Correction of optical distortions in dry depth profiling with confocal Raman microspectroscopy

We present a generalized approach to obtain improved Raman intensity profiles from in‐depth studies performed by confocal Raman microspectroscopy (CRM) with dry objectives. The approach is based on regularized deconvolution of the as‐measured confocal profile, through a kernel that simulates optical distortions due to diffraction, refraction and collection efficiency on the depth response. No specific shape or restrictions for the recovered profile are imposed. The strategy was tested by probing, under different instrumental conditions, a series of model planar interfaces, generated by the contact of polymeric films of well‐defined thickness with a substrate. Because of the aforementioned optical distortions, the as‐measured confocal response of the films appeared highly distorted and featureless. The signal computed after deconvolution recovers all the films features, matching very closely with the response expected. Copyright © 2011 John Wiley & Sons, Ltd.     

基于双螺旋点扩散函数工程的多焦点图像扫描显微

在传统共聚焦显微技术的基础上,图像扫描显微技术使用面阵探测器来代替单点探测器,结合虚拟数字针孔并利用像素重定位和解卷积图像重构算法将传统宽场显微镜的分辨率提高一倍,实现了高信噪比的超分辨共焦成像.但是,由于采用逐点扫描的方式,三维成像速度相对较慢,限制了其在活体样品成像中的应用.为了进一步提高图像扫描显微术的成像速度,...     

Numerical simulations of confocal Raman spectroscopic depth profiles of materials: a photon scattering approach

We present a simple model that uses a novel photon scattering approach to predict the depth profile response obtained when confocal Raman spectroscopy is applied both to silicon and to a number of related polymeric materials of varying optical clarity. This paper first provides an overview of the models proposed to date to demonstrate the evolution in understanding of the confocal Raman response of semi‐transparent materials, based upon geometrical optics. A new model is then described that is based upon the twin notions of a permanent extended Raman illuminated volume and the degree of extinction of the incident and Raman scattered photons from the whole of the illuminated volume as it is gradually moved further into, or defocused above, the sample. The model's predictions are compared with empirical data from previous studies of a range of semi‐crystalline polymers with different scattering properties and, by means of contrast, with that of a silicon sample. We show that, despite its inherent simplicity, the physics this model utilizes is able successfully to predict the form of the depth profile for each material, something that has not been achieved by any model previously proposed, and that the parameters used in the model scale with independent physical measurements. Finally the model is used to account for the fact that useful Raman spectra can be obtained when the laser is focused as much as 40 µm above the sample surface. Copyright © 2007 John Wiley & Sons, Ltd.     

Confocal Raman microscopy: how to correct depth profiles considering diffraction and refraction effects

A new approach to obtain corrected depth profiles by confocal Raman microscopy, which considers diffraction and refraction effects is presented. The problem of diffraction effects encountered intrinsically in the confocal configuration can be described using a linear Fredholm integral equation of the first kind, which correlates apparent and true Raman intensities with the depth resolution curve of the instrument. Refractive index differences between air and the polymer sample, which cause further errors in the obtained depth profile due to strong aberration effects have been considered. This has been carried out using an empirical variation of the depth resolution function, which is able to simulate the broadening of the depth of focus with depth and also the discrepancy between nominal and measured depth scales. It is shown that considerable differences between apparent and corrected depth profiles exist at the surface and that these depend on the gradient of the profile and the depth resolution of the Raman microscope. Copyright © 2007 John Wiley & Sons, Ltd.     

Imaging theory of nonlinear second harmonic and third harmonic generations in confocal microscopy

ItisreportedrecentlythatnonlinearopticalphenomenonofSHGandTHGhasbeenobservedinmanybiologicaltissues[16].SHGandTHGhavebeenusedtoperformthethree-dimensionalimaginginlivingtissuesandhaveattractedmuchattentionrecently.TherearemanyadvantagesofusingSHGandTHGtoperformthethree-dimensionalimaginginlivingtissues,suchasnoninvasiveandnophotobleaching,inadditiontotheimagingpropertiesofmulti-photonfluorescenceimaging[7—9].Firstly,unlikeinthesingle-andmulti-photonfluorescenceprocesses,onlyvirtualstat…     

常态中子深度分析物理模型

带电离子在样品内输运过程中的能量岐离、探测立体角有效范围及探测系统噪声都将改变中子深度分析（NDP）的多道离子能谱分布。假定上述三种因素导致的离子探测谱能量展宽都可以采用高斯函数描述,基于一般条件下的NDP装置设计,建立了预测一定深度核素产生的离子测量谱的探测器响应函数物理模型。该模型的理论分析结果与实验测量能谱的峰位、半高宽均符合良好,能够作为中子深度分析测量谱反演分析的基础。     

Micro‐Raman depth profiling on polished cross‐sections: the mapping of oxalates used in protective treatment of carbonatic substrate

We present the first depth profiling on a polished cross‐section carried out by micro‐Raman spectroscopy aimed at investigating the penetration depth of an inorganic protective material (ammonium oxalate) applied on carbonatic surfaces (ancient plaster) and to detect the distribution of mineralogical phases inside the material. The reason for such a Rapid Communication is the importance of our study for the field of conservation, in which at present the treatment with oxalates is a hot issue. Copyright © 2008 John Wiley & Sons, Ltd.     

Depth‐resolved micro‐Raman spectroscopy of tri‐layer PFSA membrane for PEM fuel cells: how to obtain reliable inner water contents

Raman depth‐profiling microspectroscopy is currently emerging as a fast and non‐invasive method for the local content measurement of water diffusing across the perfluorosulfonic acid polymer used as electrolyte in low‐temperature fuel cells. However, water depth profiles obtained thereby are affected by the gradual degradation of the Raman signal as the exciting radiation is focused deeper into the sample and, thus, usually exhibit artificial gradients. The appropriate way to rectify raw data, in order to measure reliable inner water contents, is discussed in the case of perfluorosulfonic acid membranes soaked in water. The method is tested on a tri‐layer material composed by stacked ionomers with different chemical composition, ionic exchange capacity and swelling. Comparison of Raman spectra of the different ionomers, obtained under strictly the same hydration and optical conditions, allows critical discussion of previously reported band assignments. It is shown that Raman bands arising from the polymer backbone and from the side chain can be readily discriminated. Reliable water concentration profiles can then be obtained from the ratio between the Raman signal of sorbed water and of the polymer phase. Comparison with depth profiles obtained by using the pure water signal as internal reference shows that quantitative measurement requires the careful choice of the adequate Raman bands for representing the ionomer occupation of the volume probed by the exciting radiation. Different to what usually performed, the ionomer Raman signal to be integrated for compensating optical losses should include both bands arising from the polymer backbone and side chains. Last, obtaining accurate water concentration profiles also needs the knowledge of the so‐called optical factor β, i.e. the ratio between the scattering cross sections of ionomer chemical groups and sorbed water, which has to be measured by independent Raman experiments. Copyright © 2012 John Wiley & Sons, Ltd.     

Surface‐sensitive Raman microscopy with total internal reflection illumination

A Raman microscope using a total internal reflection (TIR) annular illumination geometry through a ZnSe solid immersion lens (SIL) is described. Spectra of a thin‐film sample of the transparent organic conductor poly(3,4‐ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS) on a polyethylene terephthalate (PET) substrate are presented and compared with those from a conventional confocal Raman configuration. These spectra demonstrate a significant increase in surface selectivity upon the use of TIR illumination, as the decay length of the evanescent excitation field limits the depth of sample probed in this configuration. Spectral interference from the underlying PET substrate layer is thus greatly reduced. An increase in surface selectivity is also demonstrated for spectra acquired through the SIL with uniform illumination. Raman images of a micropatterned PEDOT:PSS film acquired with TIR illumination are also reported. Enhanced lateral resolution is realized in this configuration because of the immersion effect of the SIL, and the sampling depth is limited to 150 nm by the choice of illumination geometry. This results in analysis volumes on the order of tens of femtoliters, nearly two orders of magnitude smaller than typically achieved in conventional confocal Raman microscopes. This approach yields Raman spectra and images with surface selectivity significantly greater than can be achieved in confocal Raman, and provides a valuable tool for the microanalysis of thin surface films. Published in 2010 by John Wiley & Sons, Ltd.     

Optimizing thermography depth probing with a dynamic thermal point spread function

Derivation of two point spread functions PSFs suitable for infrared thermograms analysis is illustrated, based on two unique approaches, one based on depth decaying limit and one on diffusion limit. Experimental work using PMMA sample with back drilled holes and pulsed thermographic routine is utilized to show the effectiveness of deconvoluting pixel temperature transient history with suggested PSF’s. Synthetic second time derivative thermograms are utilized for comparison and the signal to noise ratio is used as a figure of merit for quantification.     

Non‐invasive depth profiling by space and time‐resolved Raman spectroscopy

Time‐resolved Raman spectroscopy, spatially offset Raman spectroscopy and time‐resolved spatially offset Raman spectroscopy (TR‐SORS) have proven their capability for the non‐invasive profiling of deep layers of a sample. Recent studies have indicated that TR‐SORS exhibits an enhanced selectivity toward the deep layers of a sample. However, the enhanced depth profiling efficiency of TR‐SORS, in comparison with time‐resolved Raman spectroscopy and spatially offset Raman spectroscopy, is yet to be assessed and explained in accordance to the synergistic effects of spatial and temporal resolutions. This study provides a critical investigation of the depth profiling efficiency of the three deep Raman techniques. The study compares the efficiency of the various deep Raman spectroscopy techniques for the stand‐off detection of explosive precursors hidden in highly fluorescing packaging. The study explains for the first time the synergistic effects of spatial and temporal resolutions in the deep Raman techniques and their impact on the acquired spectral data. Copyright © 2013 John Wiley & Sons, Ltd.     

Hybrid Rayleigh,Raman and two‐photon excited fluorescence spectral confocal microscopy of living cells

A hybrid fluorescence–Raman confocal microscopy platform is presented, which integrates low‐wavenumber‐resolution Raman imaging, Rayleigh scatter imaging and two‐photon fluorescence (TPE) spectral imaging, fast ‘amplitude‐only’ TPE‐fluorescence imaging and high‐spectral‐resolution Raman imaging. This multi‐dimensional fluorescence–Raman microscopy platform enables rapid imaging along the fluorescence emission and/or Rayleigh scatter dimensions. It is shown that optical contrast in these images can be used to select an area of interest prior to subsequent investigation with high spatially and spectrally resolved Raman imaging. This new microscopy platform combines the strengths of Raman ‘chemical’ imaging with light scattering microscopy and fluorescence microscopy and provides new modes of correlative light microscopy. Simultaneous acquisition of TPE hyperspectral fluorescence imaging and Raman imaging illustrates spatial relationships of fluorophores, water, lipid and protein in cells. The fluorescence–Raman microscope is demonstrated in an application to living human bone marrow stromal stem cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Fast mapping of inhomogeneities in the popular metallic perovskite Nb:SrTiO3 by confocal Raman microscopy

Confocal Raman microscopy was applied in order to investigate the homogeneity of donor doping in Nb:SrTiO₃ single crystals. Measurements of local Raman spectra revealed a systematic relation between the intensity of the Raman signal and the donor content of the crystals. We successfully elaborated a correspondence between the electronic structure and the intensity of the Raman lines using a crystal with macroscopic inhomogeneity as a demonstration sample. By mapping the distribution of the intensity of the Raman signal, we identified a characteristic inhomogeneous structure related to the presence of clusters with sizes of 5 µm to 20 µm, indicating inhomogeneous donor distribution caused by flaws introduced during crystal growth. Hence, we propose confocal Raman microscopy as a convenient technique for investigating the homogeneity and quality of doped perovskite surfaces, which are needed for various technological applications. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Calcification of aortic human valves studied in situ by Raman microimaging: following mineralization from small grains to big deposits

A Raman microimaging‐based approach has been used in the current study to evaluate formation and progression of calcification in situ in human stenotic aortic valves obtained during surgical valve replacement. The capability of the method to visualize distribution of the calcified deposits resulted in structural characterization of deposits in the various phases of development. A high spatial resolution of the method along with the confocal depth profiling enabled to identify extremely small salt inclusions (of ca. 0.5 µm in diameter), formed probably at the very early stage of calcification. Structurally, these inclusions are built from an octacalcium phosphate‐like compound that during grains' growth transforms into tricalcium phosphate, mixed with the salt containing the acidic phosphate groups (HPO₄²⁻) and, finally, into stable B‐type hydroxyapatite that is the only salt present in large‐area calcium salt deposits. Copyright © 2013 John Wiley & Sons, Ltd.     

中子半影成像的散射中子对点扩展函数的影响

基于ICF中子半影成像分辨力的要求,利用蒙特卡罗方法,模拟了4种系统模型的点扩展函数,分析半影孔装置、靶室和中子束流通道的钢管、高密度内爆靶丸的散射中子对点扩展函数的影响;模拟了平面中子源在4种模型中的半影成像,并用维纳滤波方法重建,分析了散射中子对中子半影成像诊断的影响。模拟结果表明:半影孔装置、靶室等的散射中子不会造成点扩展函数的变形,仅仅增加本底,对诊断的影响可以忽略;高密度内爆靶丸的散射中子会造成点扩展函数的平滑,但对诊断的影响有限。     

Depth profiling of porcine adipose tissue by Raman spectroscopy

Raman spectroscopy was applied on a depth profile of porcine adipose tissue (from skin to meat) with the purpose of (1) discriminating between fat layers and (2) estimating the variation in fatty acid composition as a function of fat depth and fat layer: total degree of unsaturation (iodine value), fractions of saturated, and monounsaturated and polyunsaturated fatty acids. The thickness and composition of the outer layer of porcine adipose tissue influences the final quality of backfat. A too‐thick outer layer is associated with problems such as oily appearance, rancidity development, and difficulties in separating muscle and adipose tissue when cutting. From principal component analysis on standard normal variate preprocessed Raman spectra (1800–800 cm^–1), it was possible to discriminate between the outer and the inner backfat layer. Principal component analysis loadings showed that the separation of layer was mainly explained by variation in the bands originating from vibration of double bond C = C stretching plus = C–H twisting and rocking. In the prediction of iodine value a three‐component partial least squares regression model based on full range Raman spectra showed a root mean square error of cross validation of 2.00 and R² = 0.69. Applying Cauchy–Lorentz band fitting proved that information regarding fat unsaturation was found not only in band intensity, but also in band parameters such as location and width. The results suggest Raman spectroscopy as a potential measurement technique for rapid grading of pork carcasses. Copyright © 2011 John Wiley & Sons, Ltd.     

超衍射成像中 双螺旋点扩展函数的三维定位精度

开关效应和单分子定位的结合可以实现样品的超衍射分辨成像, 双螺旋点扩展函数将单分子定位纳米分辨从二维扩展到了三维.本文对双螺旋点扩展函数的三维定位精度展开了探讨.首先, 基于费希尔信息量, 计算了双螺旋点扩展函数的无偏估计, 得出其理论定位精度, 并分析了光子数、背景噪声以及有效像元尺寸大小对其定位精度的影响; 其次, 基于单分子定位实验过程中对于数据分析通常采用的高斯拟合质心定位算法, 通过误差传递函数定律求得双螺旋点扩展函数的轴向定位精度.计算机模拟结果表明, 在光子数大于1000的条件下, 高斯拟合质心定位精度和费希尔信息量理论定位精度符合较好. 本文的讨论不仅为双螺旋点扩展函数的三维定位精度提供了理论依据, 同时也可为实验提供理论指导. 关键词： 双螺旋点扩展函数 费希尔信息量 定位精度 高斯拟合     

Micro‐Raman depth profiling of silicon amorphization induced by high‐energy ion channeling implantation

In this work, we study the silicon amorphization dependence on the crystal depth induced by 6‐MeV Al²⁺ ions implanted in the <110> and randomly oriented silicon crystal channels, which was not directly experimentally accessible in the previous similar high‐energy ion–crystal implantation cases. Accordingly, the micro‐Raman spectroscopy scanning measurements along the crystal transversal cross section of the ion implanted region were performed. The ion fluence was 10¹⁷ particles/cm². The scanning steps were 0.2 and 0.3 µm, for the channeling and random ion implantations, respectively. The obtained results are compared with the corresponding Rutherford backscattering spectra of 1.2‐MeV protons in the random and channeling orientations measured during the channeling implantation. Additionally, scanning electron microscope picture was taken on the transversal cross section of the implanted region in the channeling implantation case. We show here that the obtained silicon amorphization maxima are in excellent agreement with the corresponding estimated maxima of the aluminum concentration in silicon. This clearly indicates that the used specific micro‐Raman spectroscopy scanning technique can be successfully applied for the depth profiling of the crystal amorphization induced by high‐energy ion implantation. Copyright © 2013 John Wiley & Sons, Ltd.     

高效双螺旋点扩展函数相位片的设计与实验研究

双螺旋点扩展函数具有随离焦连续旋转变化的特性, 结合单分子定位方法可用于厚样品三维超分辨成像及分子定位追踪研究. 但双螺旋点扩展函数不足之处是光能利用率低, 对于光子数受限的荧光显微成像而言其应用受限. 本文通过对双螺旋点扩展函数在空域、频域和拉盖尔-高斯模式面等三个不同域中进行约束优化. 模拟结果表明, 优化后的双螺旋点扩展函数的光能效率提高了30多倍. 同时, 基于最优设计方案制备了相位片, 并实验验证了该设计的正确性. 与文献报道相比较, 本文结果在成像深度和光能利用率方面都有所改善. 关键词： 双螺旋点扩展函数 超分辨成像 轴向定位 优化算法