On-tissue chemical derivatization in mass spectrometry imaging

Mass spectrometry imaging (MSI) combines molecular and spatial information in a valuable tool for a wide range of applications. Matrix-assisted laser desorption/ionization (MALDI) is at the forefront of MSI ionization due to its wide availability and increasing improvement in spatial resolution and analysis speed. However, ionization suppression, low concentrations, and endogenous and methodological interferences cause visualization problems for certain molecules. Chemical derivatization (CD) has proven a viable solution to these issues when applied in mass spectrometry platforms. Chemical tagging of target analytes with larger, precharged moieties aids ionization efficiency and removes analytes from areas of potential isobaric interferences. Here, we address the application of CD on tissue samples for MSI analysis, termed on-tissue chemical derivatization (OTCD). MALDI MSI will remain the focus platform due to its popularity, however, alternative ionization techniques such as liquid extraction surface analysis and desorption electrospray ionization will also be recognized. OTCD reagent selection, application, and optimization methods will be discussed in detail. MSI with OTCD is a powerful tool to study the spatial distribution of poorly ionizable molecules within tissues. Most importantly, the use of OTCD−MSI facilitates the analysis of previously inaccessible biologically relevant molecules through the adaptation of existing CD methods. Though further experimental optimization steps are necessary, the benefits of this technique are extensive.     

Mass spectrometry imaging under ambient conditions

Mass spectrometry imaging (MSI) has emerged as an important tool in the last decade and it is beginning to show potential to provide new information in many fields owing to its unique ability to acquire molecularly specific images and to provide multiplexed information, without the need for labeling or staining. In MSI, the chemical identity of molecules present on a surface is investigated as a function of spatial distribution. In addition to now standard methods involving MSI in vacuum, recently developed ambient ionization techniques allow MSI to be performed under atmospheric pressure on untreated samples outside the mass spectrometer. Here we review recent developments and applications of MSI emphasizing the ambient ionization techniques of desorption electrospray ionization (DESI), laser ablation electrospray ionization (LAESI), probe electrospray ionization (PESI), desorption atmospheric pressure photoionization (DAPPI), femtosecond laser desorption ionization (fs‐LDI), laser electrospray mass spectrometry (LEMS), infrared laser ablation metastable‐induced chemical ionization (IR‐LAMICI), liquid microjunction surface sampling probe mass spectrometry (LMJ‐SSP MS), nanospray desorption electrospray ionization (nano‐DESI), and plasma sources such as the low temperature plasma (LTP) probe and laser ablation coupled to flowing atmospheric‐pressure afterglow (LA‐FAPA). Included are discussions of some of the features of ambient MSI for example the ability to implement chemical reactions with the goal of providing high abundance ions characteristic of specific compounds of interest and the use of tandem mass spectrometry to either map the distribution of targeted molecules with high specificity or to provide additional MS information on the structural identification of compounds. We also describe the role of bioinformatics in acquiring and interpreting the chemical and spatial information obtained through MSI, especially in biological applications for tissue diagnostic purposes. Finally, we discuss the challenges in ambient MSI and include perspectives on the future of the field. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:218–243, 2013     

基于高分辨质谱技术的整体动物体内药物成像分析新方法研究

质谱分子成像技术作为体内药物分析的新兴工具受到越来越多的关注,其中生物体内复杂基质的干扰是面临的关键问题之一。本研究利用高分辨质谱技术的优势,采用空气动力辅助离子化质谱成像方法（AFAI-MSI）,建立了高特异性的整体动物体内药物成像分析新方法,以提高体内药物成像分析结果的准确性和可靠性。以候选新药右旋娃儿藤宁碱（S-(+)-deoxytylophorinidine, CAT）为研究对象,采用高分辨质谱全扫描与靶向扫描相结合的方式,对整体动物体内药物的分布进行AFAI-MSI分析。结果表明,该方法能够准确地反映药物特异性分布和处置情况。通过一次质谱成像实验,从整体动物水平同时实现了候选新药体内分布和药物干预下内源性代谢物的成像分析,为候选新药的早期研发提供思路与手段。     

衍生化技术在MALDI质谱成像中的应用进展

近年来，基质辅助激光解吸离子化（matrix-assisted laser desorption ionization, MALDI）质谱成像（mass spectrometry imaging, MSI）技术发展迅速，在很多领域均有广泛应用。质谱成像技术虽然可以同时得到上百个化合物的数据，但是对于低丰度、离子化效率低和易受基质峰干扰的化合物的成像分析仍然极具挑战。为了提高质谱成像中目标分子的信号响应，将原位衍生化方法应用于质谱成像技术中是非常必要的。衍生化方法与质谱成像的结合已成功实现了不同类型目标分析物的成像分析，且均展现了优异的效果。本文综述了样本上原位衍生化方法在MALDI质谱成像中的应用，包括用于含有羧基、氨基、醛基等活性基团的目标分子的衍生化试剂和衍生化反应，以及衍生化的基质选择和试剂的涂覆装置，并展望了衍生化方法结合MALDI质谱成像技术的发展趋势。     

A method for the evaluation of thousands of automated 3D stem cell segmentations

There is no segmentation method that performs perfectly with any dataset in comparison to human segmentation. Evaluation procedures for segmentation algorithms become critical for their selection. The problems associated with segmentation performance evaluations and visual verification of segmentation results are exaggerated when dealing with thousands of three‐dimensional (3D) image volumes because of the amount of computation and manual inputs needed. We address the problem of evaluating 3D segmentation performance when segmentation is applied to thousands of confocal microscopy images (z‐stacks). Our approach is to incorporate experimental imaging and geometrical criteria, and map them into computationally efficient segmentation algorithms that can be applied to a very large number of z‐stacks. This is an alternative approach to considering existing segmentation methods and evaluating most state‐of‐the‐art algorithms. We designed a methodology for 3D segmentation performance characterization that consists of design, evaluation and verification steps. The characterization integrates manual inputs from projected surrogate ‘ground truth’ of statistically representative samples and from visual inspection into the evaluation. The novelty of the methodology lies in (1) designing candidate segmentation algorithms by mapping imaging and geometrical criteria into algorithmic steps, and constructing plausible segmentation algorithms with respect to the order of algorithmic steps and their parameters, (2) evaluating segmentation accuracy using samples drawn from probability distribution estimates of candidate segmentations and (3) minimizing human labour needed to create surrogate ‘truth’ by approximating z‐stack segmentations with 2D contours from three orthogonal z‐stack projections and by developing visual verification tools. We demonstrate the methodology by applying it to a dataset of 1253 mesenchymal stem cells. The cells reside on 10 different types of biomaterial scaffolds, and are stained for actin and nucleus yielding 128 460 image frames (on average, 125 cells/scaffold × 10 scaffold types × 2 stains × 51 frames/cell). After constructing and evaluating six candidates of 3D segmentation algorithms, the most accurate 3D segmentation algorithm achieved an average precision of 0.82 and an accuracy of 0.84 as measured by the Dice similarity index where values greater than 0.7 indicate a good spatial overlap. A probability of segmentation success was 0.85 based on visual verification, and a computation time was 42.3 h to process all z‐stacks. While the most accurate segmentation technique was 4.2 times slower than the second most accurate algorithm, it consumed on average 9.65 times less memory per z‐stack segmentation.     

Simultaneous two-photon fluorescence correlation spectroscopy and lifetime imaging of dye molecules in submicrometer fluidic structures

Fluorescence correlation spectroscopy (FCS) is a very sensitive technique that can be used, e.g., for the measurement of low concentrations and for the investigation of transport of fluorescent molecules. Fluorescence lifetime imaging (FLIM) provides spatially resolved information about molecular fluorescence lifetimes reflecting the interactions of the molecules with their environment. We have applied simultaneous two-photon FCS and FLIM to probe the behavior of fluorescent molecules diffusing in submicrometer silicon oxide channels. Our measurements reveal differences in fluorescence lifetimes compared to bulk solution that result from the effects of confinement and the presence of interfaces. Confinement also affects diffusional characteristics of fluorophores as reflected in fluorescence autocorrelation functions. These possible consequences of both spatial confinement and the presence of interfaces between media with different refractive indices on the diffusion and fluorescence lifetime of molecules in nanostructures are discussed in general.     

基于常压基质辅助激光解吸电离质谱成像技术原位检测三维肿瘤细胞球内代谢小分子

与传统培养的二维肿瘤细胞相比，三维肿瘤细胞球能从多个方面更好地模拟实体瘤的一些特征，如空间结构和药物抗性机理。本研究建立了常压基质辅助激光解吸电离-串联四极杆轨道离子阱质谱法原位检测三维肿瘤细胞球内代谢小分子。利用该方法检测分析了22种内源性代谢小分子在三维细胞球内的空间分布，其中4种涉及三羧酸循环代谢通路的小分子分布于整个细胞球区域，另外18种涉及甘油磷脂合成与降解通路的脂质分子在细胞球内的分布呈多样性。例如，PGP(O-30∶3)和PGP(30∶2)分布于细胞球中心区域；PG(O-36∶1)分布于细胞球外围区域；PE(18∶1/22∶6)分布于整个细胞球区域。区域分割分析表明，细胞球外围区域和中心区域的质谱峰信号强度有显著性差异。该研究可为进一步了解肿瘤模型的微环境以及肿瘤代谢的分子机制提供参考。     

An open‐source deconvolution software package for 3‐D quantitative fluorescence microscopy imaging

Deconvolution techniques have been widely used for restoring the 3‐D quantitative information of an unknown specimen observed using a wide‐field fluorescence microscope. Deconv , an open‐source deconvolution software package, was developed for 3‐D quantitative fluorescence microscopy imaging and was released under the GNU Public License. Deconv provides numerical routines for simulation of a 3‐D point spread function and deconvolution routines implemented three constrained iterative deconvolution algorithms: one based on a Poisson noise model and two others based on a Gaussian noise model. These algorithms are presented and evaluated using synthetic images and experimentally obtained microscope images, and the use of the library is explained. Deconv allows users to assess the utility of these deconvolution algorithms and to determine which are suited for a particular imaging application. The design of Deconv makes it easy for deconvolution capabilities to be incorporated into existing imaging applications.     

Multiparametric high-resolution imaging of barley embryos by multiphoton microscopy and magnetic resonance micro-imaging

Nonlinear optical microscopy and magnetic resonance imaging (MRI) address different properties of the sample and operate on different geometrical scales. MRI maps density and mobility of molecules tracking specific molecular signatures. Multiphoton imaging profits from the nonlinear absorption of light in the focus of a femtosecond laser source stimulating the autofluorescence of biomolecules. As this effect relies on a high light intensity, the accessible field of view is limited, but the resolution is very high (a few hundred nanometers). Here, we aim to link the different accessible scales and properties addressed in the different techniques to obtain a synoptic view. As model specimen we studied embryos of barley. Multiphoton stimulated autofluorescence images and images of second harmonic generation are achieved even down to low magnification (10x), low numerical aperture (N.A. 0.25) conditions. The overview images allowed morphological assignments and fluorescence lifetime imaging provides further information to identify accumulation of endogenous fluorophores. The second, complementary contribution from high-resolution MR images provides a 3D model and shows the embedding of the embryo in the grain. Images of the proton density were acquired using a standard 3D spin-echo imaging pulse sequence. Details directly comparable to the low magnification optical data are visible. Eventually, passing from the MR images of the whole grain via low magnification to high resolution autofluorescence data bridges the scale barrier, and might provide the possibility to trace transport and accumulation of, e.g., nutrients from large structure of the plant to the (sub-) cellular level.     

产品设计中生命周期评价与生命周期成本的集成与优化

生命周期评价(Life cycle assessrnent, LCA)是绿色设计中的产品环境属性的定量评价工具,但LCA的经济和时间代价制约了LCA的企业应用和发展,将LCA与生命周期成本评估(Life cycle costing, LCC)集成优化是解决这一问题的有效途径。在LCA基本架构的基础上提出LCA与LCC集成评价框架,并考虑时间因素,提出基于矩阵的集成评价算法,建立经济属性与环境属性之间的联系。基于集成评价的结果,建立环境与成本的优化模型,并基于并行子空间优化算法(Concurrent subspace optimization, CSSO)进行优化,使综合效益达到最大,实现经济性与环保性的“双赢”,为改善产品设计提供量化依据。实例研究验证了所提方法的有效性。     

Small-scale in vivo imaging of soft tissues by radioscopy using single X-ray photon counting

A method has been developed for routine laboratory visualisation of small‐scale soft tissue by means of transmission X‐ray radioscopy and tomography. Using termites as models, imaging quality with a spatial resolution of about 3 μm was achieved and 3D tomographic reconstruction was demonstrated. A termite worker individual was visualized before and after its metamorphosis towards the soldier caste. The developed methodology represents a non‐invasive and real‐time way of acquiring 3D anatomic data with a high contrast so that it is a promising candidate to become a tool for routine investigations in life sciences.     

Modeling the clay minerals–enzyme binding by fusion fluorescent proteins and under atomic force microscope

In the present study, binding of cellulase protein to different clay minerals were tested using fluorescent–protein complex and microscopic techniques. Cellulase gene (Cel5H) was cloned into three fluorescent vectors and expressed as fusion enzymes. Binding of Cel5H–mineral particles was confirmed by confocal microscopy, and enzyme assay. Among the Cel5H–fusion enzymes, green–fusion enzyme showed higher intensity compared with other red and yellow fusion–proteins. Intensity of fusion–proteins was dependent on the pH of the medium. Confocal microscopy revealed binding of the all three fusion proteins with different clay minerals. However, montmorillonite displayed higher binding capacity than kaolinite clay. Likewise, atomic force microscopy (AFM) image profile analysis showed proteins appeared globular molecules in free‐state on mica surface with an average cross sectional diameter of 110 ± 2 nm and rough surface of montmorillonite made protein appear flattened due to structural alteration. Even surface of kaolinite also exerted some strain on protein molecular conformation after binding to surface. Our results provide further evidence for 3D visualization of enzyme–soil complex and encourage furthering study of the force involved interactions. Therefore, our results indicate that binding of proteins to clay minerals was external and provides a molecular method to observe the interaction of clay minerals–enzyme complex.     

Quantification of micrometre-sized porosity in quasicrystals using coherent synchrotron radiation imaging

The ability to image phase distributions with high spatial resolution is a key capability of microscopy systems. Consequently, the development and use of phase microscopy has been an important aspect of microscopy research and development. Most phase microscopy is based on a form of interference. Some phase imaging techniques, such as differential interference microscopy or phase microscopy, have a low coherence requirement, which enables high‐resolution imaging but in effect prevents the acquisition of quantitative phase information. These techniques are therefore used mainly for phase visualization. On the other hand, interference microscopy and holography are able to yield quantitative phase measurements but cannot offer the highest resolution. A new approach to phase microscopy, quantitative phase‐amplitude microscopy (QPAM) has recently been proposed that relies on observing the manner in which intensity images change with small defocuses and using these intensity changes to recover the phase. The method is easily understood when an object is thin, meaning its thickness is much less than the depth of field of the imaging system. However, in practice, objects will not often be thin, leading to the question of what precisely is being measured when QPAM is applied to a thick object. The optical transfer function formalism previously developed uses three‐dimensional (3D) optical transfer functions under the Born approximation. In this paper we use the 3D optical transfer function approach of Streibl not for the analysis of 3D imaging methods, such as tomography, but rather for the problem of analysing 2D phase images of thick objects. We go on to test the theoretical predictions experimentally. The two are found to be in excellent agreement and we show that the 3D imaging properties of QPAM can be reliably predicted using the optical transfer function formalism.     

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.     

Application of autofluorescence robotic histology for quantitative evaluation of the 3‐dimensional morphology of murine articular cartilage

Murine models of osteoarthritis (OA) are increasingly important for understating pathogenesis and for testing new therapeutic approaches. Their translational potential is, however, limited by the reduced size of mouse limbs which requires a much higher resolution to evaluate their articular cartilage compared to clinical imaging tools. In experimental models, this tissue has been predominantly assessed by time‐consuming histopathology using standardized semi‐quantitative scoring systems. This study aimed to develop a novel imaging method for 3‐dimensional (3D) histology of mouse articular cartilage, using a robotic system—termed here “3D histocutter”—which automatically sections tissue samples and serially acquires fluorescence microscopy images of each section. Tibiae dissected from C57Bl/6 mice, either naïve or OA‐induced by surgical destabilization of the medial meniscus (DMM), were imaged using the 3D histocutter by exploiting tissue autofluorescence. Accuracy of 3D imaging was validated by ex vivo contrast‐enhanced micro‐CT and sensitivity to lesion detection compared with conventional histology. Reconstructions of tibiae obtained from 3D histocutter serial sections showed an excellent agreement with contrast‐enhanced micro‐CT reconstructions. Furthermore, osteoarthritic features, including articular cartilage loss and osteophytes, were also visualized. An in‐house developed software allowed to automatically evaluate articular cartilage morphology, eliminating the subjectivity associated to semi‐quantitative scoring and considerably increasing analysis throughput. The novelty of this methodology is, not only the increased throughput in imaging and evaluating mouse articular cartilage morphology starting from conventionally embedded samples, but also the ability to add the third dimension to conventional histomorphometry which might be useful to improve disease assessment in the model.     

Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity

Observation of highly dynamic processes inside living cells at the single molecule level is key for a better understanding of biological systems. However, imaging of single molecules in living cells is usually limited by the spatial and temporal resolution, photobleaching and the signal-to-background ratio. To overcome these limitations, light-sheet microscopes with thin selective plane illumination, for example, in a reflected geometry with a high numerical aperture imaging objective, have been developed. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-colour acquisition of -stacks. We demonstrate fast volume scanning by imaging a two-colour giant unilamellar vesicle (GUV) hemisphere. In addition, the high contrast enabled the imaging and tracking of single lipids in the GUV cap. The enhanced reflected scanning light-sheet microscope enables fast 3D scanning of artificial membrane systems and potentially live cells with single-molecule sensitivity and thereby could provide quantitative and molecular insight into the operation of cells.     

In vivo monitoring of intracellular chloroplast movements in intact leaves of c4 plants using two‐photon microscopy

Dynamic changes in the spatial distribution of chloroplasts are essential for optimizing photosynthetic capacity under changing light conditions. Light‐induced movement of chloroplasts has been widely investigated, but most studies were conducted on isolated tissues or protoplasts. In this study, a two‐photon microscopy (TPM) system was adapted to monitor the intracellular 3‐dimensional (3D) movements of chloroplasts in intact leaves of plants during dark to light transitions. The TPM imaging was based on autofluorescence of chlorophyll generated by a femto‐second Ti:Sapphire laser. All chloroplasts did not exhibit the same motion in response to irradiation variation. In the sub‐epidermal mesophyll cells, chloroplasts generally moved away from the surface following blue light treatment, however many chloroplasts did not show any movement. Such spatial heterogeneity in chloroplast motility underlines the importance of monitoring intracellular orientation and movement of individual chloroplasts across intact leaves. Our investigation shows that the 3D imaging of chloroplasts using TPM can help to understand the changes in local photosynthetic capacity in intact leaves under changing environmental conditions. Microsc. Res. Tech. 77:806–813, 2014. © 2014 Wiley Periodicals, Inc.     

Single-molecule near-field optical energy transfer microscopy with dielectric tips

The fluorescence lifetime and the fluorescence rate of single molecules are recorded as a function of the position of a Si₃N₄ atomic force microscopy tip with respect to the molecule. We observe a decrease of the excited state lifetime and the fluorescence rate when the tip apex is in close proximity to the molecule. These effects are attributed to the fact that the dielectric tip converts non‐propagating near‐fields to propagating fields within the dielectric tip effectively quenching the fluorescence. The spatial extension of the quenching area is of subwavelength dimensions. The results are discussed in terms of molecular fluorescence in a system of stratified media. The experiment provides surprising new insights into the interactions between a fluorescent molecule and a dielectric tip. The methodology holds promise for applications in ultra high‐resolution near‐field optical imaging at the level of single fluorophores.     

CATIA斜齿轮全参数化曲面法三维数字建模及精度研究

依据斜齿轮机械原理基本理论,运用CATIAV5实体和高级曲面复合建模(Hybrid modeling)先进技术,提出了一种斜齿轮全参数化曲面法三维数字建模方法,构建了三维斜齿轮理论原型的参数化数字模型,并阐述了该数模的定量几何精度检验方法.为齿轮传动系统的快速三维CAD建模、运动学和动力学分析、强度有限元分析,提供了高精度的斜齿轮全参数化数字模板.     

A light field measurement system through PSF estimation by a morphology-based method

Light field imaging technology can obtain three-dimensional (3D) information of a test surface in a single exposure. Traditional light field reconstruction algorithms not only take a long time to trace back to the original image, but also require the exact parameters of the light field system, such as the position and posture of a microlens array (MLA), which will cause errors in the reconstructed image if these parameters cannot be precisely obtained. This paper proposes a reconstruction algorithm for light field imaging based on the point spread function (PSF), which does not require prior knowledge of the system. The accurate PSF derivation process of a light field system is presented, and modeling and simulation were conducted to obtain the relationship between the spatial distribution characteristics and the PSF of the light field system. A morphology-based method is proposed to analyze the overlapping area of the subimages of light field images to identify the accurate spatial location of the MLA used in the system, which is thereafter used to accurately refocus light field imaging. A light field system is built to verify the algorithm's effectiveness. Experimental results show that the measurement accuracy is increased over 41.0％compared with the traditional method by measuring a step standard. The accuracy of parameters is also improved through a microstructure measurement with a peak-to-valley value of 25.4％and root mean square value of 23.5％improvement. This further validates that the algorithm can effectively improve the refocusing efficiency and the accuracy of the light field imaging results with the superiority of refocusing light field imaging without prior knowledge of the system. The proposed method provides a new solution for fast and accurate 3D measurement based on a light field.