Chemical mapping of a block copolymer electrolyte by low-loss EFTEM spectrum-imaging and principal component analysis

Energy-filtered transmission electron microscopy spectrum-imaging (EFTEM SI) in the low electron energy-loss range is a valuable technique for probing the chemical structure of a material with nanoscale spatial resolution using a reduced electron dose. By analyzing EFTEM SI datasets using principal component analysis (PCA), the constituent chemical phases of the material can be identified in an efficient manner without prior knowledge of the specimen. We implement low-loss EFTEM SI together with PCA to investigate thin films of the block copolymer electrolyte poly(styrene-block-ethylene oxide) (PS-b-PEO) blended with a sodium salt. PCA identifies three main phases, the first and second phases corresponding to the two blocks of the copolymer and a third phase corresponding to the salt. The low-loss spectra for these phases are extracted from a noise-reduced EFTEM SI dataset and used to generate a chemical map of the material by multiple linear least square fitting. We validate the results of the low-loss EFTEM SI/PCA technique by applying the method to a control PS-b-PEO sample that does not contain the sodium salt, and by conducting spatially resolved X-ray energy-dispersive spectrometry on the salt-containing PS-b-PEO thin film.     

Fourier transform multipixel spectroscopy for quantitative cytology

A Fourier transform multipixel spectroscopy system was set up and applied to fluorescence microscopy of single living cells. Continuous fluorescence spectra for all pixels of the cell image were recorded simultaneously by the system. Multiple frames of data were first acquired and stored as a set of interferograms for each pixel of the image; they were then Fourier transformed and used as a spatially organized set of fluorescence spectra. Practical spectral resolution of 5 nm was achieved, typically, for 10⁴ pixels in a single cell. The net result was I ( x y ,λ), the fluorescence intensity ( I ) for each pixel of the image ( x y ), as function of wavelength (λ). The present study demonstrates that multipixel spectroscopy can reveal dynamic processes of the food-digestive cycle in the unicellular Paramecium vulgaris fed with algae. Spectral variability of fluorescence intensity at different cytoplasmic sites pinpointed the location of cellular deposits of chlorophyll (630 nm) and of pheophytin (695 nm), a digestive product of the chlorophyll. Localization of compartmental spectral changes was achieved using a 'similarity mapping' algorithm, followed by enhanced image construction. Similarity mapping based on the fluorescence spectrum of native chlorophyll revealed a highlighted image of the cell cytopharynx structure where algae were ingested. Phagolysosomes, migrating vacuoles and the cytoproct, each containing different ratios of pheophytin, were similarly imaged.     

高光谱图像的分布式压缩感知成像与重构

根据高光谱数据的特点,提出了一种基于像元的分布式压缩采样模型来实现高光谱图像的有效压缩采样与重构。搭建了能实现该模型的压缩采样光谱成像系统,并研究了用于该系统成像的重构算法。在图像采集阶段,将高光谱数据分为参考像元和压缩感知像元;地面像元的辐射能通过棱镜进行谱带分离,再利用数字微镜器件实现谱带的线性编码。对压缩感知像元进行低采样率的线性编码,对参考像元进行采样率为1的线性编码。压缩采样数据重构时,不再采用传统方法直接重构高光谱数据,而是利用线性混合模型将重构高光谱数据转换成端元提取和丰度估计,然后根据重构的端元和丰度恢复原数据。对比实验表明,在压缩采样数据为总数据的20%时,重构的平均信噪比提高了10dB。所设计的成像系统应用压缩感知理论减少了采集的数据量,采样方式简单,可应用于星载或机载的高光谱压缩感知成像。     

Spectral Fluorescence Imaging for Quantitative Diagnosis and Mapping of Dental Fluorosis Affected Tooth Surfaces

Abstract

A new analytical method for quantitative diagnosis of dental fluorosis is suggested. The method is based on the spectral modifications in the fluorescence light emitted from affected enamel surfaces, relative to normal dental enamel. Imaging of the tooth surface is obtained using an imaging Fourier transform fluorescence spectrophotometer. This technique provides simultaneous fluorescence spectra at all pixels of the examined area. Images and the corresponding spectra were acquired at various tooth regions, and it was shown that normal, white opaque, brown discolored, and pitted tooth surfaces have different distinct spectral features which characterize the different degrees of the observed pathology of dental fluorosis. Criteria for quantitative assessment of the dental fluorosis stages were suggested. These were based on the measured spectral emission ratio at two wavelengths and comparison with the spectrum of normal enamel. The suggested parameters were validated against clinical observation of numerous samples affected by dental fluorosis at various pathological stages. Besides the introduction of the quantitative assessment, the method is suggested for detection of early changes in the characteristics of the tooth surface in dental fluorosis. The associated mapping capability allows for morphological characterization, which provides additional important clinical information.     

Phase differentiation via combined EBSD and XEDS

Electron backscatter diffraction (EBSD) and orientation imaging microscopy have become established techniques for analysing the crystallographic microstructure of single and multiphase materials. In certain instances, however, it can be difficult and/or time intensive to differentiate phases within a material by crystallography alone. Traditionally a list of candidate phases is specified prior to data collection. The crystallographic information extracted from the diffraction patterns is then compared with the crystallographic information from these candidate phases, and a best‐fit match is determined. Problems may arise when two phases have similar crystal structures. The phase differentiation process can be improved by collecting chemical information through X‐ray energy‐dispersive spectroscopy (XEDS) simultaneously with the crystallographic information through EBSD and then using the chemical information to pre‐filter the crystallographic phase candidates. This technique improves both the overall speed of the data collection and the accuracy of the final characterization. Examples of this process and the limitations involved will be presented and discussed.     

Observation of microdiffraction patterns with a dedicated STEM instrument

A two-dimensional detector system, designed for the observation and recording of microdiffraction patterns formed in an HB 5 scanning transmission electron microscope (STEM) is described and discussed. Possibilities are described and demonstrated for the simultaneous or successive recording of microdiffraction patterns from regions of diameter 3 å or more, bright- or dark-field STEM images, EELS spectra, secondary electron images, and in-line holograms. Applications of the system have been made to studies of catalyst particles, reflection-mode imaging of bulk surfaces, and image reconstruction from microdiffraction patterns obtained from each point of a STEM image.     

Local crystal structure analysis with several picometer precision using scanning transmission electron microscopy

We report a local crystal structure analysis with a high precision of several picometers on the basis of scanning transmission electron microscopy (STEM). Advanced annular dark-field (ADF) imaging has been demonstrated using software-based experimental and data-processing techniques, such as the improvement of signal-to-noise ratio, the reduction of image distortion, the quantification of experimental parameters (e.g., thickness and defocus) and the resolution enhancement by maximum-entropy deconvolution. The accuracy in the atom position measurement depends on the validity of the incoherent imaging approximation, in which an ADF image is described as the convolution between the incident probe profile and scattering objects. Although the qualitative interpretation of ADF image contrast is possible for a wide range of specimen thicknesses, the direct observation of a crystal structure with deep-sub-angstrom accuracy requires a thin specimen (e.g., 10 nm), as well as observation of the structure image by conventional high-resolution transmission electron microscopy.     

A high-speed area detector for novel imaging techniques in a scanning transmission electron microscope

A scanning transmission electron microscope (STEM) produces a convergent beam electron diffraction pattern at each position of a raster scan with a focused electron beam, but recording this information poses major challenges for gathering and storing such large data sets in a timely manner and with sufficient dynamic range. To investigate the crystalline structure of materials, a 16×16 analog pixel array detector (PAD) is used to replace the traditional detectors and retain the diffraction information at every STEM raster position. The PAD, unlike a charge-coupled device (CCD) or photomultiplier tube (PMT), directly images 120–200 keV electrons with relatively little radiation damage, exhibits no afterglow and limits crosstalk between adjacent pixels. Traditional STEM imaging modes can still be performed by the PAD with a 1.1 kHz frame rate, which allows post-acquisition control over imaging conditions and enables novel imaging techniques based on the retained crystalline information. Techniques for rapid, semi-automatic crystal grain segmentation with sub-nanometer resolution are described using cross-correlation, sub-region integration, and other post-processing methods.     

近红外高光谱成像技术快速鉴别国产咖啡豆品种

结合近红外高光谱成像技术和不同的判别分析模型对4种国产咖啡豆品种进行了快速无损判别。通过高光谱成像仪提取874~1 734nm波段内的光谱数据,去除首尾噪声波段后,分别基于925~1 680nm波段的全谱波段和通过连续投影算法(SPA)选择的特征波长,建立了偏最小二乘判别分析(PLS-DA)、随机森林(RF)、K最邻近算法(KNN)、支持向量机(SVM)模型和极限学习机(ELM)5种判别分析模型。基于上述判别模型对咖啡豆品种进行鉴别;然后通过准确率、命中率和否定率3个参数对鉴别结果进行了评价。实验显示,基于全谱和特征波段建立的模型均取得了较好的判别效果,其中ELM模型效果均为最优,每个品种建模集和预测集的准确率、命中率和否定率均在93.5%以上。研究结果表明,基于近红外高光谱成像技术结合模型判别分析方法可以实现对国产咖啡豆品种的识别,特征波长的选择减少了变量数,但判别效果与全谱相当。     

Secondary ion counting for surface-sensitive chemical analysis of organic compounds using time-of-flight secondary ion mass spectroscopy with cluster ion impact ionization

We report suitable secondary ion (SI) counting for surface-sensitive chemical analysis of organic compounds using time-of-flight (TOF) SI mass spectroscopy, based on considerably higher emission yields of SIs induced by cluster ion impact ionization. A SI counting system for a TOF SI mass spectrometer was developed using a fast digital storage oscilloscope, which allows us to perform various types of analysis as all the signal pulses constituting TOF SI mass spectra can be recorded digitally in the system. Effects of the SI counting strategy on SI mass spectra were investigated for C(8) and C(60) cluster ion impacts on an organically contaminated silicon wafer and on polytetrafluoroethylene targets by comparing TOF SI mass spectra obtained from the same recorded signals with different SI counting procedures. Our results show that the use of a counting system, which can cope with high SI yields, is necessary for quantitative analysis of SI mass spectra obtained under high SI yield per impact conditions, including the case of cluster ion impacts on organic compounds.     

Characterising the surface and interior chemistry of core-shell nanoparticles using scanning transmission electron microscopy

A method for extracting core and shell spectra from core-shell particles with varying core to shell volume fractions is described. The method extracts the information from a single EELS spectrum image of the particle. The distribution of O and N was correctly reproduced for a nanoparticle with a TiN core and Ti-oxide shell. In addition, the O distribution from a nanoparticle with a Cu core and a Cu-oxide shell was obtained, and the extracted Cu L_2,3-core and shell spectra showed the required change in EELS near edge fine structure. The extracted spectra can be used for multiple linear least squares fitting to the raw data in the spectrum image. The effect of certain approximations on numerical accuracy, such as treating the nanoparticle as a perfect sphere, as well as the intrinsic detection limits of the technique have also been explored. The technique is most suitable for qualitative, rather than quantitative, work.     

超光谱成像仪的精细光谱定标

为了精细标定棱镜色散超光谱成像仪1024×80光谱像元的中心波长和响应带宽,建立了一套光谱定标装置,提出了实现1nm光谱定标精度的定标方法。首先,介绍了产生谱线弯曲与谱线倾斜的原因,确定了精细光谱定标的方法和数据处理算法;然后,利用光谱定标装置测定了全部光谱响应像元的离散单色光响应值,利用高斯方程拟合了相对光谱响应曲线;最后,建立了中心波长矩阵表和带宽矩阵表,采用多项式拟合算法确定了空间视场像元的色散方程和光谱通道谱线弯曲方程,实验测定了温度变化谱线漂移结果。另外,还对光谱定标精度对辐射定标精度的影响进行了分析。光谱定标结果表明:超光谱成像仪的光谱定标精度达到了±1nm,各谱段带宽平均为8.75nm;色散方程及谱线弯曲与设计结果相符,谱线弯曲值为14～19nm,平均值为17nm;1nm的定标精度对辐射定标精度的影响分别小于1%(3000K黑体)和0.25%(6000K黑体),满足超光谱成像仪1nm光谱定标精度的要求。     

Cluster analysis of soft X-ray spectromicroscopy data

Soft X-ray spectromicroscopy provides spectral data on the chemical speciation of light elements at sub-100 nm spatial resolution. When all chemical species in a specimen are known and separately characterized, existing approaches can be used to measure the concentration of each component at each pixel. In other cases (such as often occur in biology or environmental science), some spectral signatures may not be known in advance so other approaches must be used. We describe here an approach that uses principal component analysis to orthogonalize and noise-filter spectromicroscopy data. We then use cluster analysis (a form of unsupervised pattern matching) to classify pixels according to spectral similarity, to extract representative, cluster-averaged spectra with good signal-to-noise ratio, and to obtain gradations of concentration of these representative spectra at each pixel. The method is illustrated with a simulated data set of organic compounds, and a mixture of lutetium in hematite used to understand colloidal transport properties of radionuclides.     

A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120

We describe the construction and characterization of a laser‐line‐scanning microscope capable of detection of broad fluorescence spectra with a resolution of 1 nm. A near‐infrared femtosecond pulse train at 800 nm was illuminated on a line (one lateral axis, denoted as X axis) in a specimen by a resonant scanning mirror oscillating at 7.9 kHz, and total multi‐photon–induced fluorescence from the linear region was focused on the slit of an imaging polychromator. An electron‐multiplying CCD camera was used to resolve fluorescence of different colours at different horizontal pixels and fluorescence of different spatial positions in a specimen at different vertical pixels. Scanning on the other two axes (Y and Z) was achieved by a closed‐loop controlled sample scanning stage and a piezo‐driven objective actuator. The full widths at half maximum of the point‐spread function of the system were estimated to be 0.39–0.40, 0.33 and 0.56–0.59 μm for the X (lateral axis along the line‐scan), Y (the other lateral axis) and Z axes (the axial direction), respectively, at fluorescence wavelengths between 644 and 690 nm. A biological application of this microscope was demonstrated in a study of the sub‐cellular fluorescence spectra of thylakoid membranes in a cyanobacterium, Anabaena PCC7120. It was found that the fluorescence intensity ratio between chlorophyll molecules mainly of photosystem II and phycobilin molecules of phycobilisome (chlorophyll/phycobilin), in the thylakoid membranes, became lower as one probed deeper inside the cells. This was attributable not to position dependence of re‐absorption or scattering effects, but to an intrinsic change in the local physiological state of the thylakoid membrane, with the help of a transmission spectral measurement of sub‐cellular domains. The efficiency of the new line‐scanning spectromicroscope was estimated in comparison with our own point‐by‐point scanning spectromicroscope. Under typical conditions of observing cyanobacterial cells, the total exposure time became shorter by about 50 times for a constant excitation density. The improvement factor was proportional to the length of the line‐scanned region, as expected.     

The detection of low-intensity peaks in energy dispersive x-ray spectra from particles

A critical step in the processing of energy dispersive (EDS) x-ray spectra from the automated scanning electron microscopy (ASEM) analysis of particles is the detection and identification of elemental peaks. Since there are often several hundred to several thousand spectra for each ASEM analysis, it is important that this step operate rapidly and with a minimum of interaction between analyst and the program. For peaks with large peak-to-background (P/B) ratios, most peak-find or peak-fitting methods do a reasonable job even when the spectra have low signal-to-noise (S/N) ratios. The detection of peaks with small P/B ratios is much more problematical. Peak identification and fitting procedures may not work well on low-intensity peaks, particularly in spectra with low S/N ratios. In this study, three procedures for identifying x-ray peaks, with small P/B ratios in spectra with varying S/N ratios, were evaluated. The first procedure was the identification of peaks by human analysts. The results from the analysts were then used to set a benchmark for the performance of two computer-based procedures that included three different qualitative peak identification methods, and one quantitative analysis procedure. The success of the qualitative methods in finding small peaks varied widely. In general, the quantitative analysis procedure performed as well as the best human analysts and was better than the qualitative methods.     

Characterization of Hazardous Gases Using an Infrared Hyperspectral Imaging System

A rapid algorithm for passive remote sensing of gases with an infrared hyperspectral imaging system is reported that includes approximate and precise measurements. In the former, the spectral region of interest for the analytes is identified. In the latter, fitting calculations in the selected range and for spectral similarity measurements are employed to remove erroneous pixels from the approximate measurement to better characterize the gas sample. The results verify that the algorithm reduced the time for qualitative and quantitative determination of gases compared with conventional approaches.     

Analysis of directly frozen macromolecules and tissues in the field-emission STEM

A VG Microscopes HB501 field-emission high-resolution scanning transmission electron microscope (STEM) was used to image and analyse rapidly frozen, isolated macromolecules and small organelles in tissue cryosections. Dark-field images were obtained from frozen-hydrated microtubules demonstrating that sufficient contrast is available to reveal structural information. The samples were subsequently freeze-dried in the STEM and low-dose (? 10³ e/nm²) dark-field mass maps were recorded with single electron sensitivity. Elemental analysis of individual macromolecules was achievable at high dose using parallel-detection electron energy-loss spectroscopy, albeit with some structural degradation. Detection of copper (320 atoms) in di-decameric haemocyanin molecules was easily within the limits of sensitivity. Elemental analysis of hydrated cryosections is limited by radiation damage to a resolution of approximately 1 μm². For freeze-dried sections, however, the high probe current and stable cold stage of the HB501 STEM allow energy-dispersive X-ray (EDX) microanalysis of low elemental concentrations in highly localized subcellular volumes. EDX spectra from cryosections of cerebellar cortex show that a 100-s analysis time is sufficient to quantify the calcium content of 400-nm² regions within Purkinje cell dendrites with an uncertainity of ± 2 mmol/kg dry weight, equivalent to ± 12 atoms.     

Mapping chemical and bonding information using multivariate analysis of electron energy-loss spectrum images

Electron energy-loss spectroscopy (EELS) in the transmission electron microscope (TEM) is used to obtain high-resolution information on the composition and the type of chemical bonding of materials. Spectrum imaging, where a full EEL spectrum is acquired and stored at each pixel in the image, gives an exact correlation of spatial and spectral features. However, determining and extracting the important spectral components from the large amount of information contained in a spectrum image (SI) can be difficult. This paper demonstrates that principal component analysis of EEL SIs can be used to extract chemically relevant components. With weighted or two-way scaled principal component analysis, both compositional and bonding information can be extracted. Mapping of the chemical variations in a partially reduced titanium dioxide sample and the orientation-dependent bonding in boron nitride and carbon nanotubes are given as examples.     

基于二阶校正法的多环芳烃污染物荧光检测

为了对复杂体系的多环芳烃进行定性识别和定量分析,构建了基于二阶校正法的荧光检测系统。利用二阶校正法在三维荧光数据处理中的优势,达到多种混合多环芳烃分离和鉴别的目的。采用FS920荧光光谱仪测量并分析了萘(NAP)、苊(ANA)及两者混合物的荧光光谱特性,发现NAP溶液有一个荧光峰λex/λem=290/322nm,ANA溶液存在两个荧光峰分别为λex/λem=290/322nm和λex/λem=290/336nm,NAP和ANA荧光光谱重叠严重,并且不同浓度配比混合物的荧光光谱具有差异性。通过将二阶校正法与三维荧光光谱法相结合,实现对多环芳烃混合物的浓度检测。分别采用平行因子(PARAFAC)算法和自加权交替三线性分解(SWATLD)算法对光谱数据进行分解。结果表明:两种算法对NAP和ANA混合物均有较高的分辨能力,预测平均回收率均在95%~99%、均方根误差均小于0.2μg/L。相比之下,SWATLD算法的检测效果更好。     

DART-Q-Orbitrap MS法快速检测豆制品中碱性橙Ⅱ和金胺O

采用实时直接分析(direct analysis in real time,DART)离子化技术与四极杆-静电场轨道阱高分辨质谱联用技术(Q-Orbitrap MS),建立了快速定性和定量分析方法,检测豆制品中非法添加的碱性橙Ⅱ和金胺O。在正离子模式下,采用Full MS/targetrd-MS2扫描方式,可直接将豆制品置于离子化区域内对两种染料进行快速定性分析。在定量分析中,采用80%乙腈水溶液提取样品,应用Dip-it载样方式,对实验参数进行系统性优化。采用空白基质溶液进行梯度稀释,测得碱性橙Ⅱ和金胺O在基质中的检出限均为0.2mg/L;采用内标法定量,在1～20mg/L浓度范围内线性关系良好,相关系数分别为0.997 2和0.999 4。该方法快速准确,可为食品中非法添加染料的定性和定量检测提供参考依据。