Pinhole Size Research Articles

Development of Fluorescence Label and Con-focal Laser Scanning Microscopy Method for Non-Destructive Local Impurity Distribution Analysis in Protein Crystals

A new method for quantitative analysis of the impurity concentration in protein crystals and solutions was developed. This technique utilizes fluorescence label (FL) with con-focal laser scanning microscopy (CLSM), which is more effective than SDS-PAGE analysis currently used for this purpose. The advantages of CLSM are that, it is non-destructive so that the impurity incorporation and local distribution could be observed in situ, and also that only a micro-quantity of protein solution is needed. The impurity protein is labeled with fluorescence material, and mixed with the crystallization solution. The solution and the crystal are observed by CLSM, and the fluorescence intensity from the labeled impurity is then converted to the impurity concentration by using calibration curves. A case study using Hen Egg White Lysozyme as a sample is reported. Calibration curves were obtained by comparing the fluorescence intensity and the actual impurity concentration determined by the absorbance at 280 nm and SDS-PAGE. A few factors such as the numerical aperture of the objective lens or the pinhole size were fixed. The utilization of this technique leads to the understanding of the effect of impurities on protein crystal growth.

Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes

Subtractive imaging in confocal fluorescence light microscopy is based on the subtraction of a suitably weighted widefield image from a confocal image. An approximation to a widefield image can be obtained by detection with an opened confocal pinhole. The subtraction of images enhances the resolution in-plane as well as along the optic axis. Due to the linearity of the approach, the effect of subtractive imaging in Fourier-space corresponds to a reduction of low spatial frequency contributions leading to a relative enhancement of the high frequencies. Along the direction of the optic axis this also results in an improved sectioning. Image processing can achieve a similar effect. However, a 3D volume dataset must be acquired and processed, yielding a result essentially identical to subtractive imaging but superior in signal-to-noise ratio. The latter can be increased further with the technique of weighted averaging in Fourier-space. A comparison of 2D and 3D experimental data analysed with subtractive imaging, the equivalent Fourier-space processing of the confocal data only, and Fourier-space weighted averaging is presented.

Influence of the Ar-ion irradiation on the giant magnetoresistance in Fe/Cr multilayers

The influence of 200 keV Ar-ion irradiation on the interlayer coupling in Fe/Cr multilayers exhibiting the giant magnetoresistance (GMR) effect is studied by the conversion electron Mössbauer spectroscopy (CEMS), vibrating sample magnetometer hysteresis loops, magnetoresistivity, and electric resistivity measurements and supplemented by the small-angle x-ray diffraction. The increase of Ar-ion dose causes an increase of interface roughness, as evidenced by the increase of the Fe step sites detected by CEMS. The modification of microstructure induces changes in magnetization reversal indicating a gradual loss of antiferromagnetic (AF) coupling correlated with the degradation of the GMR effect. Distinctly weaker degradation of AF coupling and the GMR effect observed for irradiated samples with a thicker Cr layer thickness suggest that observed effects are caused by pinholes creation. The measurements of temperature dependence of remanence magnetization confirm increase of pinhole density and sizes during implantation. Other effects which can influence spin dependent contribution to the resistance, such as interface roughness as well as shortening of mean-free path of conduction electrons, are also discussed.

Effects of ion beam assist on the formation of calcium phosphate film

Calcium phosphate films were formed by electron beam evaporation of β-TCP with and without simultaneous Ar ion beam bombardments. The Ca/P ratio was 0.76 without bombardments, but it was increased to 1.80 by bombarding of Ar ion beam extracted from the ion gun set to 0.8 A. Hydroxyapatite peaks were developed and became stronger with increases in beam current. The variations in chemical composition had an effect on the dissolution behaviors in isotonic saline solution showing two distinct features, pinhole and flat region. The dissolved thickness measured in the flat region of all calcium phosphate coating layers after 10 h immersion was similar with the range of 5450 and 6100 Å. However, the size of pinhole was larger for the calcium phosphate coating layer with the lower Ca/P ratio indicating more active dissolution.

Vapor pressure by DSC: extending ASTM E 1782 below 5 kPa

In 1996, American Society for Testing and Materials (ASTM) Method E 1782–96, Standard Test Method for Determining Vapor Pressure by Thermal Analysis received final approval and was published as a standard method. For vapor pressure measurements by DSC, the method requires use of hermetic-type sealable pans with a single pinhole ≤125 μm (50–75 μm recommended) in the center of the lid. Pinholes of this size produce generally acceptable results over the 5 kPa to 2 MPa operational pressure range of the method. However, as the minimum recommended pressure is approached, boiling endotherms become increasingly broad. Recent efforts using larger pinholes (175–375 μm) have shown improved peak sharpness and elimination of leading edge curvature when pinhole size is increased as pressure is reduced. This need for increased pinhole diameter at lower pressures is consistent with orifice selections used in effusion studies. Optimization of pinhole size as a function of pressure may extend the practical operational range of ASTM E 1782 to permit rapid vapor pressure determination to at least 0.2 kPa.

Diameter control and emission properties of carbon nanotubes grown using chemical vapor deposition

We grow multiwalled carbon nanotubes (CNTs) via thermal chemical vapor deposition from a sputtered 4-nm-thick nickel catalyst film on a tungsten-coated silicon substrate. CNTs grow from a mixture of nitrogen and acetylene gases at temperatures ranging from 630 to 790 °C, resulting in CNT outer diameters of 5–350 nm. CNT diameters increase exponentially with temperature. These results define regimes for template growth fabricated in catalytically active anodized aluminum oxide (AAO) with controlled pinhole sizes ranging from 10 to 50 nm. We measure a threshold electron emission field of 3 V/μm and a field enhancement factor β=5230 on randomly oriented 10-nm diameter CNTs.

Pinhole detection system for glass wool paper by electric discharge

A pinhole detection system for glass wool paper has been developed based on electric discharge phenomenon. The main part of the system consists of two electrodes, i.e. anode and cathode, through which the glass wool paper passes. The line cathode consists of many needles with an equidistant separation of 6 mm. Three sets of line cathode, with needles at intervals of 6 mm, were shifted 2 mm from each other. A high resistance of 100 MΩ was used in the electric circuit to control the discharge current, i.e. damage to the paper. The detectable pinhole size using this system was between 0.1 and 1 mm. The detection rate was above 70%. The production rate of satisfactory goods was increased to approximately 33% as demonstrated in a manufacturing plant.

Micro X-ray fluorescence using a pinhole aperture in quasi-contact mode

A cone shaped pinhole aperture (inner diameter, 0.3 mm and outer cone slope, 60 degrees) was developed to reduce the primary X-ray beam size of a conventional XRF setup. This aperture was used in combination with a single capillary. The pinhole was placed very close to the sample surface, that is, in quasi-contact mode. The gap size between the aperture and the sample was adjusted to be less than 0.5 mm. The X-ray fluorescence produced by the sample was measured from this gap at a small takeoff angle. The lateral resolution was evaluated using a tungsten wire placed at different gap sizes. The best resolution, which is almost equal to the pinhole size, was obtained in quasi-contact mode. The X-ray fluorescence signals of Au Lα and Co Kα from a Au-Co grating were analyzed while scanning the sample in front of the conical aperture. From this line scan, it was evident that the best resolution and the highest intensities of X-ray fluorescence were obtained in quasi-contact mode.

Understanding Dark Spot Formation and Growth in Organic Light-Emitting Devices by Controlling Pinhole Size and Shape

We report on our in-situ experimental observations of dark spots in organic light-emitting diodes using optical microscopy. Uniformly sized silica microparticles are used to intentionally create size- and shape-controllable pinholes on the cathode protective layer. Subsequently, the pinholes trigger the initial formation of dark spots, which we then monitor. Due to the use of particles of various diameters, we are able to linearly associate the growth rate with pinhole size. This allows us to estimate the original pinhole sizes that give rise to the dark spots and to study their distribution. Our studies verify that pinholes on the protective layer create pathways for water or oxygen diffusion, which controls the dark spot growth rate. The pinhole size dependence illustrates that the pinhole perimeter (not the area) determines the amount of water or oxygen diffusing into the diodes at a certain time.

Optic fibre bundle contact imaging probe employing a laser scanning confocal microscope.

A small diameter (600 micro m) fused optic fibre imaging bundle was used as a probe to compare fluorescent specimens by direct contact imaging using both a conventional fluorescence microscope and a laser scanning confocal microscope (LSCM) system. Green fluorescent polyester fibres placed on a green fluorescent cardboard background were used to model biological tissue. Axial displacement curves support the hypothesis that pinhole size in the LSCM system reduces the contribution of non-focal plane light. Qualitative comparison showed that the LSCM system produced superior image quality and contrast over the conventional system. The results indicate that the new LSCM-probe combination is an improvement over conventional fluorescence-probe systems. This study shows the feasibility of employing such a small diameter probe in the investigation of biological function in difficult to access areas.

Film model for coated cement concrete

The performance of a polymer concrete coated cement concrete under sulfuric acid environment was studied for 3 years. Both dry and wet concrete specimens were used in this study. The mass transfer coefficient ratio of cement concrete to polymer concrete was over 12. Coated cement concrete cylinders with pinholes were used to study the chemical resistance of the coated concrete under sulfuric acid environments to represent the worst sewer condition. Effects of pinhole sizes on the performance of coated concrete were studied, and changes in weight of the coated concrete specimens were measured regularly. The weight change in coated cement concrete was modeled using a film model and the effect of the pinhole sizes on the performance of the coated concrete was quantified.

Quantification in highly scattering/absorbing layered samples using photon time-of-flight measurements and confocal optical geometry

A method for depth-resolved quantification in scattering/absorbing layered samples is described. Confocal optical geometry was used in conjunction with time gated detection for non-invasive quantification of sub-surface absorbing constituents. For analysis, the time-resolved diffuse reflectance intensities from a series of layered absorbing samples were ratioed to that of a similar, non-absorbing specimen. Using this approach, absorbance information sensitive to changing sample composition was obtained. The effect of confocal optical geometry focussing on depth-resolved quantification was characterized using different portions of the time profile. In addition, imaging performance is assessed with varying focal depth, numerical aperture and effective pinhole size. Results indicate that the effect of multiple scattering on the detected signal may be reduced by using a larger numerical aperture objective and small effective pinhole size. Also, the sample absorbance was found to be more linear over a wider concentration range when compared with a large pinhole. When time information was included, the initial rising portion of the time profile was found to enhance sample absorbance linearity when a large pinhole is used. However, little enhancement was observed when the imaging pinhole size was small. Similar effects were seen at each focal depth in the sample. This finding suggests that although including time information may be beneficial, it is not needed when confocal light collection is employed in the analysis of macroscopic scattering samples.

Influence of Image Acquisition Parameters on Quantitative Measurements of Biofilms using Confocal Laser Scanning Microscopy

The influence of confocal laser scanning microscope (CLSM) image acquisition on quantitative measurements of entrapped fluorescent microbeads and biofilms was studied. The effects of acquisition settings such as pinhole size, detector gain, amplifier offset/gain, scan speed/average, zoom, and laser intensity on microbead area and fluorescence intensity were measured. Prior to the acquisition of biofilm images, microbeads were immobilized within biofilm samples as calibration controls. Pinhole size, detector gain, and amplifier offset/gain had significant influence on microbead area and intensity measurements, while scan speed/average, zoom, and laser intensity influenced intensity only. An optimal setting was selected based on the experiment in order to obtain the measurements with minimum error. Quantification of biofilm components was performed under optimal conditions and two non-optimal conditions (with respect to pinhole size, detector gain and amplifier gain), along with microbeads. Significant difference in percentage coverage of biofilm and microbeads were observed among these three conditions (P <0.0001). The non-optimal settings resulted in an increase in biofilm percentage area coverage of 43-88% as compared to optimal settings. The immobilized bead error was minimum (9.2%) under optimal conditions when compared to non-optimal conditions (55-96%). The results clearly show that immobilized microbeads serve as a useful control to determine the error in biofilm quantification caused by sub-optimal image acquisition settings. It is suggested that microscopic evaluations of biofilm parameters should be accompanied by calibration control using microbeads.

Simulation of confocal microscopy through scattering media with and without time gating

An efficient and fast simulation technique is presented to calculate characteristic features of confocal imaging through scattering media. The simulation can predict the time-resolved confocal response to pulsed illumination that allows optimizing of imaging contrast when time-gating techniques are applied. Modest computational effort is sufficient to obtain contrast predictions for arbitrary numerical aperture, focus depth, pinhole size, and scattering density, while the simulation accuracy is independent of scattering density and pinhole size. In the case of isotropic scattering, our results indicate that reflection-mode confocal imaging through scattering media is limited to μd≈3.5 optical thicknesses for continuous-wave illumination. If time-gating is applied, imaging through scattering densities of μd≈8 is theoretically possible.

How to design perforated polymeric films for modified atmosphere packs (MAP)

AbstractIncreasing proportions of fresh produce are being sold in modified atmosphere packs (MAP) with the aim of preserving product quality longer and reducing freight costs. A rigorous theoretical analysis was made of the transport phenomena across packaging film (composite, perforated, etc.) in order to find out whether polymeric film will permit a stationary modified atmosphere (MA) inside the pack, and if so when, and to investigate the effect of the size and shape of the holes in the perforated film. The continuity equations of the pack, for all diffusing species, were written and solved for stationary conditions, with the boundary conditions that species not involved in metabolic processes do not diffuse across polymeric film. After a detailed analysis of the transport phenomena across both continuous and perforated film, and of the metabolic rate processes, it transpires that no stationary conditions compatible with any MA can be found for continuous film, owing to the permeation characteristics of the film and the rate of the metabolic processes. With perforated film it is possible to find, at least for certain metabolic process rates, a stationary state where a constant MA is maintained inside the pack. A proposal is given, provided the rate of the metabolic process is known, for the design of a pack in terms of polymeric materials and of the pinhole size. Two case studies, strawberry and cabbage, are presented and discussed, along with the optimization of the polymeric film and the size and length of the pinholes of the packs. Another point raised deals with the advantages of using perforated film and/or of making holes or openings along the edges where the polymeric film is welded. Copyright © 2001 John Wiley &amp; Sons, Ltd.

3D micro-inspection goes DMD

Digital image-projection and video-projection are the classical applications of Digital Micromirror Devices (DMD) but further applications in the field of optical metrology are also possible. Operated with certain patterns, a DMD can function, for instance, as an array of pinholes that may substitute the Nipkow disk presently used for lateral scanning in confocal microscopes. The various process parameters that influence the measurement (e.g. pinhole size, pinhole shape, lateral step-size and the number of pinholes used simultaneously, etc.) can be configured easily for individual measurements by simply programming the DMD. Furthermore, price-drops for metrological precision instruments may be achieved by implementation of the mass article DMD. The paper explains the basics of DMD technology, illustrates the principle setup of a newly designed instrument, shows results achieved with it and gives examples for its application.

Design and testing of a novel microscopic photon correlation spectrometer with higher accuracy

A novel microscopic photon correlation spectrometer has been designed and developed in order to employ photon correlation spectroscopy to investigate dynamic properties within a microscopic volume. It shows a spatial resolution scale of about 0.5 micrometres and a relaxation timescale of milliseconds. The variance of the scattering vector is less than 5% even at a smaller scattering angle. Furthermore, a standard performance test using 94±13 nm polystyrene latex beads has been performed on the apparatus. The autocorrelation function value at the first channel is higher than 0.8, that indicates a higher signal-to-noise ratio and a good coherent detection. CONTIN analysis gives the latex beads size as 94±2 nm. Different angle scattering results give the translation diffusion coefficient (4.57±0.08)×10-8 cm2 s-1 at 293.15 K, that agrees with the theory very well. Besides, scattering spectroscopy using different pinhole sizes determines the optimized configuration for the spectrometer with higher accuracy. All these document that the designed system works much more accurately than previous systems and may probe dynamic information from different local sites of a single living cell non-invasively.

Correlation between dark spot growth and pinhole size in organic light-emitting diodes

Our in situ experimental observations of dark spot growth in organic light-emitting diodes using optical microscopy show a linear rate of growth for the area of all the dark spots. We used uniformly sized silica micro particles to intentionally create size-controllable pinholes on the cathode protective layer. Subsequently, we observed initial formation of dark spots as a result of these pinholes and then monitored their growth. Due to usage of particles of various diameters, we were able to linearly correlate the growth rate with pinhole size. This allows us to estimate the original pinhole sizes that gave rise to the dark spots, which we believe were initiated by “dust” particles. Our studies verify that dark spot formation is due to pinholes on the protective layer that creates pathways for water or oxygen permeation, and that dark spot growth is dependent on the pinhole sizes.

Characterization of defects in the formation process of self-assembled thiol monolayers by electrochemical impedance spectroscopy

Abstract Self-assembled monolayers of octadecanethiol (ODT) on gold have been studied by electrochemical impedance spectroscopy (EIS). The fractional coverage, pinhole size and separation have been examined as a function of immersion time of Au in the ODT deposition solution. The fractional coverage of ODT monolayer increases sharply from zero to more than 99% of its maximum within the first minute. However, it takes hours or a day for the fractional coverage to approach its final value. The pinhole separation increases from ca. 1.8 μm to 53 μm as the assembling time increases from 5 s to 24 h, indicating that the number of pinholes decreases. The pinhole radius increases slowly from ca. 0.3 μm to 1.6 μm in the same time interval. The slight trend of increasing pinhole size with adsorption time is attributed to the increasing influence of collapsed sites in the ODT monolayer.

Scapholunate ligamentous communicating defects in symptomatic and asymptomatic wrists: characteristics.

To determine whether the sizes and locations of scapholunate ligamentous communicating defects are different in symptomatic and asymptomatic cases. Bilateral wrist arthrograms were reviewed for 30 consecutive patients with a history of wrist trauma and unilateral wrist pain who had at least one scapholunate ligamentous communicating defect and unremarkable conventional radiographs. The location and size of each ligamentous defect was recorded. Differences between symptomatic and asymptomatic wrists were analyzed with the chi(2) or Fisher exact test. Most communicating defects in both groups were incomplete and ranged from pinhole size to large. There was a higher frequency of complete disruption in the symptomatic wrists (nine [32%] of 28 wrists) than in the asymptomatic wrists (two [10%] of 20 wrists; P: =.092). Communicating defects involved the dorsal portion in 18 (64%) of the 28 symptomatic cases and in five (25%) of the 20 asymptomatic cases (P: =.007). The data suggest that the demonstration of a complete ligamentous disruption or involvement of the dorsal portion of the ligament may indicate a traumatic cause rather than a degenerative change.