Quantitative structured-illumination phase microscopy

We introduce a quantitative phase imaging method for homogeneous objects with a bright field transmission microscope by using an amplitude mask and a digital processing algorithm. A known amplitude pattern is imaged on the sample plane containing a thick phase object by placing an amplitude mask in the field diaphragm of the microscope. The phase object distorts the amplitude pattern according to its optical path length (OPL) profile, and the distorted pattern is recorded in a CCD detector. A digital processing algorithm then estimates the object's quantitative OPL profile based on a closed form analytical solution, which is derived using a ray optics model for objects with small OPL gradients.     

Quantitative second-harmonic generation microscopy in collagen

The second-harmonic signal in collagen, even in highly organized samples such as rat tail tendon fascicles, varies significantly with position. Previous studies suggest that this variability may be due to the parallel and antiparallel orientation of neighboring collagen fibrils. We applied high-resolution second-harmonic generation microscopy to confirm this hypothesis. Studies in which the focal spot diameter was varied from approximately 1 to approximately 6 microm strongly suggest that regions in which collagen fibrils have the same orientation in rat tail tendon are likely to be less than approximately 1 microm in diameter. These measurements required accurate determination of the focal spot size achieved by use of different microscope objectives; we developed a technique that uses second-harmonic generation in a quartz reference to measure the focal spot diameter directly. We also used the quartz reference to determine a lower limit (dXXX > 0.4 pm/V) for the magnitude of the second-order nonlinear susceptibility in collagen.     

Quantitative aspects of grain size measurement

Abstract

Grain size measurement is directly dependent on the ability of the microstructure to be revealed in a form that is representative of the material. A single phase, equiaxed ferritic steel was used throughout the present investigative work, this material being chosen because of the apparent simplicity of the microstructure. The lineal intercept, circular intercept, and planimetric measurement techniques were used. All the results are reported using the ASTM grain size number, G. Two aspects of grain size measurement are reported in the present paper. The first is the impact of missing boundaries on grain size measurements. The etching techniques established within industry to reveal microstructures often only partially reveal grain boundaries. An experiment is reported where the impact of missing grain boundaries on grain size measurements is assessed and hence the importance of revealing all grain boundaries is determined. An image analysis system was used to completely reconstruct the microstructure in a binary form, then to remove a known percentage of the boundaries, followed by measuring the grain size using the different techniques. The selection of the boundaries to be removed was done randomly to allow for any bias. The results reported show that, even with up to 20% missing boundaries, the impact on the grain size measurement was not significant, giving a difference of ～0.5 grain size units. Sampling is the second factor studied. In order for measurements to be representative the number of grains within a field of view from each specimen, the number of fields of view per specimen, and the number of specimens have to be considered. From an analysis of the results of the characterisation of the ferritic steel it was clear that the number of specimens used for measurement was the most important factor regarding microstructural representation.     

Quantitative modelling in scanning probe microscopy

Significant progress has been made in the theoretical modelling of scanning probe microscopy. The models available now are sufficiently refined to provide information not only about the surface, but also the probe tip, and the physical changes occurring during the scanning process. This has significantly improved the quantitative analysis of experimental and theoretical results. Scanning probe microscopes can now be reliably used to analyse events on the level of single atoms and single electrons.     

Quantitative phase measurements using optical quadrature microscopy

Imaging of phase or optical path length is becoming more important with the development of better imaging systems, computational algorithms, faster computers, and a greater interest in the imaging of transparent objects. Early phase imaging involved qualitative imaging of phase gradients. New computational algorithms can be used to extract some quantitative phase imaging from these techniques. In contrast, new hardware has enabled full-field quantitative phase imaging on a practical and cost-effective scale. We explore a quantitative comparison between two techniques for imaging phase. In the first technique, phase is recovered from a pair of differential interference contrast images, and in the second technique, phase is measured pixel-by-pixel interferometrically. It is shown, experimentally, that the overall results are similar, but each technique has its own advantages and disadvantages.     

Quantitative analysis of historical mortars using optical microscopy

As a part of the activities of the RILEM TC 167-COM committee two methods of quantitative microscopy have been developed for analysing historical mortars. This paper gives background information for the application of these methods. The methods concern the determination of mix proportions and aggregate size distribution using microscopical methods. A method is also described for correcting determinations of mix proportions based on chemical analysis of acid-soluble calcium oxide for the presence of carbonate in the aggregate.

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Résumé Dans le cadre des activités de la Commission RILEM TC 167-COM, deux méthodes de microscopie quantitative ont été développées pour l’analyse des mortiers historiques. Ce document donne des informations sur les antécédents de l’application de ces méthodes. Celles-ci concernent la détermination des proportions de mélanges et la répartition des tailles des appareils en utilisant la méthode microscopique. Une méthode est également décrite pour la correction des déterminations des proportions de mélanges, à partir de l’analyse chimique de l’oxyde de calcium soluble dans l’acide pour déterminer la présence de carbonate dans l’appareil.

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TC MEMBERSHIP: Chairman: Caspar Groot, the Netherlands.Secretary: Geoff Ashall, UK.Members: Giulia Baronio, Italy; Peter Bartos, UK; Luigia Binda, Italy; Kristof Callebaut, Jan Elsen, Belgium; Rob van Hees, the Netherlands; John Hughes, UK; Loek van der Klugt, the Netherlands; Jan Erik Lindqvist, Sweden; Elisabeth Marie-Victorie, France; Bernhard Middendorf, Germany; Ioanna Papayianni, Greece; Margaret Thomson, USA; Eleni-Eva Toumbakari, Greece; Alf Waldum, Norway.     

Quantitative phase microscopy using dual-plane in-line digital holography

We present detailed theoretical evaluation and thorough experimental investigation of quantitative phase imaging using our previously demonstrated dual-plane in-line digital holographic microscopy technique [Opt. Lett. 35, 3426 (2010)]. This evaluation is based on the recording of two interferograms at slightly different planes and numerically reconstructing the object information. The zero-order diffracted wave is eliminated by using the method of subtraction of average intensity of the entire hologram, and the twin-image diffracted wave is removed by Fourier domain processing of the two recorded holograms. Experiments are performed using controlled amplitude and phase objects and human muscle cells to demonstrate the potential of this technique.     

Quantitative measurements of apoptotic cell properties using acoustic microscopy

Time-resolved acoustic microscopy was used to measure properties of cells such as the thickness, sound velocity, acoustic impedance, density, bulk modulus, and attenuation, before and after apoptosis. A total of 12 cells were measured, 5 apoptotic and 7 non-apoptotic. Measurements made at 375 MHz showed a statistically significant increase in the cell thickness from 13.6 ± 3.1 μm to 17.3 ± 1.6 μm, and in the attenuation from 1.08 ± 0.21 dB/cm/MHz to 1.74 ± 0.36 dB/cm/MHz. The other parameters, such as the sound velocity, density, acoustic impedance, and bulk modulus remained similar within experimental error. Acoustic images obtained at 1.0 GHz showed increased RF-signal backscatter and a clear delineation of the nucleus and cytoplasm from apoptotic cells compared with non-apoptotic cells. Extensive activity was observed optically and acoustically within apoptotic cells. Acoustic measurements made one minute apart showed variations in the ultrasonic backscatter but not attenuation in the cells, which indicated rapid structural changes were occurring but not changes in bulk composition. The normalized crosscorrelation coefficient was used to quantify the variations in the backscatter RF-signal during apoptosis by comparing the first RF signal measured to each successive RF signal every 10 s. A coefficient of 1 indicates strong correlation, whereas a coefficient of 0 indicates no correlation. An average correlation coefficient of 0.93 ± 0.05 was measured for non-apoptotic cells, compared with 0.68 ± 0.17 for apoptotic cells, indicating that the RF signal as a function of time varied rapidly during apoptosis.     

Quantitative phase imaging in confocal microscopy by optical differentiation

The technique of optical differentiation is applied to confocal microscopy for the purpose of quantitative phase imaging. One-dimensional absorptive filters are placed in the pupil of the microscope objective to produce images related to the local phase slope in the object. With suitable signal processing and integration, a quantitative phase profile is obtained. This method is demonstrated in a reflection-based surface-profiling instrument.     

Quantitative fluorescence microscopy to determine molecular occupancy of phospholipid vesicles

Encapsulation of molecules in phospholipid vesicles provides unique opportunities to study chemical reactions in small volumes as well as the behavior of individual proteins, enzymes, and ribozymes in a confined region without requiring a tether to immobilize the molecule to a surface. These experiments generally depend on generating a predictable loading of vesicles with small numbers of target molecules and thus raise a significant measurement challenge, namely, to quantify molecular occupancy of vesicles at the single-molecule level. In this work, we describe an imaging experiment to measure the time-dependent fluorescence from individual dye molecules encapsulated in ~130 nm vesicles that are adhered to a glass surface. For determining a fit of the molecular occupancy data to a Poisson model, it is critical to count empty vesicles in the population since these dominate the sample when the mean occupancy is small, λ ≤ ~1. Counting empty vesicles was accomplished by subsequently labeling all the vesicles with a lipophilic dye and reimaging the sample. By counting both the empty vesicles and those containing fluors, and quantifying the number of fluors present, we demonstrate a self-consistent Poisson distribution of molecular occupancy for well-solvated molecules, as well as anomalies due to aggregation of dye, which can arise even at very low solution concentrations. By observation of many vesicles in parallel in an image, this approach provides quantitative information about the distribution of molecular occupancy in a population of vesicles.     

Quantitative characterization of pores in transparent ceramics by coupling electron microscopy and confocal laser scanning microscopy

This work is dedicated to the implementation of a new characterization method of porosity in transparent ceramics. This quantitative method couples scanning electron microscopy with confocal laser scanning microscopy. From this original method, the volume fraction of pores has been determined for different sintered Nd:YAG specimens with an accuracy of about 10%. This technique appears to be promising because it leads to both the pore size distribution and the residual porosity for fully dense samples. For example, it becomes possible to reach porosity levels ranging from 0.09% to 0.0004% for transparent Nd:YAG specimens which could not be measured by using conventional techniques. Finally, correlations between the residual porosity of these full dense samples and their optical properties could be established.     

Quantitative characterization of clay dispersion in polypropylene-clay nanocomposites by combined transmission electron microscopy and optical microscopy

This paper presents a novel method to describe the microstructure of polymer/clay nanocomposites quantitatively. Based on the image analyses of transmission electron microscopy (TEM) and optical microscopy micrographs, two parameters, degree of dispersion (χ) and mean interparticle distance per unit volume of clay (λ_V) are proposed to describe the level of clay dispersion. The degree of dispersion gives the percentage of exfoliation, and λ_V is a measure of spatial separation between particles relative to clay loading. A polypropylene/clay system was chosen as an example to show the effects of processing conditions and biaxial stretching on clay dispersion using the proposed quantifiers. It provides insights into the ‘real’ clay dispersion using a combination of both microscopical and macroscopical aspects.     

Quantitative subsurface contact resonance force microscopy of model polymer nanocomposites

We present experimental results on the use of quantitative contact resonance force microscopy (CR-FM) for mapping the planar location and depth of 50?nm diameter silica nanoparticles buried beneath polystyrene films 30-165?nm thick. The presence of shallowly buried nanoparticles, with stiffness greater than that of the surrounding matrix, is shown to locally affect the surface contact stiffness of a material for all depths investigated. To achieve the necessary stiffness sensitivity, the CR-FM measurements are obtained utilizing the fifth contact eigenmode. Stiffness contrast is found to increase rapidly with initial increases in force, but plateaus at higher loads. Over the explored depth range, stiffness contrast spans roughly one order of magnitude, suggesting good depth differentiation. Scatter in the stiffness contrast for single images reveals nonuniformities in the model samples that can be explained by particle size dispersity. Finite element analysis is used to simulate the significant effect particle size can have on contact stiffness contrast. Finally, we show how measurements at a range of forces may be used to deconvolve particle size effects from depth effects.     

Quantitative phase imaging of live cells using fast Fourier phase microscopy

Using the decomposition of an image field in two spatial components that can be controllably shifted in phase with respect to each other, a new quantitative-phase microscope has been developed. The new instrument, referred to as the fast Fourier phase microscope (f-FPM), provides a factor of 100 higher acquisition rate compared with our previously reported Fourier phase microscope. The resulting quantitative-phase images are characterized by diffraction limited transverse resolution and path-length stability better than 2 nm at acquisition rates of 10 frames/s or more. These features make the f-FPM particularly appealing for investigating the structure and dynamics of live cells over a broad range of time scales. In addition, we demonstrate the possibility of examining subcellular structures by digitally processing the amplitude and phase information provided by the instrument. Thus we developed software that can emulate phase contrast and differential interference contrast microscopy images by numerically processing FPM images. This approach adds the flexibility of digitally varying the phase shift between the two interfering beams. The images obtained appear as if they were recorded by variable phase contrast or differential interference contrast microscopes that deliver an enhanced view to the subcellular structure when compared with the typical commercial microscope.     

Quantitative analysis of electrodeposited tin film morphologies by atomic force microscopy

This study of the electrodeposition of tin on steel substrates demonstrates that it is possible to obtain quantitative information on the thin film growth at industrially relevant substrates using atomic force microscopy (AFM) to monitor the film morphology and X-ray fluorescence (XRF) to measure the average film thickness. The effects of current density and electrolyte temperature on the film morphology, surface roughness, and grain size distribution (GSD) are reported. While the roughness of the substrates used in this study can vary by several hundred nanometers to a micrometer, we are interested in quantitative characterization of the tin films with thickness varying from a few tens of nanometers to several hundred nanometers. This study shows that for the range of film thickness and length scale studied, analysis of the AFM images can provide quantitative characterization of the thin film roughness and grain size distribution at various stages of growth with little interference from the substrate morphological inhomogeneities.     

Characterization of ohmic contacts to InP

The results of a study of the electrical and metallurgical properties of thin metallic layers deposited on InP for use as ohmic contacts are presented. The layers were heat treated at temperatures up to 550°C and were examined with Auger electron spectroscopy. For contact to n-type InP three thin film systems were investigated: gold, nickel and a composite Ni/Au/Ge layer. Nickel was found to produce ohmic behavior in the Ni/Au/Ge/InP system with a minimum specific contact resistance r_c of 3×10^?5 Ω cm² for a net doping of 3×10¹⁶ cm^?3. For contact to p-type InP a film consisting of Au/Mg was investigated. For heat treatment of the Au/Mg/InP system above 350°C, r_c decreased as the temperature of the heat treatment increased and the surface morphology exhibited increasing signs of alloying at higher temperatures. The smoothest surface was obtained at 446°C for 50 min with r_c≈1×10^?4Ω cm² for a net doping of 6×10¹⁷ cm^?3.     

Self-heating effect at graphite ohmic heating

We present the numerical modeling results of an experiment where the self-heating process was observed. The results confirm the supposition (put forward earlier) that self-heating occurs during the transmission of an electric current through a graphite specimen at temperatures above 3000 K and is the consequence of a combination of low thermal conductivity values and strong temperature dependence of specific electrical resistivity.     

ZnO上欧姆接触的研究进展

为制备高性能的ZnO基器件如UV光发射器，探测器、场效应晶体管，在ZnO上形成优良的金属电极是十分必要的。回顾了近年来ZnO上制备欧姆接触的新进展，对在n型ZnO上制备欧姆接触的Al，A1/Pt，A1/Au，Ti/Al，Ti，AU，Ti/A1/Pt/Au，Re/Ti/Au等金属化方案的性能与特点，以及影响欧姆接触电阻率和热稳定性的因素，如表面处理和退火等进行了分析与归纳。同时，对P型ZnO上难以获得低接触电阻的原因进行了讨论。文章还简要说明了ZnO上透明欧姆接触的研究现状，指出获得低阻、高导电、高透光和高热稳定性的接触是未来ZnO基光电器件的发展方向。     

Quantitative phase and refractive index measurements with point-source digital in-line holographic microscopy

Point-source digital in-line holographic microscopy with numerical reconstruction is ideally suited for quantitative phase measurements to determine optical path lengths and to extract changes in refractive index within accuracy close to 0.001 on the submicrometer length scale. This is demonstrated with simulated holograms and with detailed measurements on a number of different micrometer-sized samples such as suspended drops, optical fibers, as well as organisms of biological interest such as E. coli bacteria, HeLa cells, and fibroblast cells.