Phase identification of individual crystalline particles by electron backscatter diffraction

Recently, an electron backscatter diffraction (EBSD) system was developed that uses a 1024 × 1024 CCD camera coupled to a thin phosphor. This camera has been shown to produce excellent EBSD patterns. In this system, crystallographic information is determined from the EBSD pattern and coupled with the elemental information from energy or wavelength dispersive X-ray spectrometry. Identification of the crystalline phase of a sample is then made through a link to a commercial diffraction database. To date, this system has been applied almost exclusively to conventional, bulk samples that have been polished to a flat surface. In this investigation, we report on the application of the EBSD system to the phase identification analysis of individual micrometre and submicrometre particles rather than flat surfaces.     

Energy filtering transmission electron microscopy using the new JEM-2010FEF

The new JEM-2010FEF electron microscope provides useful techniques based on energy filtering as an omega-type energy filter is integrated into a thermal field-emission 200 kV transmission electron microscope. For example, the zero-loss imaging improves the contrast of high resolution lattice images as well as images of precipitates or lattice defects in alloys. The acquisition time for elemental mapping with core-loss electrons is one order in magnitude shorter than with energy-dispersive X-ray spectroscopy. The removal of inelastically scattered electrons enables us to observe weak lines in convergent-beam electron diffraction patterns from a thicker specimen with a probe size 1–2 nm in diameter. A combination of the field emission gun and sensitive recording media such as an imaging plate and a slow-scan CCD camera makes the energy filtering more powerful.     

Processing of secondary ion microscope images: an example of application to the thyroid

Ion microscopy is a microanalytical method by which one can obtain distribution images of any chemical element with isotope discrimination even at very low local concentrations, in successive slices of the specimen. These images are obtained at the price of progressive erosion of the specimen, so that the analysis may not be replayed and it is necessary to record the maximum amount of information during specimen erosion. We present an improvement of this method using a highly sensitive camera connected to a video analog-digital converter. The images are acquired and digitized on line and may be processed by an image computer. We illustrate the technique described with an application of ion microscopy that is made possible by digital recording and processing of images. This application concerns the precise comparison of iodine isotopes and phosphorus distributions in sections of the thyroid gland of rats which were submitted to an iodine-deficient diet followed by an injection of ¹²⁹ I.     

φFLIM: a new method to avoid aliasing in frequency-domain fluorescence lifetime imaging microscopy

In conventional wide‐field frequency‐domain fluorescence lifetime imaging microscopy (FLIM), excitation light is intensity‐modulated at megahertz frequencies. Emitted fluorescence is recorded by a CCD camera through an image intensifier, which is modulated at the same frequency. From images recorded at various phase differences between excitation and intensifier gain modulation, the phase and modulation depth of the emitted light is obtained. The fluorescence lifetime is determined from the delay and the decrease in modulation depth of the emission relative to the excitation. A minimum of three images is required, but in this case measurements become susceptible to aliasing caused by the presence of higher harmonics. Taking more images to avoid this is not always possible owing to phototoxicity or movement. A method is introduced, φFLIM, requiring only three recordings that is not susceptible to aliasing. The phase difference between the excitation and the intensifier is scanned over the entire 360° range following a predefined phase profile, during which the image produced by the intensifier is integrated onto the CCD camera, yielding a single image. Three different images are produced following this procedure, each with a different phase profile. Measurements were performed with a conventional wide‐field frequency‐domain FLIM system based on an acousto‐optic modulator for modulation of the excitation and a microchannel‐plate image intensifier coupled to a CCD camera for the detection. By analysis of the harmonic content of measured signals it was found that the third harmonic was effectively the highest present. Using the conventional method with three recordings, phase errors due to aliasing of up to ± 29° and modulation depth errors of up to 30% were found. Errors in lifetimes of YFP‐transfected HeLa cells were as high as 100%. With φFLIM, using the same specimen and settings, systematic errors due to aliasing did not occur.     

Compact water-window transmission X-ray microscopy

We demonstrate sub-100 nm resolution water-window soft X-ray full-field transmission microscopy with a compact system. The microscope operates at λ = 3.37 nm and is based on a 100 Hz table-top regenerative debris-free droplet-target laser-plasma X-ray source in combination with normal-incidence multilayer condenser optics for sample illumination. High-spatial-resolution imaging is performed with a 7.3% efficiency nickel zone plate and a 1024 × 1024 pixel CCD detector. Images of dry test samples are recorded with exposure times of a few minutes and show features smaller than 60 nm.     

A confocal laser microscope scanner for digital recording of optical serial sections

A confocal laser microscope scanner developed at our institute is described. Since an ordinary microscope is used, it is easy to view the specimen prior to scanning. Confocal imaging is obtained by laser spot illumination, and by focusing the reflected or fluorescent light from the specimen onto a pinhole aperture in front of the detector (a photomultiplier tube). Two rotating mirrors are used to scan the laser beam in a raster pattern. The scanner is controlled by a microprocessor which coordinates scanning, data display, and data transfer to a host computer equipped with an array processor. Digital images with up to 1024 × 1024 pixels and 256 grey levels can be recorded. The optical sectioning property of confocal scanning is used to record thin (～ 1 μm) sections of a specimen without the need for mechanical sectioning. By using computer-control to adjust the focus of the microscope, a stack of consecutive sections can be automatically recorded. A computer is then used to display the 3-D structure of the specimen. It is also possible to obtain quantitative information, both geometric and photometric. In addition to confocal laser scanning, it is easy to perform non-confocal laser scanning, or to use conventional microscopic illumination techniques for (non-confocal) scanning. The design has proved reliable and stable, requiring very few adjustments and realignments. Results obtained with this scanner are reported, and some limitations of the technique are discussed.     

A procedure to determine the correct thickness of an object with confocal microscopy in case of refractive index mismatch

A difference in refractive index (n) between immersion medium and specimen results in increasing loss of intensity and resolution with increasing focal depth and in an incorrect axial scaling in images of a confocal microscope. Axial thickness measurements of an object on such images are therefore not exact. The present paper describes a simple procedure to determine the correct axial thickness of an object with confocal fluorescence microscopy. We study this procedure for a specimen that has a higher refractive index than the immersion medium and with a thickness up to 100 µm. The measuring method was experimentally tested by comparing the thickness of polymer layers measured on axial images of a confocal microscope in case of a water–polymer mismatch to reference values obtained from an independent technique, i.e. scanning electron microscopy. The case when the specimen has a lower refractive index than the immersion medium is also shown by way of illustration. Measured thickness data of a water layer and an oil layer with the same actual thickness were obtained using an oil-immersion objective lens with confocal microscopy. Good agreement between theory and experiment was found in both cases, consolidating our method.     

Automated high-throughput electron tomography by pre-calibration of image shifts

Electron tomography is a versatile method for obtaining three‐dimensional (3D) images with transmission electron microscopy. The technique is suitable to investigate cell organelles and tissue sections (100–500 nm thick) with 4–20 nm resolution. 3D reconstructions are obtained by processing a series of images acquired with the samples tilted over different angles. While tilting the sample, image shifts and defocus changes of several µm can occur. The current generation of automated acquisition software detects and corrects for these changes with a procedure that incorporates switching the electron optical magnification. We developed a novel method for data collection based on the measurement of shifts prior to data acquisition, which results in a five‐fold increase in speed, enabling the acquisition of 151 images in less than 20 min. The method will enhance the quality of a tilt series by minimizing the amount of required focus‐change compensation by aligning the optical axis to the tilt axis of the specimen stage. The alignment is achieved by invoking an amount of image shift as deduced from the mathematical model describing the effect of specimen tilt. As examples for application in biological and materials sciences 3D reconstructions of a mitochondrion and a zeolite crystal are presented.     

Automated microscopy for electron tomography.

Instrumentation and methodology for the automatic collection of tomographic tilt series data for the three-dimensional reconstruction of single particles is described. The system consists of a Philips EM 430 TEM, with a Gatan 673 cooled slow-scan CCD camera and a Philips C400 microscope computer control unit attached. The procedure for data collection includes direct digital recording of the images on the CCD camera and the automatic measurement and correction of (a) image shifts resulting from tilting the specimen, (b) variation of defocus and (c) the eucentric height position of the specimen. Experiments are described illustrating the possibilities and limitations of automatic data collection. Data collection at a magnification of 30k shows that the exposure time of the specimen to the beam is reduced by a factor of 10-100 compared to manual operation of the TEM.     

A simple video method for the quantification of microscopic objects

The design and operation of a simple, semi-automatic video system for the analysis of geometric parameters of microscopic specimens is described. The system consists of standard video equipment and a custom built Electronic Integrator. The video signal from a camera attached to a microscope is mixed with the video signal from a second camera focused on a drawing board. A contour outline of the specimen to be analysed is drawn with the aid of a video monitor display of the combined images. The contour image is passed into the Electronic Integrator and analysed automatically. Logic circuitry in the Electronic Integrator permits analysis of complex contours independent of their orientation in the video raster. The area or volume of complex, overlapping specimens with restricted grey scale range can be rapidly analysed. The Electronic Integrator also is suitable for densitometric and specimen motion analyses.     

Three-dimensional spectral imaging by Hadamard transform spectroscopy in a programmable array microscope

We report the acquisition and deconvolution of three-dimensional spectrally resolved images in a programmable array microscope implementing a Hadamard transform fluorescence spectroscopy system with adjustable spectral resolution. A stack of 16 two-dimensional spectral images was collected at 400 nm intervals along the optical axis. The specimen consisted of a polytene chromosome spread from Drosophila melanogaster doubly labelled for the Polyhomeotic protein by indirect immunofluorescence labelling with Alexa594 and for DNA with YOYO-1. The resulting four-dimensional data set consisted of the xyz spatial dimensions (898 × 255 × 16) with a 26-point spectrum at each spatial location. The total exposure time to the sample was 34 min. The system requires the acquisition of multiple images, and thus works best with fluorophores that are resistant to photobleaching. Image deconvolution reduced the amount of out-of-focus blur by up to a factor of 8, resulting in a dramatic improvement in the visualization of the chromosome backbone and localization of the specific Polyhomeotic domains.     

Three dimensional image restoration in fluorescence lifetime imaging microscopy

A microscope set-up and numerical methods are described which enable the measurement and reconstruction of three-dimensional nanosecond fluorescence lifetime images in every voxel. The frequency domain fluorescence lifetime imaging microscope (FLIM) utilizes phase detection of high-frequency modulated light by homodyne mixing on a microchannel plate image intensifier. The output signal at the image intensifier's phosphor screen is integrated on a charge coupled device camera. A scanning stage is employed to obtain a series of phase-dependent intensity images at equally separated depths in a specimen. The Fourier transform of phase-dependent data gives three-dimensional (3D) images of the Fourier coefficients. These images are deblurred using an Iterative Constrained Tikhonov–Miller (ICTM) algorithm in conjunction with a measured point spread function. The 3D reconstruction of fluorescence lifetimes are calculated from the deblurred images of the Fourier coefficients. An improved spatial and temporal resolution of fluorescence lifetimes was obtained using this approach to the reconstruction of simulated 3D FLIM data. The technique was applied to restore 3D FLIM data of a live cell specimen expressing two green fluorescent protein fusion constructs having distinct fluorescence lifetimes which localized to separate cellular compartments.     

Masking a CCD camera allows multichord charge exchange spectroscopy measurements at high speed on the DIII-D tokamak

Charge exchange spectroscopy is one of the standard plasma diagnostic techniques used in tokamak research to determine ion temperature, rotation speed, particle density, and radial electric field. Configuring a charge coupled device (CCD) camera to serve as a detector in such a system requires a trade-off between the competing desires to detect light from as many independent spatial views as possible while still obtaining the best possible time resolution. High time resolution is essential, for example, for studying transient phenomena such as edge localized modes. By installing a mask in front of a camera with a 1024 × 1024 pixel CCD chip, we are able to acquire spectra from eight separate views while still achieving a minimum time resolution of 0.2 ms. The mask separates the light from the eight spectra, preventing spatial and temporal cross talk. A key part of the design was devising a compact translation stage which attaches to the front of the camera and allows adjustment of the position of the mask openings relative to the CCD surface. The stage is thin enough to fit into the restricted space between the CCD camera and the spectrometer endplate.     

Imaging 'intact' myofibrils with a near-field scanning optical microscope

Fluorescently labelled myofibrils were imaged in physiological salt solution by near-field scanning optical microscopy and shear-force microscopy. These myofibrils were imaged in vitro , naturally adhering to glass while retaining their ability to contract. The Z-line protein structure of the myofibrils was antibody labelled and easily identified in the near-field fluorescence images. The distinctive protein banding structure of the myofibril was also seen clearly in the shear-force images without any labelling requirement. With the microscope in the transmission mode, resolution of the fluorescence images was degraded significantly by excessive specimen thickness (>1 μm), whereas the shear-force images were less affected by specimen thickness and more affected by poor adherence to the substrate. Although the exact mechanism generating contrast in the shear-force images is still unknown, shear-force imaging appears to be a promising new imaging modality.     

X-ray microscopy and imaging of Escherichia coli, LPS and DNA

Ultrastructural examination by transmission and scanning electron microscopy involves a series of specialized preparation steps which may introduce artefacts in the micrographs. X-ray microscopy can take instant images of speci-mens but is mostly restricted to a few synchrotron X-ray sources. We have utilized a bench-top nanosecond laser-plasma to produce a single-shot source of nanosecond X-rays tuned for maximum contrast with carbon-rich material. To examine the ultrastructure by absorption profiles, we utilized a laser-produced plasma generated by a single-shot laser (1.06 μm wavelength, 5 × 10¹² W cm⁻² intensity) focused on to a silicon target as an X-ray source for high-resolution X-ray microscopy. This approach eliminates the specimen preparation steps. Whole hydrated cells of Escherichia coli and purified preparations of lipopolysaccharide (LPS) and chromosomal DNA (cDNA) were streaked onto poly(methyl methacrylate) (PMMA)-coated grids (resist). This resist was exposed to X-rays under vacuum at a distance of 2.5 cm from the target disc. The silicon plasma produced by a 10-ns burst of laser energy (at 20 J) radiates strong emission lines in the region of 300 eV. The X-rays penetrate the sample and their absorption profile is transferred on to the resist where PMMA acts as a negative to generate an image. By atomic force microscopy imaging of this photoresist we have visualized layers around cells of E. coli , darker areas inside the cell probably corresponding to cDNA, and preliminary images of LPS and DNA molecules. This technique has resolution at the 100 Å level, produces images similar to the space-filling models of macromolecules and may be of great value in the study of the ultrastructure of hydrated live biological specimens.     

Microtomography from limited projections in conventional TEM for 3D reconstruction of an intact cell

It is possible to generate three-dimensional reconstructions of whole, non-sectioned biological cells in conventional TEM using an 80 kV tungsten source. A TEM specimen stage was modified to accommodate a precise single-axis tilting mechanism controlled by a digital stepping motor interfaced to a computer. For image collection, a video camera was optically coupled to the TEM phosphorescent screen, and the video image was digitized by a frame buffer interfaced to a computer. Specimen tilt and projection image collection were fully computer-automated. This microtomography system design could be readily adapted for most TEMs. Image reconstruction was achieved through computation on projection images from limited tilts; typically less than thirty projection images were needed for a coarse 3D reconstruction. The iterative reconstruction algorithm used certain statistical assumptions about the distribution of image gray values. Since microtomography was performed on non-sectioned whole mount cells viewed under an 80 kV electron beam, methods of embedment-free specimen preparation with chemical fixation and extraction were employed. These methods were utilized successfully to permit good image formation of the entire cell mitotic nucleus a few micrometers in thickness. The 3D reconstruction of a single kidney cell mitotic nucleus was carried out and shown to produce a reasonable microtomogram of gross features like the condensed chromosomes.     

Spot-scan imaging of microcrystals of an influenza neuraminidase-antibody fragment complex

Electron micrographs of two-dimensional microcrystals of a complex of an avian influenza virus neuraminidase and an antibody Fab fragment, termed 32/3, have been recorded using the spot-scan method of imaging. The crystals have a large unit cell (159.5 A x 159.5 A x 130.5 A) and a high solvent content (approximately 71% by volume) and are a challenging specimen for testing the spot-scan methodology. Crystalline order was preserved to beyond 4 A resolution as demonstrated by electron diffraction, using an embedding medium of a mixture of glucose and neutral potassium phosphotungstate. Using a Philips C400 computer control system interfaced to an EM420 electron microscope, and with the inclusion of additional software in the system, we have been able to record micrographs at low temperature with a relatively narrow (1500 A diameter) moving beam. There is evidence that the use of such a spot-scan beam reduces the effects of beam-induced specimen motion on the quality of micrographs. Conventional low-dose "flood-beam" images showed good isotropic optical diffraction in only 15% of cases whereas 30% of spot-scan images showed good diffraction. The best flood-beam images gave phases to only 15 A resolution after computer processing, whereas the best spot-scan images gave phases to 7 A resolution. Electron diffraction patterns were also recorded at low temperature, and the resulting diffraction amplitudes combined with phases from spot-scan images to yield a projection map of the structure. A 7 A resolution projection map of the complex is presented, and is compared with the projection map of the same avian influenza neuraminidase complexed with a different monoclonal Fab fragment, NC41, which has been solved to high resolution by X-ray diffraction.     

Measurement of dynamic fracture toughness and failure behavior for explosive mock materials

In this work, a pre-cracked semi-circular shaped explosive simulant was loaded using a split Hopkinson pressure bar (SHPB). A high-speed camera was used to capture the deformation and fracture process of the specimen in situ. The digital images were processed using the digital image correlation (DIC) method. Next, full displacement and strain fields were obtained. The displacement vector field shows that the specimen fractured under tensile stress action. The strain field can be used to predict the crack propagation. Results show that the method of combined DIC and SHPB is effective to study the dynamic deformation and fracture behavior of explosive simulants. In addition, the specimen was loaded using a drop weight. The fracture toughness of the specimen was preliminary measured.     

Direct observation of a Ti/Cu interface with atomic displacement under electron irradiation

A Ti film was deposited onto a Cu substrate by means of a radio frequency magnetron sputtering method. Cross-sectional thin foils for TEM observation were prepared using a focused ion beam. Electron irradiation was carried out using a high-resolution high-voltage electron microscope operated at 1.25 MV . The Cu/Ti interface of the foils was irradiated at 623 K. In-situ observation images during electron irradiation were recorded by a CCD camera with a digital video cassette. The (020)_Cu plane on the Cu/Ti interface preferentially moved towards the Ti film with irradiation. Composition analysis of the diffused region showed that its composition corresponded to Ti₃Cu₂.     

利用数字图像处理技术测量直齿圆柱齿轮几何尺寸

研究了采用数字图像处理技术实现对直齿圆柱齿轮几何尺寸的非接触式测量.首先对CCD摄像机拍摄的图像进行畸变校正与滤波去噪,然后对图像进行阈值分割,采用基于数学形态法的四邻域腐蚀来获得单像素宽的图像边缘.针对齿轮的边缘轮廓形状特征,运用重心法、最小二乘拟合、Bresenham画圆法和Hough变换等原理,建立了测量齿轮齿数、模数、中心孔半径、齿顶圆半径、齿根圆半径和变位系数等齿轮几何尺寸参数的测量算法.使用1280×1024的CCD摄像机及黑白采集卡对分度圆直径为50mm的直齿圆柱齿轮进行测量实验,与人工测量值对比,绝对误差小于一个像素,在各测量参数中,最大尺寸的齿顶圆半径绝对误差值为13μm.研究结果表明:因为CCD摄像设备的分辨率越高、被测物体的尺寸越小,则测量精度越高,且为线性关系,所以,使用高分辨率CCD摄像设备并配备光学放大系统,可实现对高精度和微小直齿圆柱齿轮几何尺寸参数的测量.如使用1280×1024像素以上的CCD摄像设备测量分度圆直径5mm以下的直齿圆柱齿轮时,测量精度可在2μm以下.