这项研究是由美国国家科学基金会（DBI 0227541）的支持。

<ol>
	<li>Brewer, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+controlled+vocabulary+and+software+application+to+analyze+fruit+shape+variation+in+tomato+and+other+plant+species.">Development of a controlled vocabulary and software application to analyze fruit shape variation in tomato and other plant species.</a> Plant Physiol. 141 (1), 15-25 (2006).</li><li>Gonzalo, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+Fruit+Shape+Analysis+Using+Morphometric+and+Morphology+Attributes+Implemented+in+Tomato+Analyzer+Software+Program.">Tomato Fruit Shape Analysis Using Morphometric and Morphology Attributes Implemented in Tomato Analyzer Software Program.</a> Journal of the American Society for Horticultural Science. 134 (1), 77-87 (2009).</li><li>Darrigues, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+analyzer-color+test:+A+new+tool+for+efficient+digital+phenotyping.">Tomato analyzer-color test: A new tool for efficient digital phenotyping.</a> Journal of the American Society for Horticultural Science. 133 (4), 579-586 (2008).</li><li>Brewer, M. T., Moyseenko, J. B., Monforte, A. J., van der Knaap, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+variation+in+tomato:+a+comprehensive+study+of+quantitative+trait+loci+controlling+fruit+shape+and+development.">Morphological variation in tomato: a comprehensive study of quantitative trait loci controlling fruit shape and development.</a> J Exp Bot. 58 (6), 1339-1349 (2007).</li><li>Gonzalo, M. J., Knaap, E. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+analysis+into+the+genetic+bases+of+morphology+in+tomato+varieties+exhibiting+elongated+fruit+shape.">A comparative analysis into the genetic bases of morphology in tomato varieties exhibiting elongated fruit shape.</a> Theor Appl Genet. 116 (5), 647-656 (2008).</li><li>Rodr&#237;guez, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+SUN,+OVATE+and+FAS+in+the+tomato+germplasm+and+their+effect+on+fruit+morphology+(in+preparation.">Diversity of SUN, OVATE and FAS in the tomato germplasm and their effect on fruit morphology (in preparation.</a> , .</li><li>Wu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Masters Thesis. , (2009).</li><li>Orsi, C. H., Tanksley, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+variation+in+an+ABC+transporter+gene+associated+with+seed+size+evolution+in+tomato+species.">Natural variation in an ABC transporter gene associated with seed size evolution in tomato species.</a> PLoS Genet. 5 (1), e1000347-e1000347 (2009).</li><li>Yue, B., Cai, X., Yuan, W., Vick, B., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+quantitative+trait+loci+(QTL)+controlling+seed+morphology+and+disk+diameter+in+sunflower+(Helianthus+annuus+L).">Mapping quantitative trait loci (QTL) controlling seed morphology and disk diameter in sunflower (Helianthus annuus L).</a> Helia. 32, 17-35 (2009).</li><li>Zeng, Z. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+Basis+for+Separation+of+Multiple+Linked+Gene+Effects+in+Mapping+Quantitative+Trait+Loci.">Theoretical Basis for Separation of Multiple Linked Gene Effects in Mapping Quantitative Trait Loci.</a> Proceedings of the National Academy of Sciences of the United States of America. 90 (23), 10972-10976 (1993).</li><li>Zeng, Z. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+Mapping+of+Quantitative+Trait+Loci.">Precision Mapping of Quantitative Trait Loci.</a> Genetics. 136 (4), 1457-1468 (1994).</li><li>Jiang, C. J., Zeng, Z. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple-Trait+Analysis+of+Genetic-Mapping+for+Quantitative+Trait+Loci.">Multiple-Trait Analysis of Genetic-Mapping for Quantitative Trait Loci.</a> Genetics. 140 (3), 1111-1127 (1995).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> IPGRI Descriptors for Tomato (Lycopersicon spp.). , 47-47 (1996).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> UPOV Guidelines for the Conduct of Tests for Distinctness, Uniformity, and Stability. TOMATO (Lycopersicon Lycopersicum (L.) Karsten Ex Farw.). , 51-51 (2001).</li><li>Depypere, L., Chaerle, P., Mijnsbrugge, K. V., Goetghebeur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stony+endocarp+dimension+and+shape+variation+in+Prunus+section+Prunus.">Stony endocarp dimension and shape variation in Prunus section Prunus.</a> Annals of Botany. 100 (7), 1585-1597 (2007).</li><li>Depypere, L., Chaerle, P., Breyne, P., Mijnsbrugge, K. V., Goetghebeur, P., P, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combined+morphometric+and+AFLP+based+diversity+study+challenges+the+taxonomy+of+the+European+members+of+the+complex+Prunus+L.+section+Prunus.+.">A combined morphometric and AFLP based diversity study challenges the taxonomy of the European members of the complex Prunus L. section Prunus. .</a> Plant systematics and evolution. 279 (1-4), 219-231 (2009).</li><li>Xiao, H., Jiang, N., Schaffner, E., Stockinger, E. J., van der Knaap, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+retrotransposon-mediated+gene+duplication+underlies+morphological+variation+of+tomato+fruit.">A retrotransposon-mediated gene duplication underlies morphological variation of tomato fruit.</a> Science. 319 (5869), 1527-1530 (2008).</li></ol>

植物器官的形态和色彩的高吞吐量和准确的评价是困难的，因为其量化的性质往往是主观的量化。 TA几个水果形态学和色度的特征，在高通量和半自动的方式提供客观，准确的测量。电讯局长的效用已被证实的研究，导致鉴定番茄果实形状的QTL 2,4,5 。此外，cultivars6分类和一组物种的分类进行了评估使用TA 的 15,16输出。番茄果实的形状基因孙结果拉长水果 17的高表达。使用电讯管理局局长，叶和子叶形状是确定一个高层次的7表达孙线，示范，应用程序可以有效地用来衡量其他植物器官形态以及。此外，电讯局长一直在8,9西红柿和向日葵的种子大小确定 ​​的遗传基础非常重要的。最后，色彩测试模块，电讯管理局局长实施更精确，更准确，比其他的方法来分析在番茄果实的颜色更便宜。总之，电讯局长已成为植物器官的形态和色彩变化的客观和可靠的评估的重要工具。

tomato analyzer: a useful software application to collect accurate and detailed morphological and colorimetric data from two-dimensional objects

测量这些特性的详细的表型分析在一个客观的和可重复性的方式蔬菜和水果作物的果实形态和颜色特征是很重要的。番茄分析仪（TA）是一个软件程序，措施37在半自动和可重复性的方式 1,2的二维形状的属性。许多这些属性，如缩进水果和地区的远端和近端的角度，是难以量化手动。在软件的十大类：基本测量，果形指数，块效应，同质化，近端水果尾形状，远端水果尾形状，不对称，内部偏心，纬度组和形态测量的属性是组织。最后一类既不需要事先了解，也没有预定的形状属性的概念，形态分析提供了一个不带偏见的选项可能比属性分析，以更好地适应高通量分析。电讯局长亦提供色彩测试应用程序的目的是收集从扫描图像的颜色测量和允许扫描设备进行校准，使用的颜色标准3。 TA提供了几个选项，以出口和分析形状属性，形态，颜色数据。出口数据可能会在批处理模式下的Excel文件（100多个图像在同一时间），或作为单独的图像出口。用户可以选择输出之间，平均为每幅图像（包括标准差），或输出，用于显示图像上的每个对象的属性值的对象的每个属性显示。电讯管理局局长已被鉴别和确认番茄果实形状的数量性状位点（QTL），以及关键的水果形状的基因，在植物形态的影响的深入分析执行了一个宝贵和有效的的工具。此外，电讯局长可以用来客观地分类成各种形状的类别的水果。最后，水果的形状和其他植物的叶子和种子等，以及其他植物器官的颜色性状，可以与TA进行评估。

Watch this Scientific Journal Video about Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects at JoVE.com

Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects

番茄仪（TA）在重复性好，准确地量化的二维形状和颜色的属性。一步一步的过程是一个获得高质量的数字化图像的番茄果实，形态和色彩分析这些图像，然后使用几个应用程序，通过该软件生成的数据。

番茄分析器：一个有用​​的软件应用，收集准确和详细的二维物体的形态和色度数据

Measuring fruit morphology and color traits of vegetable and fruit crops in an objective and reproducible way is important for detailed phenotypic ...

tomato-analyzer-useful-software-application-to-collect-accurate

Research

JoVE Journal

Biology

26.2K 次观看.  The Ohio State University. 番茄仪（TA）在重复性好，准确地量化的二维形状和颜色的属性。一步一步的过程是一个获得高质量的数字化图像的番茄果实，形态和色彩分析这些图像，然后使用几个应用程序，通过该软件生成的数据。

视频: Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

评估电子晶体结构生物学研究的小膜蛋白的二维结晶试验

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

科研

生物学

Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living cells. The proposed instrument extends the sensitivity of label-free optical microscopy to nanoscale changes in organelle size and shape and can be used to accelerate the study of the structure-function relationship pertaining to organelle dynamics underlying fundamental biological processes, such as programmed cell death or cellular differentiation. The microscope can be easily implemented on existing microscopy platforms, and can therefore be disseminated to individual laboratories, where scientists can implement and use the proposed methods with unrestricted access.
The proposed technique is able to characterize subcellular structure by observing the cell through two-dimensional optical Gabor filters. These filters can be tuned to sense with nanoscale (10's of nm) sensitivity, specific morphological attributes pertaining to the size and orientation of non-spherical subcellular organelles. While based on contrast generated by elastic scattering, the technique does not rely on a detailed inverse scattering model or on Mie theory to extract morphometric measurements. This technique is therefore applicable to non-spherical organelles for which a precise theoretical scatter description is not easily given, and provides distinctive morphometric parameters that can be obtained within unstained living cells to assess their function. The technique is advantageous compared with digital image processing in that it operates directly on the object's field transform rather than the discretized object's intensity. It does not rely on high image sampling rates and can therefore be used to rapidly screen morphological activity within hundreds of cells at a time, thus greatly facilitating the study of organelle structure beyond individual organelle segmentation and reconstruction by fluorescence confocal microscopy of highly magnified digital images of limited fields of view.
In this demonstration we show data from a marine diatom to illustrate the methodology. We also show preliminary data collected from living cells to give an idea of how the method may be applied in a relevant biological context.

We demonstrate a dark-field microscopy method based on Gabor-like filtering to measure subcellular dynamics within single living cells. The technique is sensitive to alterations in the structure of organelles, such as mitochondrial fragmentation.

基于二维Gabor滤波器的光散射显微镜

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living ...

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort mycelial pellets using a Complex Object Parametric Analyzer and Sorter (COPAS). Detailed instructions are given for the use of the instrument and the basic statistical analysis of the data. We furthermore describe how pellets can be sorted according to user-defined settings, which enables downstream processing such as the analysis of the RNA or protein content. Using this methodology the mechanism underlying heterogeneous growth can be tackled. This will be instrumental for improving streptomycetes as a cell factory, considering the fact that productivity correlates with pellet size.

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Liquid-grown Streptomyces cultures are characterized by mycelial pellets that are heterogeneous in size. We here describe a method to analyze and sort such pellets in a high-throughput manner. These pellets can be used for further analyses, which will provide leads to understand and control growth heterogeneity.

排序<em&gt;链霉菌</em&gt;细胞沉淀用一个复杂的对象参数分析仪和分选机

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

维甲酸的应用获取从初级小鼠成骨细胞骨细胞培养

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

肿瘤细胞球体的高通量图像分析:一个用户友好的软件应用来测量球体的大小自动准确

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

工作流的高含量，荧光标记的万能工具显微镜数据，支持开源软件的单个细胞定量

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

完整的工作流程为组蛋白翻译后修饰的分析采用自下而上质谱法:从组蛋白提取数据分析

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

使用软件和冷却装置进行鱼类精子评估

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

用化学干燥进行扫描电镜 (sem) 形态分析的原核生物和真核生物的制备

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

番茄分析器：一个有用​​的软件应用，收集准确和详细的二维物体的形态和色度数据

番茄分析器：一个有用的软件应用，收集准确和详细的二维物体的形态和色度数据