM0662223.

Photoacoustic and high-frequency power Doppler ultrasound biomicroscopy: a comparative study

Both photoacoustic imaging and power Doppler ultrasound are capable of producing images of the vasculature of living subjects, however, the contrast mechanisms of the two modalities are very different. We present a quantitative and objective comparison of the two methods using phantom data, highlighting relative merits and shortcomings. An imaging system for combined photoacoustic and high-frequency power Doppler ultrasound microscopy is presented. This system uses a swept-scan 25-MHz ultrasound transducer with confocal dark-field laser illumination optics. A pulse-sequencer enables ultrasonic and laser pulses to be interlaced so that photoacoustic and power Doppler ultrasound images can be coregistered. Experiments are performed on flow phantoms with various combinations of vessel size, flow velocity, and optical wavelength. For the task of blood volume detection, power Doppler is seen to be advantageous for large vessels and high flow speeds. For small vessels with low flow speeds, photoacoustic imaging is seen to be more effective than power Doppler at the detection of blood as quantified by receiver operating characteristic analysis. A combination of the two modes could provide improved estimates of fractional blood volume in comparison with either mode used alone.

A method of laser dark-field illumination for equal-probability excitation and photobleaching of randomly oriented fluorescent molecules

Calculations have been done that allow developing a new type of illuminators optimized for dark-field microscopy and fluorescent nanoscopy, a new type of ultrahigh-resolution microscopy for which we have patent priority, and its later analogs. The concept proposed here substantiates the feasibility of uniformly illuminating different parts of an object to ensure equal probability of excitation and, in due time, simultaneous photobleaching of fluorescent molecules despite the random orientation of their light-absorbing oscillators. The latter is important because non-bleached fluorophores that accumulate in the specimen produce background noise and make it too difficult to locate the centers of spots produced by new fluorescent molecules.

A structured mechanism development and experimental parameter selection of laser scattering for the surface inspection of flat-panel glasses

The surface inspection of flat-panel glass is important in manufacturing processes. In this study, an experimental analysis based on dark-field laser scattering was performed to inspect micro-defects in flat-panel glass for infrared cut-off filters. The laser scattering parameters were investigated and scattering light paths analysed. On the basis of these results, a structured mechanism was designed and an experimental inspection system developed using the mechanism. By examining the effect of changing parameter values, it was found that two dark-field laser scattering parameters—the incident angle of the laser and the viewing angle of the camera—are critical for the intensity and distribution of the scattering light for accurate and robust surface inspection. Also, the difference of these angles is very important. Based on the design of experiment, previous experimental results are evaluated and predominant parameters are optimally determined. The results of the experimental inspection system demonstrate the effectiveness of the proposed method.

High-Resolution Laser Schlieren Imaging of Coronal Plasma Evolution in 80-kA $X$-Pinches

X-pinches driven by an 80-kA current are investigated by using high-resolution dark-field laser Schlieren imaging. Results demonstrate the evolution of the low-density coronal plasma and the formation of an axial jet. Quantitative measurements of plasma expansion rates and the jet velocity are carried out for several materials.