Depth sectioning in scanning transmission electron microscopy based on core-loss spectroscopy

Recent and ongoing improvements in aberration correction have opened up the possibility of depth sectioning samples using the scanning transmission electron microscope in a fashion similar to the confocal scanning optical microscope. We explore questions of principle relating to image interpretability in the depth sectioning of samples using electron energy loss spectroscopy. We show that provided electron microscope probes are sufficiently fine and detector collection semi-angles are sufficiently large we can expect to locate dopant atoms inside a crystal. Furthermore, unlike high angle annular dark field imaging, electron energy loss spectroscopy can resolve dopants of smaller atomic mass than the supporting crystalline matrix.     

An optical sectioning programmable array microscope implemented with a digital micromirror device

The defining feature of a programmable array microscope (PAM) is the presence of a spatial light modulator in the image plane. A spatial light modulator used singly or as a matched pair for both illumination and detection can be used to generate an optical section. Under most conditions, the basic optical properties of an optically sectioning PAM are similar to those of rotating Nipkow discs. The method of pattern generation, however, is fundamentally different and allows arbitrary illumination patterns to be generated under programmable control, and sectioning strategies to be changed rapidly in response to specific experimental conditions. We report the features of a PAM incorporating a digital micromirror device, including the axial sectioning response to fluorescent thin films and the imaging of biological specimens. Three axial sectioning strategies were compared: line scans, dot lattice scans and pseudo-random sequence scans. The three strategies varied widely in light throughput, sectioning strength and robustness when used on real biological samples. The axial response to thin fluorescent films demonstrated a consistent decrease in the full width at half maximum (FWHM), accompanied by an increase in offset, as the unit cells defining the patterns grew smaller. Experimental axial response curves represent the sum of the response from a given point of illumination and cross-talk from neighbouring points. Cross-talk is minimized in the plane of best focus and when measured together with the single point response produces a decrease in FWHM. In patterns having constant throughput, there appears to be tradeoff between the FWHM and the size of the offset. The PAM was compared to a confocal laser scanning microscope using biological samples. The PAM demonstrated higher signal levels and dynamic range despite a shorter acquisition time. It also revealed more structures in x - z sections and less intensity drop-off with scanning depth.     

Construction of a Microcomputer Controlled Photoacoustic Spectrometer

ABSTRACT

We have developed a UV-Visible photoacoustic spectrophotometer using relatively inexpensive individual optical components. The instrument was initially developed due to the absence of a commercial photoacoustic instrument in the UV-Visible range. We have found that this custom built instrument allows numerous software generated options that are unavailable on any commercial spectrometric instruments. In addition to recording routine intensity versus wavelength spectra, we can record the following: 1) resolution of the spectra in layered samples; 2) quantitative depth profiles in samples; 3) spectra and spectral fragments can be smoothed and joined into continuous spectra; and 4) signal averaging of spectra can be carried out. The construction of this custom built instrument allowed the maximum flexibility in experimental application with minimum cost to the investigator.     

Reconstruction and display of curvilinear objects from optical section data using 3-D curve fitting algorithms

Biological objects resembling filaments are often highly elongated while presenting a small cross-sectional area. Examination of such objects requires acquisition of images from regions large enough to contain entire objects, but at sufficiently high resolution to resolve individual filaments. These requirements complicate the application of conventional optical sectioning and volume reconstruction techniques. For example, objective lenses used to acquire images of entire filaments or filament networks may lack sufficient depth ( Z ) resolution to localize filament cross-sections along the optical axis. Because volume reconstruction techniques consider only the information represented by a single volume element (voxel), views of filament networks reconstructed from images obtained at low Z -resolution will not accurately represent filament morphology. A possible solution to these problems is to simultaneously utilize all available information on the path of an object by fitting 3-D curves through data points localized in 2-D images. Here, we present an application of this approach to the reconstruction of microtubule networks from 2-D optical sections obtained using confocal microscopy, and to synthesized curves which have been distorted using a simple mathematical model of optical sectioning artefacts. Our results demonstrate that this strategy can produce high resolution 3-D views of filamentous objects from a small number of optical sections.     

Local phase measurements of light in a one-dimensional photonic crystal

For the first time the local optical phase evolution in and around a small, one-dimensional photonic crystal has been visualized with a heterodyne interferometric photon scanning tunnelling microscope. The measurements show an exponential decay of the optical intensity inside the crystal, which consists of a periodic array of subwavelength air rods fabricated in a conventional ridge waveguide. In addition it is found that the introduction of the air rods has a counterintuitive effect on the phase development inside the structure. The heterodyne detection scheme allows the detection of low-intensity scattered waves. In the vicinity of the scattering air rods phase singularities are found with a topological charge of plus or minus one.     

Three-dimensional capillary geometry in gut tissue

Accurate characterization of capillary geometry is of the utmost importance for physiological tissue studies such as oxygen transport. We show that 3D microscopy can be used to measure tissue capillary geometry both in normal and disease states. We imaged fluorescently labeled gut mucosa capillary beds of three control rats and three rats 4 hours after i.p. injection of 9 mg/kg endotoxin. We used serial optical sectioning microscopy coupled with deconvolution to reconstruct 3D capillary geometry. Theoretical point spread functions accounting for depth into the specimen resulted in better reconstructions than experimentally measured point spread functions. We next derived the distribution of the shortest distances to the nearest capillary from all extravascular tissue voxels. In normal rats the shortest-distance distributions were remarkably constant despite widely varying capillary geometry. Furthermore, the mean of the shortest-distance distributions increased significantly for endotoxemic rats (4.8+/-0.4 microm) compared to controls (4.3+/-0.3 microm, P<0.05). Hence, serial sectioning microscopy provides an accurate venue for measuring physiologically relevant 3D capillary structure.     

保偏光纤模场直径和数值孔径测试研究

基于光纤测量远场法原理测量光纤远场光强分布。通过改进远场法得到光纤的远场光强分布后,同时计算得到保偏光子晶体光纤的模场直径和数值孔径,使测量装置实现集成化和简便化。通过光束测试仪测量光纤的出射光强分布,光束测试仪测量的是光纤中心最大光强点的两个垂直方向上的光强分布。保偏光子晶体光纤的出射光斑是椭圆的,每个方向的模场直径、数值孔径分布并不相同。传统的测试方法不能解决这个问题。通过旋转光纤测量光纤各个方向上的光强分布,然后计算各个方向上的模场直径,最后通过拟合各个方向上的模场直径得到保偏光子晶体光纤椭圆形的模场分布。实验测得保偏光子晶体光纤椭圆光斑长短轴的模场直径分别为7.5μm和4.2μm,数值孔径分别为0.159和0.276。     

Time-domain whole-field fluorescence lifetime imaging with optical sectioning

A whole-field time-domain fluorescence lifetime imaging (FLIM) microscope with the capability to perform optical sectioning is described. The excitation source is a mode-locked Ti:Sapphire laser that is regeneratively amplified and frequency doubled to 415 nm. Time-gated fluorescence intensity images at increasing delays after excitation are acquired using a gated microchannel plate image intensifier combined with an intensified CCD camera. By fitting a single or multiple exponential decay to each pixel in the field of view of the time-gated images, 2-D FLIM maps are obtained for each component of the fluorescence lifetime. This FLIM instrument was demonstrated to exhibit a temporal discrimination of better than 10 ps. It has been applied to chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment. Initially, standard fluorescent dyes were studied and then this FLIM microscope was applied to the imaging of biological tissue, successfully contrasting different tissues and different states of tissue using autofluorescence. To demonstrate the potential for real-world applications, the FLIM microscope has been configured using potentially compact, portable and low cost all-solid-state diode-pumped laser technology. Whole-field FLIM with optical sectioning (3D FLIM) has been realized using a structured illumination technique.     

Resolution and optical sectioning in the confocal microscope

In this short review, we present a self-contained discussion of the image formation properties of the fluorescent confocal microscope. The optical sectioning or depth discrimination property is discussed in detail and new analytic formulae are presented, which relate the optical sectioning strength to the wavelength, numerical aperture and pinhole aperture size in a particularly simple fashion.     

A study of abrasive waterjet cutting of alumina ceramics with controlled nozzle oscillation

This paper presents and discusses an experimental investigation of abrasive waterjet (AWJ) cutting of alumina ceramics with controlled nozzle oscillation. Particular attention is paid to the effect of small oscillation angles on the various cutting performance measures. It is found that nozzle oscillation at small angles can equally improve the major cutting performance measures, if the cutting parameters are correctly selected. However, under high water pressures, high nozzle traverse speeds and large oscillation frequencies, nozzle oscillation may cause a decrease in some major cutting performance measures, such as surface finish. Plausible trends of cutting performance with respect to the process parameters are further considered. Finally, a predictive mathematical model for the depth of cut is developed and verified.     

ENHANCING THE DEPTH OF CUT IN ABRASIVE WATERJET CUTTING OF ALUMINA CERAMICS BY USING MULTIPASS CUTTING WITH NOZZLE OSCILLATION

An experimental investigation is presented to increase the depth of cut in abrasive waterjet (AWJ) cutting of alumina ceramics by introducing a new cutting technique combining multipass operations with controlled nozzle oscillation. Plausible trends of the depth of cut per pass and total depth of cut with respect to the number of passes and the parameters in each pass are discussed. It shows that cutting with nozzle oscillation can significantly increase the depth of cut in the single-pass cutting mode, while further gains in the depth of cut can be made by using multipass cutting with nozzle oscillation. While multipass cutting can be used to increase the total depth of cut for machining thicker materials, it has been found that an average increase of 50.8% in the total depth of cut can be expected by using multipass cutting with nozzle oscillation as compared to single-pass cutting without nozzle oscillation within the same cutting time. Recommendations are finally made as a practical guide for the selection of process parameters in multipass AWJ cutting of alumina ceramics with controlled nozzle oscillation.     

Some effects of electron channeling on electron energy loss spectroscopy

As an electron beam (of order 100 keV) travels through a crystalline solid it can be channeled down a zone axis of the crystal to form a channeling peak centered on the atomic columns. The channeling peak can be similar in size to the outer atomic orbitals. Electron energy loss spectroscopy (EELS) measures the losses that the electron experiences as it passes through the solid yielding information about the unoccupied density of states in the solid. The interaction matrix element for this process typically produces dipole selection rules for small angle scattering. In this paper, a theoretical calculation of the EELS cross section in the presence of strong channeling is performed for the silicon L23 edge. The presence of channeling is found to alter both the intensity and selection rules for this EELS signal as a function of depth in the solid. At some depths in the specimen small but significant non-dipole transition components can be produced, which may influence measurements of the density of states in solids.     

Growth simulation for 3D surface and through-thickness cracks using SGBEM-FEM alternating method

An SGBEM-FEM alternating method had been proposed by Nikishkov, Park and Atluri for the analysis of three-dimensional planar and non-planar cracks and their growth. The proposed method is an effective method for fatigue or stress corrosion crack growth simulation. During crack growth simulation, however, an oscillation phenomenon is observed in crack advance or stress intensity factor distribution. If oscillating amplitude in SIF or crack advance does not decrease during next increment steps, the crack growth simulation fails. In this paper several methods are examined to remove the oscillation phenomenon. As a result, it is found that smoothing in stress intensity factor distribution or in crack front geometry can remove or weaken the oscillation phenomenon. Using the smoothing techniques, stress corrosion crack growth simulation is performed for a semi-elliptical surface crack and a through-thickness crack embedded in a plate. Crack front shape and stress intensity factor distribution are obtained after each increment during the crack growth. And the depth and length of a crack are obtained as a function of time. It is noted that the SGBEM-FEM alternating method is a very effective method for SCC growth simulation for a surface crack and a through-thickness crack.     

Fiber laser based two-photon FRET measurement of calmodulin and mCherry-E(0)GFP proteins

The speed and accuracy of Förster resonance energy transfer (FRET) measurements can be improved by rapidly alternating excitation wavelengths between the donor and acceptor fluorophore. We demonstrate FRET efficiency measurements based on a fiber laser and photonic crystal fiber as the source for two‐photon excitation (TPE). This system offers the potential for rapid wavelength switching with the benefits of axial optical sectioning and improved penetration depth provided by TPE. Correction of FRET signals for cross excitation and cross emission was achieved by switching the excitation wavelength with an electrically controlled modulator. Measurement speed was primarily limited by integration times required to measure fluorescence. Using this system, we measured the FRET efficiency of calmodulin labeled with Alexa Fluor 488 and Texas Red dyes. In addition, we measured two‐photon induced FRET in an E⁰GFP‐mCherry protein construct. Results from one‐photon and two‐photon excitation are compared to validate the rapid wavelength switched two‐photon measurements. Microsc. Res. Tech. 75:837–843, 2012. © 2011 Wiley Periodicals, Inc.     

Apertureless scanning near-field fluorescence microscopy in liquids

We show that apertureless scanning near-field optical microscopes that use sharp vibrating conical tips can be operated in liquid environments. We have investigated the damping of the tip oscillation as a function of its shape and as a function of its depth under the liquid surface. The degradation of the quality factor from 150 in air down to 15 in liquid does not impede to perform topographic and optical measurements with a very good sensitivity. As an example of application, we present near-field fluorescence images of dye-doped polystyrene spheres immersed in a liquid.     

Adaptive optics in spinning disk microscopy: improved contrast and brightness by a simple and fast method

Multiconfocal microscopy gives a good compromise between fast imaging and reasonable resolution. However, the low intensity of live fluorescent emitters is a major limitation to this technique. Aberrations induced by the optical setup, especially the mismatch of the refractive index and the biological sample itself, distort the point spread function and further reduce the amount of detected photons. Altogether, this leads to impaired image quality, preventing accurate analysis of molecular processes in biological samples and imaging deep in the sample. The amount of detected fluorescence can be improved with adaptive optics. Here, we used a compact adaptive optics module (adaptive optics box for sectioning optical microscopy), which was specifically designed for spinning disk confocal microscopy. The module overcomes undesired anomalies by correcting for most of the aberrations in confocal imaging. Existing aberration detection methods require prior illumination, which bleaches the sample. To avoid multiple exposures of the sample, we established an experimental model describing the depth dependence of major aberrations. This model allows us to correct for those aberrations when performing a z‐stack, gradually increasing the amplitude of the correction with depth. It does not require illumination of the sample for aberration detection, thus minimizing photobleaching and phototoxicity. With this model, we improved both signal‐to‐background ratio and image contrast. Here, we present comparative studies on a variety of biological samples.     

Low-temperature near-field spectroscopy of CdTe quantum dots

A near-field optical microscope has been developed for operation at low temperature. This microscope is used to study the photoluminescence of CdTe-based quantum dots. Spectra collected upon approaching the optical tip into the near-field region of the sample reveal the evolution from a broad far-field luminescence band − that is typical for a large ensemble of dots − to a near-field structure made up of a few sharp peaks originating from individual dots. Experiments carried out in the excitation-collection mode through the optical tip allow study of the effect of an increase in excitation power on the near-field spectra. It is found that upon increasing the excitation by two orders of magnitude, a spatially resolved spectrum progressively transforms back into a broad 'far-field-like' spectrum. Photoluminescence images taken by scanning the sample under the tip are used to discriminate various contributions coming from individual dots.     

Scaling study of cavitation pitting from cavitating jets and ultrasonic horns

Cavitation erosion prediction and characterization of cavitation field strength are of interest to industries suffering from cavitation erosion detrimental effects. One means to evaluate cavitation fields and materials is to examine pitting rates during the incubation period, where the test sample undergoes localized permanent deformations shaped as individual pits. In this study, samples from three metallic materials, an Aluminum alloy (Al 7075), a Nickel Aluminum Bronze (NAB) and a Duplex Stainless Steel (SS A2205) were subjected to a vast range of cavitation intensities generated by cavitating jets at different driving pressures and by an ultrasonic horn. The resulting pitted sample surfaces were examined and characterized with a non-contact 3D optical scanner and the resulting damage computer-analyzed. A statistical analysis of the pit population and its characteristics was then carried out. It was found that the various cavitation field strengths can be correlated to the measured pit distributions and that two characteristic quantities: a characteristic number of pits per unit surface area and unit time, and a characteristic pit diameter or a characteristic pit depth can be attributed to a given “cavitation intensity level”. This characterization concept can be used in the future to study the cavitation intensity of the full scale and to develop methods of full scale predictions based on model scale erosion data.     

Predictive depth of jet penetration models for abrasive waterjet cutting of alumina ceramics

A study of the depth of jet penetration (or depth of cut) in abrasive waterjet (AWJ) cutting of alumina ceramics with controlled nozzle oscillation is presented and discussed. An experimental investigation is carried out first to study the effects of nozzle oscillation at small angles on the depth of cut under different combinations of process parameters. Based on the test conditions, it is found that nozzle oscillation at small angles can improve the depth of cut by as much as 82% if the cutting parameters are correctly selected. Depending on the other cutting parameters in this study, it is found that a high oscillation frequency (10–14 Hz) with a low oscillation angle (4–6°) can maximize the depth of cut. Using a dimensional analysis technique, predictive models for jet penetration when cutting alumina ceramics with and without nozzle oscillation are finally developed and verified. It is found that the model predictions are in good agreement with the experimental results with the average percentage errors of less than 2.5%.