TLM-Converter: reorganization of long time-lapse microscopy datasets for downstream image analysis

Automated microscopy enables in vivo studies in developmental biology over long periods of time. Time-lapse recordings in three or more dimensions to study the dynamics of developmental processes can produce huge data sets that extend into the terabyte range. However, depending on the available computational resources and software design, downstream processing of very large image data sets can become highly inefficient, if not impossible. To address the lack of available open source and commercial software tools to efficiently reorganize time-lapse data on a desktop computer with limited system resources, we developed TLM-Converter. The software either fragments oversized files or concatenates multiple files representing single time frames and saves the output files in open standard formats. Our application is undemanding on system resources as it does not require the whole data set to be loaded into the system memory. We tested our tool on time-lapse data sets of live Drosophila specimens recorded by laser scanning confocal microscopy. Image data reorganization dramatically enhances the productivity of time-lapse data processing and allows the use of downstream image analysis software that is unable to handle large data sets of ≥2 GB. In addition, saving the outputs in open standard image file formats enables data sharing between independently developed software tools.     

Hardware acceleration of processing of mass spectrometric data for proteomics

MOTIVATION: High-resolution mass spectrometers generate large data files that are complex, noisy and require extensive processing to extract the optimal data from raw spectra. This processing is readily achieved in software and is often embedded in manufacturers' instrument control and data processing environments. However, the speed of this data processing is such that it is usually performed off-line, post data acquisition. We have been exploring strategies that would allow real-time advanced processing of mass spectrometric data, making use of the reconfigurable computing paradigm, which exploits the flexibility and versatility of Field Programmable Gate Arrays (FPGAs). This approach has emerged as a powerful solution for speeding up time-critical algorithms. We describe here a reconfigurable computing solution for processing raw mass spectrometric data generated by MALDI-ToF instruments. The hardware-implemented algorithms for de-noising, baseline correction, peak identification and deisotoping, running on a Xilinx Virtex 2 FPGA at 180 MHz, generate a mass fingerprint over 100 times faster than an equivalent algorithm written in C, running on a Dual 3 GHz Xeon workstation.     

imzML--a common data format for the flexible exchange and processing of mass spectrometry imaging data

The application of mass spectrometry imaging (MS imaging) is rapidly growing with a constantly increasing number of different instrumental systems and software tools. The data format imzML was developed to allow the flexible and efficient exchange of MS imaging data between different instruments and data analysis software. imzML data is divided in two files which are linked by a universally unique identifier (UUID). Experimental details are stored in an XML file which is based on the HUPO-PSI format mzML. Information is provided in the form of a 'controlled vocabulary' (CV) in order to unequivocally describe the parameters and to avoid redundancy in nomenclature. Mass spectral data are stored in a binary file in order to allow efficient storage. imzML is supported by a growing number of software tools. Users will be no longer limited to proprietary software, but are able to use the processing software best suited for a specific question or application. MS imaging data from different instruments can be converted to imzML and displayed with identical parameters in one software package for easier comparison. All technical details necessary to implement imzML and additional background information is available at www.imzml.org.     

A software program for more reliable precursor ion assignation from LC-MS analysis using LTQ ultra zoom scan

We developed a software program (titled Precursor Ion Calibration software for LTQ or, in short, PICsL) that increases the reliability of precursor ion assignations from LC-MS analysis using ultra zoom scanning of LTQ linear ion trap MS and automatically corrects the assignations. Although existing software calculates the theoretical isotopic distribution according to m/z with a computational algorithm, our method simply searches for ions close to the theoretical mass value using both MS/MS raw data and Mascot search result files, followed by a second database search that identifies the proteins using the regenerated peak list files. Our software program mimics the manual inspection of the spectral data of precursor ions and is expected to be applicable not only for low resolution MS, such as LTQ, but also for a wide variety of MS instruments.     

System Matrix Analysis for Computed Tomography Imaging

In practical applications of computed tomography imaging (CT), it is often the case that the set of projection data is incomplete owing to the physical conditions of the data acquisition process. On the other hand, the high radiation dose imposed on patients is also undesired. These issues demand that high quality CT images can be reconstructed from limited projection data. For this reason, iterative methods of image reconstruction have become a topic of increased research interest. Several algorithms have been proposed for few-view CT. We consider that the accurate solution of the reconstruction problem also depends on the system matrix that simulates the scanning process. In this work, we analyze the application of the Siddon method to generate elements of the matrix and we present results based on real projection data.     

微波热声成像技术用于人体甲状腺检测

本文首次通过人体实验验证了微波热声成像技术用于人体甲状腺检测的可行性.文章首先讨论了该技术用于人体甲状腺检测的可行性;然后对3名志愿者的健康甲状腺进行了微波热声成像实验.结果表明:微波热声成像能够对人体健康甲状腺进行清晰成像,能够真实反映皮肤、甲状腺和气管等不同组织的结构特征;并且一次完整的检测过程时间约5 s,系统操作简单成像快速.综上所述,微波热声成像技术有望为甲状腺疾病的基础研究和临床诊断提供一种新的影像学参考.     

Interpolated Compressed Sensing for 2D Multiple Slice Fast MR Imaging

Sparse MRI has been introduced to reduce the acquisition time and raw data size by undersampling the k-space data. However, the image quality, particularly the contrast to noise ratio (CNR), decreases with the undersampling rate. In this work, we proposed an interpolated Compressed Sensing (iCS) method to further enhance the imaging speed or reduce data size without significant sacrifice of image quality and CNR for multi-slice two-dimensional sparse MR imaging in humans. This method utilizes the k-space data of the neighboring slice in the multi-slice acquisition. The missing k-space data of a highly undersampled slice are estimated by using the raw data of its neighboring slice multiplied by a weighting function generated from low resolution full k-space reference images. In-vivo MR imaging in human feet has been used to investigate the feasibility and the performance of the proposed iCS method. The results show that by using the proposed iCS reconstruction method, the average image error can be reduced and the average CNR can be improved, compared with the conventional sparse MRI reconstruction at the same undersampling rate.     

Accelerated Optical Projection Tomography Applied to In Vivo Imaging of Zebrafish

Optical projection tomography (OPT) provides a non-invasive 3-D imaging modality that can be applied to longitudinal studies of live disease models, including in zebrafish. Current limitations include the requirement of a minimum number of angular projections for reconstruction of reasonable OPT images using filtered back projection (FBP), which is typically several hundred, leading to acquisition times of several minutes. It is highly desirable to decrease the number of required angular projections to decrease both the total acquisition time and the light dose to the sample. This is particularly important to enable longitudinal studies, which involve measurements of the same fish at different time points. In this work, we demonstrate that the use of an iterative algorithm to reconstruct sparsely sampled OPT data sets can provide useful 3-D images with 50 or fewer projections, thereby significantly decreasing the minimum acquisition time and light dose while maintaining image quality. A transgenic zebrafish embryo with fluorescent labelling of the vasculature was imaged to acquire densely sampled (800 projections) and under-sampled data sets of transmitted and fluorescence projection images. The under-sampled OPT data sets were reconstructed using an iterative total variation-based image reconstruction algorithm and compared against FBP reconstructions of the densely sampled data sets. To illustrate the potential for quantitative analysis following rapid OPT data acquisition, a Hessian-based method was applied to automatically segment the reconstructed images to select the vasculature network. Results showed that 3-D images of the zebrafish embryo and its vasculature of sufficient visual quality for quantitative analysis can be reconstructed using the iterative algorithm from only 32 projections—achieving up to 28 times improvement in imaging speed and leading to total acquisition times of a few seconds.     

IMIRS: a high-resolution 3D reconstruction package integrated with a relational image database

Recent advances in electron cryomicroscopy instrumentation and single particle reconstruction have created opportunities for high-throughput and high-resolution three-dimensional (3D) structure determination of macromolecular complexes. However, it has become impractical and inefficient to rely on conventional text file data management and command-line programs to organize and process the increasing numbers of image data required in high-resolution studies. Here, we present a distributed relational database for managing complex datasets and its integration into our high-resolution software package IMIRS (Image Management and Icosahedral Reconstruction System). IMIRS consists of a complete set of modular programs for icosahedral reconstruction organized under a graphical user interface and provides options for user-friendly, step-by-step data processing as well as automatic reconstruction. We show that the integration of data management with processing in IMIRS automates the tedious tasks of data management, enables data coherence, and facilitates information sharing in a distributed computer and user environment without significantly increasing the time of program execution. We demonstrate the applicability of IMIRS in icosahedral reconstruction toward high resolution by using it to obtain an 8-A 3D structure of an intermediate-sized dsRNA virus.     

A general-purpose system for the rapid acquisition and computer processing of optical and scanning electron microscope images—II. Software

The design and implementation of software for the operation of a general-purpose optical and electron microscope image processing system is described. The software is a group of programs, controlled by a command-line interpreter called IPR (Image PRocessing). The interpreter may be used interactively, or groups of commands may be issued indirectly after placing them in files. Programs for specialized image processing applications may obtain the services of the system's memory-resident programs, through the same interprogram communication methods that are used by the command-line interpreter.     

Exploring skyline for both MSE‐based label‐free proteomics and HRMS quantitation of small molecules

The MS^E (where MS^E is low energy (MS) and elevated energy (E) mode of acquisition) acquisition method commercialized by Waters on its Q‐TOF instruments is regarded as a unique data‐independent fragmentation approach that improves the accuracy and dynamic range of label‐free proteomic quantitation. Due to its special format, MS^E acquisition files cannot be independently analyzed with most widely used open‐source proteomic software specialized for processing data‐dependent acquisition files. In this study, we established a workflow integrating Skyline, a popular and versatile peptide‐centric quantitation program, and a statistical tool DiffProt to fulfill MS^E‐based proteomic quantitation. Comparison with the vendor software package for analyzing targeted phosphopeptides and global proteomic datasets reveals distinct advantages of Skyline in MS^E data mining, including sensitive peak detection, flexible peptide filtering, and transparent step‐by‐step workflow. Moreover, we developed a new procedure such that Skyline MS1 filtering was extended to small molecule quantitation for the first time. This new utility of Skyline was examined in a protein–ligand interaction experiment to identify multiple chemical compounds specifically bound to NDM‐1 (where NDM is New Delhi metallo‐β‐lactamase 1), an antibiotics‐resistance target. Further improvement of the current weaknesses in Skyline MS1 filtering is expected to enhance the reliability of this powerful program in full scan‐based quantitation of both peptides and small molecules.     

2dx_merge: data management and merging for 2D crystal images

Electron crystallography of membrane proteins determines the structure of membrane-reconstituted and two-dimensionally (2D) crystallized membrane proteins by low-dose imaging with the transmission electron microscope, and computer image processing. We have previously presented the software system 2dx, for user-friendly image processing of 2D crystal images. Its central component 2dx_image is based on the MRC program suite, and allows the optionally fully automatic processing of one 2D crystal image. We present here the program 2dx_merge, which assists the user in the management of a 2D crystal image processing project, and facilitates the merging of the data from multiple images. The merged dataset can be used as a reference to re-process all images, which usually improves the resolution of the final reconstruction. Image processing and merging can be applied iteratively, until convergence is reached. 2dx is available under the GNU General Public License at http://2dx.org.     

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples² (Fig.1). A 12 μm thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity². Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson''s disease (PD). PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age^3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA⁵. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)^1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA⁷. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control^7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson''s disease and L-DOPA induced dyskinesia. MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin''s opioid-receptor binding capacity⁹. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.     

Revisiting rat spermatogenesis with MALDI imaging at 20-microm resolution

Matrix-assisted laser desorption/ionization (MALDI) molecular imaging technology attracts increasing attention in the field of biomarker discovery. The unambiguous correlation between histopathology and MALDI images is a key feature for success. MALDI imaging mass spectrometry (MS) at high definition thus calls for technological developments that were established by a number of small steps. These included tissue and matrix preparation steps, dedicated lasers for MALDI imaging, an increase of the robustness against cell debris and matrix sublimation, software for precision matching of molecular and microscopic images, and the analysis of MALDI imaging data using multivariate statistical methods. The goal of these developments is to approach single cell resolution with imaging MS. Currently, a performance level of 20-μm image resolution was achieved with an unmodified and commercially available instrument for proteins detected in the 2-16-kDa range. The rat testis was used as a relevant model for validating and optimizing our technological developments. Indeed, testicular anatomy is among the most complex found in mammalian bodies. In the present study, we were able to visualize, at 20-μm image resolution level, different stages of germ cell development in testicular seminiferous tubules; to provide a molecular correlate for its well established stage-specific classification; and to identify proteins of interest using a top-down approach and superimpose molecular and immunohistochemistry images.     

Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy

In this protocol, we describe a 3D imaging technique known as 'volume electron microscopy' or 'focused ion beam scanning electron microscopy (FIB/SEM)' applied to biological tissues. A scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and a backscattered electron (BSE) detector, used to image the milled surfaces, generates a large series of images that can be combined into a 3D rendered image of stained and embedded biological tissue. Structural information over volumes of tens of thousands of cubic micrometers is possible, revealing complex microanatomy with subcellular resolution. Methods are presented for tissue processing, for the enhancement of contrast with osmium tetroxide/potassium ferricyanide, for BSE imaging, for the preparation and platinum deposition over a selected site in the embedded tissue block, and for sequential data collection with ion beam milling; all this takes approximately 90 h. The imaging conditions, procedures for alternate milling and data acquisition and techniques for processing and partitioning the 3D data set are also described; these processes take approxiamtely 30 h. The protocol is illustrated by application to developing chick cornea, in which cells organize collagen fibril bundles into complex, multilamellar structures essential for transparency in the mature connective tissue matrix. The techniques described could have wide application in a range of fields, including pathology, developmental biology, microstructural anatomy and regenerative medicine.     

Teaching computer interfacing with virtual instruments in an object-oriented language.

LabVIEW is a graphic object-oriented computer language developed to facilitate hardware/software communication. LabVIEW is a complete computer language that can be used like Basic, FORTRAN, or C. In LabVIEW one creates virtual instruments that aesthetically look like real instruments but are controlled by sophisticated computer programs. There are several levels of data acquisition VIs that make it easy to control data flow, and many signal processing and analysis algorithms come with the software as premade VIs. In the classroom, the similarity between virtual and real instruments helps students understand how information is passed between the computer and attached instruments. The software may be used in the absence of hardware so that students can work at home as well as in the classroom. This article demonstrates how LabVIEW can be used to control data flow between computers and instruments, points out important features for signal processing and analysis, and shows how virtual instruments may be used in place of physical instrumentation. Applications of LabVIEW to the teaching laboratory are also discussed, and a plausible course outline is given.     

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.     

Prospective image planning in radiation therapy for optimization of image quality and reduction of patient dose

IntroductionCT simulation data in image-guided radiation therapy (IGRT) provides patient-specific subject contrast. This information can be exploited to establish, a priori, a suitable imaging goal and to select patient-specific imaging acquisition parameters that optimize the similarity between reference and daily set-up images and reduce imaging dose. This study aims to describe and clinically validate a computerized algorithm designed to provide such optimization.Material and methodsAn image planning system (IPS) was developed to assist in planar kV imaging technique selection for radiation therapy. The system's patient-specific image quality and dose reduction capabilities were validated herein. Anthropomorphic phantom and clinical data were acquired. Mutual information (MI) was used to compare simulated and measured images in both phantom and clinical tests. Variations in contrast resolution resulting from imaging panel underexposure, saturation and a contrast plateau were investigated. For evaluation of patient-specific imaging dose reduction, the IPS was used to modify acquisition settings for six patients.ResultsPhantom data confirmed the IPS's predictive capability regarding image contrast. Measured and simulated images showed similar progressions from under-exposure, image quality peak, and loss of contrast due to detector saturation. Clinical data demonstrated that contrast resolution and imaging dose could be prospectively improved without loss of image contrast. The algorithm reduced imaging dose by an average of 47%, and a maximum of 80%.ConclusionsLoss of image contrast resulting from under-exposure or over-exposure, as well as a contrast plateau can be predicted by use of a prospective image planning algorithm. Image acquisition parameters can be predicted that reduce patient dose without loss of useful contrast.     

高光谱成像技术对人体面部和手掌的成像及光谱分析

目的：应用高光谱成像技术对正常人体不同部位图像进行采集,分析这些部位的光谱特征,得到正常值数据,并为该技术用于疾病诊断和中医面诊和手诊奠定基础。方法：使用高光谱成像仪,采集10例（男5女5）健康人的面部和双手掌图像,应用高光谱处理分析软件,对面和手掌划分15个分析区域,统计各分区从450-900 nm每间隔10 nm一个光谱段的各个分析区光强度值和光谱特征,并分析个体特点。结果：在面和手掌的高光谱图像上可以清晰地显示面部器官和手掌的不同部位,以580-830 nm段图像更为清晰,530 nm以下图像杂波较多。面部颧、颧下、鼻尖、眉间、额等部位和四指指丘、大小鱼际部位的反光较强,而眼、眉、嘴角和五指末端等则较弱。面部光谱双侧基本对称,而左右手的对称性在不同的个体上不尽相同。在光谱曲线上可以见到某一或者某些波长处光谱出现突变的细节。结论：高光谱成像技术可以清晰地显示人体面和手的图像,显示各部位（器官）的光谱特征;本文得到的面、手高光谱正常数据和特征将为该技术用于疾病诊断和中医辨证提供参考依据。