1。仪器设置<ol><li>打开分光光度计，让温暖1小时。 </li><li>使用Excel中创建一个文件的标准和样本数据，如图1所示，它保存在逗号分隔值“CSV”格式。 <img src="/files/ftp_upload/2465/2465fig1.jpg" alt="图1" /> 图1。标准品和样品数据。 </li><li>设置荧光如下： <ol><li>点击<img src="/files/ftp_upload/2465/2465icon1.jpg" alt="图标1" />按钮，这将打开窗口，如图2所示的分析方法。 <img src="/files/ftp_upload/2465/2465fig2.jpg" alt="图2" /> 图2。分析法窗口，“常规”选项卡。 进行以下选择： <ul><li>在“测量”字段，从下拉菜单选择的光度测定。 </li><li>在“操作”栏，输入相应的操作员的名字。 </li><li>有没有必要的“工具”栏位输入信息。这是由软件自动选择。 </li><li> “采样”领域处于非活动状态时使用酶标仪。 </li><li>输入您可能希望包含在报告中的“备注”字段的任何意见。 </li><li>在“附件”字段中，输入用于此测试的配件的相关信息。 </li><li>请注意在右侧窗口中的按钮。 </li><li> “Load”按钮允许加载以前保存的分析方法。 </li><li> “保存”按钮允许更新测试参数的设置或以前保存和加载的分析方法，使用相同的名称。 </li><li> “另存为”按钮允许保存一套合适的名称下的新的测试参数或分析方法。 </li></ul> 现在点击“定量分析”选项卡上，见图3，并做出以下选择。 <img src="/files/ftp_upload/2465/2465fig3.jpg" alt="图3" /> 图3。窗口分析方法，定量分析“选项卡。 <ul><li>在“定量型”字段，选择波长。这表明将使用选定的波长的荧光读数。 </li><li>在“校准类型”字段，选择第一顺序。 </li><li>在“波长数”字段中，选择1。 </li><li>在“浓度单位”字段中，输入纳克/毫升。 </li><li>保留未选中“手动校准”和“力曲线通过零点”框。 </li><li>在“小数点后的数字”的领域，选择2。 </li><li>在“低浓度限制”字段中，输入0。 </li><li>在“上浓度限制字段中，输入10000。 </li></ul> 现在点击“工具”选项卡，并做出以下选择 。 <img src="/files/ftp_upload/2465/2465fig4.jpg" alt="图4" /> 图4。分析方法“窗口，仪器标签 <ul><li>在“数据模式：”字段中，选择荧光。 </li><li> “斩速度”领域处于非活动状态，在这个时候，它是仅用于磷光测量。 </li><li>在“波长模式”的领域，选择两者WL固定。 </li><li>在“EX / WL1”字段输入480nm。 </li><li>在“EM / WL1”字段输入520nm。 </li><li>在这个时候处于非活动状态下的所有其他领域的EX和EM。 </li><li>在“前缝”的领域，从下拉菜单选择5.0nm。 </li><li>在“新兴市场缝隙”字段从下拉菜单选择5.0nm。 </li><li>保留未选中的复选框前面的“PMT电压1 - 1000V”字段。 </li><li>在“PMT电压”字段，从下拉菜单选择700V。 </li><li>在“自动统计钙的数量”字段中选择2。 </li><li>在“复制”字段中，选择1。 </li><li>在“集成时间”字段选择0.1秒。/ LI&gt; <li>在“延迟”字段输入或选择“0”。 </li></ul> 点击“监视器”标签。 <img src="/files/ftp_upload/2465/2465fig5.jpg" alt="图5" /> 图5。分析方法“窗口中，监视器标签。 <ul><li>在“Y轴”，“最大：”字段中，选择10000 </li><li>在“Y轴”，“闵：”字段中，选择0。 </li><li>选择框左侧的“打开数据采集处理后的数据”。 </li><li>保留未选中框左侧的“数据采集后打印报告”，除非你想有一个读数完成后自动打印报告。 </li></ul> 点击“报告”标签，见图6。 <img src="/files/ftp_upload/2465/2465fig6.jpg" alt="图6" /> 图6。分析方法“窗口中，报告选项卡。 <ul><li>在“输出”栏，从下拉菜单选择“打印报告。您也可以选择“使用Microsoft Excel”如果你想导出到Excel的数据，或“使用打印生成表”，如果使用可选的附加方案的报告发生器，这将允许生成自定义报告。 </li><li>点击您想在报告中包括的项目框。 </li><li>为了节省不同的选项卡的“分析方法”窗口中的所有选定的参数，再次单击“常规”标签，并保存在一个文件名，和他们在一个位置，在那里你可以找到以供将来使用，并点击“确定“按钮关闭此窗口。 </li><li>这样就完成了设置仪器测试参数。 </li><li>现在，我们将成立酶标仪。 </li><li>点击<img src="/files/ftp_upload/2465/2465icon2.jpg" alt="图标2" />按钮，这将带来顶部的MPR设置窗口。 </li></ul> 选择MPR设置窗口/板标签的标准和样品的井，如图7所示 。 <img src="/files/ftp_upload/2465/2465fig7.jpg" alt="图7" /> 图7。 MPR设置窗口中，选项卡板。 <ul><li> A1位置上点击并拖动鼠标至E1，然后在<img src="/files/ftp_upload/2465/2465icon3.jpg" alt="图标3" />按钮。这将选择要使用标准的立场。这些职位将突出绿色。 </li><li>现在，我们需要选择F1至H3样品的位置。单击并拖动鼠标的位置F1出发，H3的位置，然后点击<img src="/files/ftp_upload/2465/2465icon4.jpg" alt="图示4" />按钮，这将突出显示在黄色这些职位。 </li><li>然后重复相同的操作位置A2至E3，选择样品测量这些位置。 </li><li>现在，让我们的负载文件以逗号分隔变量（CSV）文件的标准和样品的信息提前做好准备。 </li></ul> 点击“好信息”选项卡“MPR设置窗口”如图8所示，。 <img src="/files/ftp_upload/2465/2465fig8.jpg" alt="图8" /> 图8。 MPR设置“窗口，那么信息”选项卡。 <ul><li>然后点击<img src="/files/ftp_upload/2465/2465icon5.jpg" alt="图标5" />按钮。 Windows资源管理器窗口将打开，在图9所示，这将使定位的“好information.csv”文件，并加载它。 <img src="/files/ftp_upload/2465/2465fig9.jpg" alt="图9" /> 图9。载入以及样品信息。 装载的“好消息”文件后，“好信息”将如下图10所示 <img src="/files/ftp_upload/2465/2465fig10.jpg" alt="图10" /> 图10。装样品以及信息。 </li></ul></li></ol></li></ol> 2。 Picogreen检测样品制备<ol><li>准备加入1毫升20 × TE缓冲液1X TE缓冲液（10毫米的Tris - HCl，1 mM EDTA的）至19毫升DI H 2 O。 </li><li> PicoGreen试剂溶液9.95毫升1 × TE缓冲液加入50μL的200 × PicoGreen试剂准备。 </li><li>准备通过股票双链DNA（100微克/毫升）的系列稀释的标准（LAMBDA双链）。首先准备50μg/ mL的双链DNA溶液，加入25μL股票双链DNA（100微克/毫升）和25μL1 × TE缓冲液。然后准备加入表示卷的dsDNA和缓冲下表列出的标准的解决方案： <table border="1"><tbody><tr><td>集中的dsDNA </td><td>音量的dsDNA </td><td>卷缓冲区</td></tr><tr><td> 1μg/ mL的</td><td> 20μL50μg/ mL的双链DNA </td><td> 980μL1 × TE缓冲液</td></tr><tr><td> 0.1微克/毫升</td><td> 100μL，1μg/ mL的双链DNA </td><td> 900μL1 × TE缓冲液</td></tr><tr><td> 0.01微克/毫升</td><td> 100μL，0.1μg/ mL的双链DNA </td><td> 900μL1 × TE缓冲液</td></tr><tr><td> 0.001微克/毫升</td><td> 100μL，0.01μg/ mL的双链DNA </td><td> 900μL1 × TE缓冲液</td></tr></tbody></table></li></ol> 3。测量标准和样品的荧光<ol><li>移液器A1 - E1的成井150μL每个标准96孔板，每图7，并根据下表： <table border="1"><tbody><tr><td>以及</td><td>标准</td></tr><tr><td> A1 </td><td> 1μg/ mL的</td></tr><tr><td> B1 </td><td> 0.1微克/毫升</td></tr><tr><td> C1 </td><td> 0.01微克/毫升</td></tr><tr><td>首长级薪级第1点</td><td> 0.001微克/毫升</td></tr><tr><td> E1 </td><td> 1 × TE缓冲液</td></tr></tbody></table></li><li>移取150μL的未知样品的成井F1 H3，如图7所示</li><li>倒入PicoGreen的解决方案，在步骤1.2制备成设计用于多通道移液器使用低谷。使用多道移液器提供150μLPicoGreen解决方案A1 - H3井。混合解决方案和标准与吸管轻轻地。 </li><li>将板在暗区，并等待2-5分钟，以发展为反应。 </li></ol> 4。代表性的成果一个良好的校准曲线是使用不同的参数测量软件校准曲线的标准和计算后的F - 7000软件提供的评估。 决定系数表示的衡量标准的适合度和计算的校准曲线。越接近这个值是“1”，更好的测量值和校准曲线拟合。 如果该值是从“1”，标准的，必须重新测量，再准备，或者改变校准曲线的健身。 图11显示了一个校准曲线计算出的决心系数= 0.99993获得的dsDNA使用PicoGreen染料定量，的例子。 <img src="/files/ftp_upload/2465/2465fig11.jpg" alt="图11" /> 图11。双链校准曲线的例子 。

<ol>
	<li>Singer, V. L., Jones, L. J., Yue, S. T., Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+PicoGreen+reagent+and+development+of+a+fluorescence-based+solution+assay+for+double-stranded+DNA+quantitation.">Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation.</a> Analytical Biochemistry. 249 (2), 228-238 (1997).</li></ol>

路易斯A.莫雷诺和肯德拉L.考克斯是受雇于日立高新技术美国，生产在这篇文章中使用的仪器。

仔细地吹打在样品制备，以及使用一个稳定的和敏感的荧光分光光度计是为取得良好的和可重复性的结果至关重要。 在本次测试使用的荧光分光光度计的情况下，建议使用300μL，最后的样本量在酶标仪。降低音量降低的灵敏度，量也较大，可能会导致油井之间的交叉污染。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Distilled H2O</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Quant-iT Picogreen dsDNA Assay Kit from Invitrogen</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>P7589</td>
<td>&nbsp;</td>
<tr>
<td>Nalge Nunc Fluorescence Microplates p/n 237108</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>237108</td>
<td>&nbsp;</td>
<tr>
<td>12 Channel Eppendorf Micropipette</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>3516677</td>
<td>&nbsp;</td>
<tr>
<td>1000 &mu;L Eppendorf Pipette</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>200 &mu;L Eppendorf Pipette </td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 mL serological pipet</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Aluminum foil</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipette tips</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hitachi F-7000 Fluorescence Spectrophotometer with FL Solutions 2.1</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>5J1-0003</td>
<td>&nbsp;</td>
<tr>
<td>Microplate Reader Accessory for F-7000</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>5J0-0139</td>
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quantification of dsdna using the hitachi f-7000 fluorescence spectrophotometer and picogreen dye

定量，尤其是在低浓度的DNA，是一种与生物应用范围广泛，包括DNA的合成和纯化，如DNA扩增产物的定量诊断应用，DNA分子药物检测的标准，如分子生物学实验的重要任务准备工作。在这段视频中，我们将展示日立F - 7000型荧光分光光度计的功能配备执行双链DNA定量使用QUANT PicoGreen染料试剂盒分子探针与微酶标仪配件。 F - 7000型荧光分光光度计，提供高灵敏度和高速测量。它是一个高度灵活的系统，能够测量荧光，发光和磷光。几种测量模式可供选择，包括波长扫描，扫描时间，测光和3 - D扫描测量。使用300μL的样品量微孔板分光光度计灵敏度范围在50荧光picomoles时，是能够测量为60000海里/分钟的扫描速度。它也有很宽的动态范围高达5个数量级，校准曲线的浓度范围内使用，允许订单的。光学系统采用反射光学最大的能源和灵敏度。标准的波长范围是200至750纳米，可以延长至900纳米，当使用可选的近红外光电倍增管。该系统可以从5至60度摄氏使用一个可选的外部温度控制液体循环酶标仪可选的温度控制。酶标仪允许使用96孔板，96口井的测量速度小于60秒时，使用的动力学模式。 为F - 7000和酶标仪的软件控制也非常灵活。样品可以在任一列或行格式设置，选择样品测量和水井的任意组合可。这使得微孔板的最佳利用。此外，该软件允许进口微板样品配置，在Excel中创建和保存在逗号分隔的值，或“CSV”格式。微孔板测量配置可以保存和回忆说，为方便起见，软件和提高生产力。数据结果可以输出到一个标准的报告，Excel，或一个可选的报表生成器程序。

Watch this Scientific Journal Video about Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye at JoVE.com

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

示范的dsDNA定量使用分子探针PicoGreen染料和日立F - 7000型荧光分光光度计，配备酶标仪配件。

双链DNA定量，使用日立F - 7000荧光分光光度计和PicoGreen染料

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

quantification-dsdna-using-hitachi-f-7000-fluorescence

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24.4K 次观看.  Hitachi High Technologies America. 示范的dsDNA定量使用分子探针PicoGreen染料和日立F - 7000型荧光分光光度计，配备酶标仪配件。

视频: Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

微量法测定蛋白浓度使用NanoDrop 2000C分光光度计

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

科研

生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

使用微量的生物规格的纳米分光光度计核酸和蛋白的浓度测定

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

粉末样品的绝对量子产量测量

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

可视化线虫角质层结构的亲脂性的重要染料，直接投资收益

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

观察与端粒的定量和重复序列用荧光<em&gt;原位</em&gt;杂交（FISH）与PNA探针<em&gt;秀丽隐杆线虫</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

使用[18F]FDG微PET / MR成像对小鼠中的棕色和米色脂肪组织进行可视化和定量

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

使用单细胞荧光成像对菌际竞争进行量化

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...