Determination of the size distribution of lecithin liposomes: a comparative study using freeze fracture, cryoelectron microscopy and dynamic light scattering

The size distribution of liposomes is often determined using freeze fracture, cryoelectron microscopy or dynamic light scattering. However, the resulting size distributions do not directly coincide owing to the different weighting of the techniques. We present several methods which correct for these effects and allow a comparison of liposome size distributions as obtained by freeze fracture, cryoelectron microscopy or dynamic light scattering. These methods are based on theoretical models for the weighting of the size distribution of liposomes, which result from the preparation procedure for freeze fracture electron microscopy and from the measurement by dynamic light scattering. The proposed transformation methods are then experimentally tested with a sample of lecithin liposomes, whose size distribution was determined by dynamic light scattering, freeze fracture and cryoelectron microscopy. Furthermore, the weaknesses of the experimental techniques and hence of the resulting size distributions are discussed.     

The refractive index and dry mass distribution of mammalian erythrocytes

Quantitative microscopical methods are now of outstanding importance in the diagnosis and assessment of mammalian red blood cells. After release from the erythropoetic organs, all red blood cells in the peripheral blood of mammals pass through the reticulocyte stage before becoming mature erythrocytes. Therefore, the determination of reticulocyte number is used as an index of erythropoetic activity in clinical hæmatology. The corpuscular solid concentration, the corpuscular dry weight, the osmotic resistance and the size of red blood cells can be determined by use of microscopical methods. For the determination of the corpuscular solid concentration of erythrocytes, cell refractometry by the use of phase-contrast microscopy has proved to be a very sensitive and accurate method. The dry weight of single erythrocytes may likewise be obtained by the use of interference microscopy with relatively high accuracy. Both of these methods might also be of use in clinical hæmatology. In the experimental work described in this paper, both methods mentioned above are used to answer the question: whether and under what conditions the refractive index as a criterion of measurement of the solid concentration and the relative dry weight can be determined separately for reticulocytes and erythrocytes from the same blood sample by use of microscopical methods alone. The frequency distributions of refractive indices and relative dry weights determined on human reticulocytes and mature erythrocytes show significantly lower mean values for the reticulocyte generation in accordant with findings published by other authors and obtained by use of sometimes entirely different methods. The additional information gained by the determination of the frequency distribution of refractive index and dry weight of reticulocytes and mature erythrocytes separately in a blood sample and the necessity for simplifying and automating such methods for hæmatological diagnosis are discussed.     

Moving EM: the Rapid Transfer System as a new tool for correlative light and electron microscopy and high throughput for high-pressure freezing

In this paper, the Rapid Transfer System (RTS), an attachment to the Leica EMPACT2 high‐pressure freezer, is described as a new tool for special applications within the cryofixation field. The RTS is an automated system that allows for fast processing of samples (<5 s) that make it possible for the first time to use high‐pressure freezing in combination with high time resolution correlative light and electron microscopy. In addition, with a working cycle of 30 s this rapid turn over time allows one to acquire more samples of biopsy material before it deteriorates than with other HPF machines with longer cycle times. With the use of the RTS it was possible to obtain three samples each of four different tissues in 6 min. Together with the finding that 90% of samples of cells grown on sapphire discs were well frozen, the RTS was both fast and reliable. Most important, together with other newly developed accessories, the RTS made it possible to capture specific events occurring live in the cell as observed by light microscopy, to cryofix that sample/event within 4 s, and then to analyze that event at high resolution in the electron microscope with excellent preservation of ultra‐structure. These developments should give us the tools to unravel intracellular processes that can be observed by live cell imaging but are too rare or fast to be picked up by routine EM methods or where the history of a structure is necessary to be able to discern its nature.     

Nanoscale characterization of gold nanoparticles created by in situ reduction at a polymeric surface

Transmission Electron Microscopy is used as a quantitative method to measure the shapes, sizes and volumes of gold nanoparticles created at a polymeric surface by three different in situ synthesis methods. The atomic number contrast (Z‐contrast) imaging technique reveals nanoparticles which are formed on the surface of the polymer. However, with certain reducing agents, the gold nanoparticles are additionally found up to 20 nm below the polymer surface. In addition, plan‐view high‐angle annular dark‐field scanning transmission electron microscopy images were statistically analyzed on one sample to measure the volume, height and effective diameter of the gold nanoparticles and their size distributions. Depth analysis from high‐angle annular dark‐field scanning transmission electron microscopy micrographs also gives information on the dominant shape of the nanoparticles.     

一种平面运动位姿的并联组合测量方法研究

基于特殊的测量环境需要，提出了用于平面运动位姿测量的并联组合测量方法。介绍了并联组合测量方法的测量机构组成和测量原理，并进行了可行性论证；通过建立误差模型，对几何误差源与原始测量参数的映射关系及其对最终位姿测量误差的影响进行了分析，仿真结果和实际应用测量数据验证了分析的正确性。所述并联组合测量方法构思新颖，结构合理，适用于具有一定特殊测量条件的高精度平面大范围运动过程位姿测量。如果在工程应用中有效地控制几何误差源的影响，该方法则具有一定的推广应用价值。     

Comparison of quartz crystallographic preferred orientations identified with optical fabric analysis,electron backscatter and neutron diffraction techniques

Three techniques are used to measure crystallographic preferred orientations (CPO) in a naturally deformed quartz mylonite: transmitted light cross‐polarized microscopy using an automated fabric analyser, electron backscatter diffraction (EBSD) and neutron diffraction. Pole figure densities attributable to crystal‐plastic deformation are variably recognizable across the techniques, particularly between fabric analyser and diffraction instruments. Although fabric analyser techniques offer rapid acquisition with minimal sample preparation, difficulties may exist when gathering orientation data parallel with the incident beam. Overall, we have found that EBSD and fabric analyser techniques are best suited for studying CPO distributions at the grain scale, where individual orientations can be linked to their source grain or nearest neighbours. Neutron diffraction serves as the best qualitative and quantitative means of estimating the bulk CPO, due to its three‐dimensional data acquisition, greater sample area coverage, and larger sample size. However, a number of sampling methods can be applied to FA and EBSD data to make similar approximations.     

Edge-corrected estimators of the nearest-neighbour distance distribution function for three-dimensional point patterns

In multiphase systems consisting of ‘particles’ embedded in a matrix the three-dimensional spatial distribution of the particles may represent important structural information. In systems where the matrix is transparent or translucent recent developments in microscopy allow the three-dimensional location of particles to be recorded. Using these data a spatial statistical, or second-order stereological, analysis can be carried out. In second-order stereology functions of interparticle distances are used as summary statistics of the spatial distributions. These statistics show whether the particles are randomly arranged or, more commonly, either clustered together or inhibited from close approach to each other. This paper focuses on the estimation of one of these spatial statistics, the nearest-neighbour distance distribution function or G-function. In practice, estimation of the G-function is plagued by an ‘edge-effect’ bias introduced by the sampling process itself. There exist a number of G-function estimators that tackle this edge effect problem; for single sample ‘bricks’ it can be shown that these estimators become increasingly accurate as the brick size increases, i.e. they are consistent. However, in many practical cases the size of a sampling brick is fixed by experimental constraints and in these circumstances the only way to increase sample size is to take replicated sampling regions. In this paper we review a number of existing G-function estimators and propose a new estimator. These estimators are compared using the criterion of how well they overcome the edge-effect when they are applied to replicated samples of a fixed size of brick. These comparisons were made using Monte-Carlo simulation methods; the results show that three existing estimators are clearly unsuitable for estimating the G-function from replicated sample bricks. Of the other estimators the recommended estimator depends upon the number of replicates taken; however, we conclude that if a total of more than about 800 points are analysed then the bias in the pooled estimate of the G-function can be reduced to tolerable levels.     

An on-line computer system for point counting stereology

Efficient application of point counting stereological methods in quantitative microscopy in biology and materials science depends on the availability of powerful and rapid counting and computing facilities. An on-line computer system is described which is easily programmed without knowledge of a special programming language to perform counting, computation of parameters, and statistical tests automatically. By means of a master program, provided with the system, specific user's programs are compiled on a teletype in conversation with the computer by following simple instructions. The program is distinguished by great flexibility; it provides for statistical tests on the reliability of the sample analysed at any time during analysis. The final results are printed in tabular form as means and standard errors of the mean.     

Mapping piezoelectric-field distribution in gallium nitride with scanning second-harmonic generation microscopy

Taking advantage of the electric field-enhanced second-harmonic generation effect in bulk gallium nitride (GaN) and indium gallium nitride (InGaN) quantum wells, we demonstrated the piezoelectric field distribution mapping in bulk GaN and InGaN multiple-quantum-well (MQW) samples using scanning second-harmonic generation (SHG) microscopy. Scanning SHG microscopy and the accompanying third-harmonic generation (THG) microscopy of the bulk GaN sample were demonstrated using a femtosecond Cr:forsterite laser at a wavelength of 1230 nm. Taking advantage of the off-resonant electric field-enhanced SHG effect and the bandtail state-resonance THG effect, the second- and third-harmonic generation microscopic images obtained revealed the piezoelectric field and bandtail state distributions in a GaN sample. Combined with 720 nm wavelength excited two-photon fluorescence microscopy in the same sample, the increased defect density around the defect area was found to suppress bandedge photoluminescence, to increase yellow luminescence, to increase bandtail state density, and to decrease residue piezoelectric field intensity. Scanning SHG microscopy of the InGaN MQW sample was resonant excited with 800 nm femtosecond pulses from a Ti:sapphire laser in order to suppress SHG contribution from the bulk GaN substrate. Taking advantage of the strong piezoelectric field inside the InGaN quantum well, the wavelength resonant effect, and the electric field-enhanced SHG effect of InGaN quantum wells, resonant scanning SHG microscopy revealed the piezoelectric field distribution inside the wells. Combined with accompanying three-photon fluorescence microscopy from the bulk GaN substrate underneath the quantum wells, the direct correspondence between the piezoelectric field strength inside the quantum well and the substrate quality can be obtained. According to our study, the GaN substrate area with bright bandedge luminescence corresponds to the area with strong SHG signals indicating a higher stained-induced piezoelectric field. These scanning harmonic generation microscopies exhibit superior images of the piezoelectric field and defect state distributions in GaN and InGaN MQWs not available before. Combining with scanning multiphoton fluorescence microscopy, these techniques open new ways for the physical property study of this important material system and can provide interesting details that are not readily available by other microscopic techniques.     

Six common errors cause dangerous mistakes in interpretation of electron micrographs

The highly complex techniques of electron microscopy made it bound to the sensitive and critical micrograph analysis. The accurately interpreted micrographs are of paramount values in basic investigations. Interpretation skills and quality of the micrographs are the two fundamental keys in accomplishment of these goals but there are many mistakes and errors that can happen during the sample preparation, sectioning, EM operation, and photo publishing. The mentioned mistakes and errors effect directly in the final result which is a micrograph and can lead the analyzer who can be a pathologist to an interpretation followed by serious danger for patient. Artifacts caused by any given stimuli expected to be bothersome for investigators. Even best qualified equipments can be regarded as source of artifact generation. In this article, seven serious errors in electron micrographs which usually occur in transmission electron microscopy are addressed.     

Studies of supported metal catalysts using high resolution secondary electron imaging in a STEM

An additional technique for use in the characterization of catalysts by electron microscopy is presented. High resolution secondary electron images obtained in a VG HB501 scanning transmission electron microscope have been used to study the surface topography of catalysts consisting of small metal particles on high surface area carbon supports. Surface features down to nanometre dimensions can be seen, allowing the examination of micropores in the support as well as larger pore structures. The results are compared with pore size distributions determined by gas adsorption methods, and are shown to yield valuable additional information. In addition, the method in principle allows examination of the locations of small metal catalyst particles on the support.     

DEVELOPMENT AND ANALYSIS OF TIME VARIANT DISCRETE FOURIER TRANSFORM ORDER TRACKING

Several of the common order tracking methods for solving noise and vibration problems of rotating machinery are reviewed. These methods include the fast Fourier transform (FFT) and resampling based order tracking algorithms. The time variant discrete Fourier transform (TVDFT) is developed as a new and alternative order tracking method. This method contains many advantages which the current order tracking methods do not possess. The TVDFT has the advantage of being very computationally efficient as well as the ability to minimise leakage errors. The basic TVDFT method may also be extended to a more complex method through the use of an orthogonality compensation matrix which can separate closely spaced orders as well as separate the contributions of crossing orders. The basic TVDFT is a combination of the FFT and the resampling based methods, a special case of the chirp-z transform. This method can be formulated in several different manners, one of which will give results matching the resampling based methods very closely. Both analytical and experimental data are used to establish the behavioural characteristics of this new method.     

A fast reciprocal space method for image simulation

In this paper, we introduce a fast reciprocal space method for image simulation. It is well known that the scattering matrix (SM) P with NxN elements consists of N different structure factors and N different excitation errors. However, most structure factors of the SM P are extremely small so that they can be neglected. Therefore, the size of the SM P is reduced drastically. On the other hand, the structure factors have two-dimensional space group symmetries, so that by reducing the symmetry related structure factors to symmetrically independent structure factors, the size of the SM P can be reduced further. The calculation speed based on the simplified SM P will be several hundred times faster than that by other conventional methods. In this paper, we describe the method for how to reduce the SM P in detail and give an example of implementation.     

Errors in the evaluation of the coverage factor as a criterion of applications of approximate methods of evaluation of expanded uncertainty

The paper presents the results of the research on the accuracy of the chosen, approximate methods of evaluation of expanded uncertainty most often used for a simple, direct measurement when there are two standard uncertainties and the sample is small. The research was conducted on the basis of the knowledge of the convolution of the probability distributions of component errors.     

原子力显微镜管式扫描器运动学建模与误差分析

针对样本扫描模式原子力显微镜,对其管式扫描器-样本-探针系统进行了运动学分析,建立了该系统的运动学模型,该模型表明:对于给定原子力显微镜扫描器,样本上与探针接触点的横向和纵向位移取决于探针尖端相对于扫描管轴心的偏置量、所加电压(或名义扫描范围)及样本厚度。据此模型,对由于弯曲运动模式所产生的两种重要误差—交叉耦合误差及扫描范围误差进行了定量分析,分析表明:扫描范围误差主要受样本厚度及名义扫描范围影响,而Z向交叉耦合误差主要受探针偏置量及名义扫描范围影响,实验验证了所建立的运动学模型和误差计算公式的正确性;另外,还提出了相应的减小误差的方法。     

Heat transfer simulation and thermal measurements of microfabricated x-ray transparent heater stages

A microfabricated amorphous silicon nitride membrane-based nanocalorimeter is proposed to be suitable for an x-ray transparent sample platform with low power heating and built-in temperature sensing. In this work, thermal characterization in both air and vacuum are analyzed experimentally and via simulation. Infrared microscopy and thermoreflectance microscopy are used for thermal imaging of the sample area in air. While a reasonably large isothermal area is found on the sample area, the temperature homogeneity of the entire sample area is low, limiting use of the device as a heater stage in air or other gases. A simulation model that includes conduction, as well as radiation and convection heat loss, is presented with radiation and convection parameters determined experimentally. Simulated temperature distributions show that the homogeneity can be improved by using a thicker thermal conduction layer or reducing the pressure of the gas in the environment but neither are good solutions for the proposed use. A new simple design that has improved temperature homogeneity and a larger isothermal area while maintaining a thin thermal conduction layer is proposed and fabricated. This new design enables applications in transmission x-ray microscopes and spectroscopy setups at atmospheric pressure.     

Quantification of small, convex particles by TEM

It is shown how size distributions of arbitrarily oriented, convex, non-overlapping particles extracted from conventional transmission electron microscopy (TEM) images may be determined by a variation of the Schwartz-Saltykov method. In TEM, particles cut at the surfaces have diminished projections, which alter the observed size distribution. We represent this distribution as a vector and multiply it with the inverse of a matrix comprising thickness-dependent Scheil or Schwartz-Saltykov terms. The result is a corrected size distribution of the projections of uncut particles. It is shown how the real (3D) distribution may be estimated when particle shape is considered. Computer code to generate the matrix is given. A log-normal distribution of spheres and a real distribution of pill-box-shaped dispersoids in an Al-Mg-Si alloy are given as examples. The errors are discussed in detail.     

Total internal reflection fluorescence imaging using an upconverting cover slip for multicolour evanescent excitation

Total internal reflection fluorescence microscopy is well known as a means of studying surface‐bound structures in cell biology. It is usually measured either by coupling a light source to the sample using a prism or with a special objective where light passing through the periphery of the lens illuminates the contact region beyond the critical angle. In this study we present a new and simple approach to total internal reflection fluorescence microscopy where the sample is mounted on a cover slip prepared from a high‐index upconverting glass‐ceramic. Excitation of the cover slip with a low‐cost near‐infrared laser diode generates intense narrow‐band visible emission within the cover slip, some of which is totally internally reflected. This emission gives rise to an evanescent wave at the interface and hence can excite surface‐bound fluorescent species. Depending on the excitation conditions the cover slip can generate violet, green and red emission and hence can excite a wide range of fluorescent labels. Fluorescence emission from the sample can be detected in spectral regions where the direct emission from the cover slip is very weak. The advantages and limitations of the technique are discussed in comparison with conventional total internal reflection fluorescence microscopy measurements and prospects for novel total internal reflection fluorescence microscopy geometries are considered.     

Detection of Gross Measurement Errors Using the Grey System Method

A novel method using the grey system to detect the gross errors involved in a measurement process is proposed. Although the gross errors should be avoided completely, they can occur and on many occasions are not apparent. The grey system theory is used to characterise the geometric aspects in the measurement. Theoretical analysis of the principle of gross error detection is presented and a detection criterion is proposed. An experimental test shows that the proposed method compares favourably with statistical methods which are difficult to implement when the distribution of the data is unknown and the sample size is small.     

Scaling-index method as an image processing tool in scanning-probe microscopy

The scaling-index method (SIM) is a novel tool for image processing in scanning-probe microscopy. Originating from the theory of complex systems, the SIM can be used in order to extract structural information from arbitrary data sets. This method can readily be applied to the analysis of digital atomic-force microscopy (AFM) images. Especially for biomedical diagnostics, where genetic material is investigated by various microscopic methods, a reliable image segmentation based on the SIM algorithm is helpful. As a first application, AFM-images of GTG-banded human metaphase chromosomes (with G bands obtained by Trypsin using Giemsa) are compared with micrographs from conventional light microscopy by means of a scaling-index analysis. While the grey-level distributions of the optical and the AFM-images are largely different from each other, the scaling-index images are remarkably similar. Using this method, a fingerprint of an image can be produced which helps in the classification and interpretation of the measured data.