这项研究得到了从美国国立卫生研究所的资助，并在美国德州阿灵顿大学设立基金的MM。

1。植物生长和准备细菌<ol><li>要开始此过程母猪拟南芥种子1:1:1 v：V：V生长介质的混合物（REDI地球插件和幼苗组合，孙格罗），精细的蛭石，和珍珠岩。 </li><li>成长的植物在生长室（22℃，12小时100μmol/毫米2 /秒的日常光照和65 ± 5％湿度）和水需要。植物是准备在4-6周使用，当他们年轻的完全展开叶前抽薹。 </li><li>在实验开始前两天准备的细菌培养。甘油对修改后的LB培养基（10克/ L，蛋白胨，5克/ L酵母提取物，5 g / L的NaCl的pH值7.0，和1.5％琼脂）和24小时的孵育28 ° C。股票数假单胞菌丁香使用新鲜的文化准备的接种，总是从甘油股票的培养板作为分培养后变得不那么致命的细菌。 </li><li>使用细菌，盘上的增长，启动了10毫升的液体文化体育丁香在50 mL锥形瓶中。培养过夜孵育28 ° C剧烈摇晃，直到达到0.8和1之间的600 nm的光密度或外径。 </li><li>测量外径在600和1360 XG为10分钟，离心收获细胞。重悬的细胞，因此，最后外径为0.2在蒸馏水。此外径10 8集落形成单位（CFU）/ mL的对应。与细菌的准备，着手准备的叶子，并进行检测。 </li></ol> 2。叶染色和细菌的孵化<ol><li>为了色斑的叶子先准备20μM的碘化丙啶或PI在水溶液中。 10毫升的溶液是足够的染色时间3小叶子。 </li><li>检索从生长室和钳分离3年轻，完全展开的叶子的植物。在PI溶液浸泡5分钟整个叶片。然后取出树叶，并用蒸馏水冲洗一下。 </li><li>下一个叶片具有较低的表面面临载玻片。叶用锋利的刀片切成四大块，不包括中静脉使叶平放在幻灯片上。 </li><li>孵化与叶中的细菌，添加300μL菌悬液，在幻灯片上的叶件。确保较低的叶表面接触接种。 </li><li>孵育相同的环境条件下，被用来种植植物的样品。当时在显微镜下观察叶片，叶片转移到一个较低的表面朝上的新幻灯片。注意，安装的幻灯片时，不使用盖玻片。相同的叶样品进行成像孵化过程中多次，根据研究目标，可以设立一个时间过程。 </li></ol> 3。显微镜，测量和数据分析<ol><li>在这里，激光扫描共聚焦显微镜（LSM 510 META，卡尔蔡司公司）是用来研究的叶子。观察较低的叶子表面检测PI（激发453 nm，发射543和620 nm）的荧光。 </li><li>使用相同的树叶样品采集气孔的图像随着时间的推移。保存图像测量气孔的宽度。 </li><li>测量气孔宽度为每个使用LSM的图像浏览器在每个时间点的治疗至少60个气孔。 </li><li>气孔宽度计算平均值和标准错误。可以使用2尾，配对聪明的学生的t -检验计算结果的统计意义。 </li></ol> 4。气孔反应孵化代表性的图像与细菌<ol><li>这里是一个典型的显微图像（使用20倍的目标）激光下的拟南芥叶表面扫描共聚焦显微镜，在明亮的视场（图1）。 </li><li>碘化丙啶污渍的增加，除了允许识别数据收集（图2）完好的细胞保卫细胞的知名度的活细胞的细胞壁。可以看出，在这些显微气孔孔径开口，从完全封闭的气孔敞开气孔。 </li><li>全封闭的气孔保卫细胞的形状（图3）确定。光圈宽度被认为是0微米。 </li><li>对于开放气孔（图4）显示的光圈宽度测量使用最广泛的气孔孔径面积绘制一条直线横跨。 </li><li>这些都是典型的实验结果，这个气孔试验。Arabidopis叶假单胞菌丁香有三个不同的菌株，在黑暗中孵育。只有细菌的Pst DC3000能够打开黑暗的封闭气孔，随机选取60％的菌株孔（图5）的光圈宽度测量。 </li></oL&gt; <img src="/files/ftp_upload/2185/2185fig1.jpg" alt="图1" /> 图1。 拟南芥的叶子表面的显微照片。叶下激光扫描共聚焦显微镜（LSCM）使用20倍的目标表面的领域之一。请注意，气孔不资助的荧光，通过碘化丙啶染色（图2）相比，明显。 <img src="/files/ftp_upload/2185/2185fig2.jpg" alt="图2" /> 图2。一个碘化丙啶染色拟南芥叶表面的显微照片 。叶下激光扫描共聚焦显微镜（LSCM）使用20倍的目标表面的领域之一。注意气孔孔道的开放范围。黄色箭头指出完全关闭气孔和绿色箭头指出敞开气孔。 <img src="/files/ftp_upload/2185/2185fig3.jpg" alt="图3" /> 图3。完全关闭气孔的形状和保卫细胞之间的开放确定 。光圈宽度被认为是0微米。 <img src="/files/ftp_upload/2185/2185fig4.jpg" alt="图4" /> 图4。打开气孔微米孔径的宽度 。测量是使用浏览器基于跨最广泛的气孔孔径地区绘制一条直线的激光共聚焦显微镜。 <img src="/files/ftp_upload/2185/2185fig5.jpg" alt="图5" /> 图5。在实验的时候， 被蒙在鼓里。孵育3株假单胞菌丁香光伏番茄的拟南芥叶片气孔：DC3118，DB29，和DC3000植物。结果显示平均（N = 50-70）±标准误差。双尾学生的t -检验检测统计学意义。实验重复三次类似的结果。

<ol>
	<li>Brooks, D. M., Hernandez-Guzman, G., Kloek, A. P., Alarcon-Chaidez, F., Sreedharan, A., Rangaswamy, V., Penaloza-Vazquez, A., Bender, C. L., Kunkel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+well-defined+series+of+coronatine+biosynthetic+mutants+of+Pseudomonas+syringae+pv.+tomato+DC3000.">Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonas syringae pv. tomato DC3000.</a> Mol Plant-Microbe Interact. 17, 162-174 (2004).</li><li>Katagiri, F., Thilmony, R., He, S. Y., Somerville, C. R., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+thaliana-Pseudomonas+syringae+interaction.">The Arabidopsis thaliana-Pseudomonas syringae interaction.</a> The Arabidopsis Book. , (2002).</li><li>Ma, S. W., Morris, V. L., Cuppels, D. A. <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+a+DNA+region+required+for+production+of+the+phytotoxin+coronatine+by+Pseudomonas+syringae+pv.+tomato.">Characterization of a DNA region required for production of the phytotoxin coronatine by Pseudomonas syringae pv. tomato.</a> Mol Plant-Microbe Interact. 4, 69-74 (1991).</li><li>Melotto, M., Underwood, W., Koczan, J., Nomura, K., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+stomata+function+in+innate+immunity+against+bacterial+invasion.">Plant stomata function in innate immunity against bacterial invasion.</a> Cell. 126, 969-980 (2006).</li></ol>

我们已经提出了直线前进的程序，以衡量完整的叶片气孔反应不同的治疗方法易于评估的组织气孔。 虽然我们已经介绍了使用的模型系统拟南芥 / 假单胞菌丁香 ，完整的叶片气孔法可以与任何植物细菌组合潜在的执行。该协议可以很容易地进行修改，以适应其他植物和细菌病原体的生长要求。总体原则和程序保持不变。此外，这种方法可能有利于研究人员希望研究保卫细胞，不仅要活的微生物的功能输出，但也给其他刺激和化学药剂的条件下，保持叶片的自然环境。 全叶可由于其厚度不均匀地形的形象很难。删除中旬叶静脉，所以它平放在幻灯片上，使用共聚焦显微镜，可以缓解这个问题。然而，其他类型的荧光显微镜可用于叶面​​获得高分辨率的图像。

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Propidium iodide</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P4864</td>
<td>&nbsp;</td>
<tr>
<td>Tryptone</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP1421-1</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>0127-01-7</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>7647-14-5</td>
<td>&nbsp;</td>
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Bio Express</td>
<td>J637-2500G</td>
<td>&nbsp;</td>
<tr>
<td>Plant growth chamber</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Shaker incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Laser scanning confocal microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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assessing stomatal response to live bacterial cells using whole leaf imaging

气孔在植物表皮负责植物室内和环境之间的气体交换的自然开口。他们是由一对保卫细胞，它能够关闭一些外部因素，包括光照强度，二氧化碳的浓度，和相对湿度（RH）的气孔孔径形成。气孔孔径也病原体进入叶子，为疾病的发生发展的关键的一步的主要途径。最近的研究，推出了封闭的孔隙，能有效地减少在拟南芥中的细菌性疾病的发展;植物先天免疫系统的组成部分。在此之前，我们已经使用了表皮剥落评估气孔反应的活细菌（Melotto等人，2006年）;然而维持有利的环境条件对植物表皮的果皮和细菌细胞，已具有挑战性。叶表皮，可以保持活着，健康与MES缓冲液（氯化钾10毫米，25毫米的MES KOH，pH值6.15）保卫细胞的电生理实验。然而，这个缓冲区是不适合获得菌悬液。另一方面，细菌细胞可以保持水是不妥当的长期维持表皮换肤活着。当对水的表皮剥离彩车，在4个小时，暴露在空气干燥的果皮细胞限制时间进行实验。保卫细胞中的一个特定的刺激效果评估的一个理想的方法应该气孔生理和尽可能多的植物自然环境的干扰极小。因此，我们开发出一种新的方法来评估气孔反应活叶伤人及操纵大大降低，旨在提供一个容易复制和可靠的气孔检测细菌。该协议是基于完整的叶与碘化丙啶（PI）染色叶菌悬液的潜伏期，和叶下激光扫描共聚焦显微镜观察染色。最后，此方法允许在同一时间延长期间使用的条件下，紧密地模仿自然条件下，植物病原体攻击，活叶样品的观察。

Watch this Scientific Journal Video about Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging at JoVE.com

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

我们已经开发出一种简单，重复性的协议来访问气孔反应活的细菌。这种方法最大限度地减少以前相比，使用表皮换肤叶伤人及操纵报告。

评估气孔反应活细菌细胞，采用全叶成像

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

assessing-stomatal-response-to-live-bacterial-cells-using-whole-leaf

Research

JoVE Journal

Immunology and Infection

18.2K 次观看.  University of Texas at Arlington. 我们已经开发出一种简单，重复性的协议来访问气孔反应活的细菌。这种方法最大限度地减少以前相比，使用表皮换肤叶伤人及操纵报告。

视频: Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

科研

免疫与感染

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

生物发光细菌成像<em在体内</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

可视化的活细菌细胞蛋白-DNA相互作用使用光活化的单分子跟踪

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

细菌性叶渗透试验使用的植物防御反应精细表征<em&gt;拟南芥，丁香假单胞菌</em&gt;病害系统

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

测量的生理反应<em&gt;果蝇</em&gt;感觉神经元脂质费洛蒙使用实时钙成像

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

为了应对抑菌活性的实时成像人力初级中性粒细胞和单核细胞<em&gt;一种。曲霉</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

果蝇、果蝇细胞免疫应答的评估

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

细菌细胞的三维成像，实现精确的细胞表现和精确的蛋白质定位

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...