Name: fluorescently labeled bacteria as a tracer to reveal novel pathways of organic carbon flow in aquatic ecosystems
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Description: Watch this Scientific Journal Video about Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems at JoVE.com

这项研究得到了捷克科学基金会的支持，分别向K.和D.S.颁发了13-00243S和19-16554S的研究补助金。本文还得到由欧洲区域发展基金资助的"生物操作作为改善水坝水库水质的工具"项目（无CZ.02.1.01/0.0/0.0/0.0/16_025/0007417）项目的支持，该项目由欧洲区域发展基金资助，用于业务方案研究、开发和教育。

1. 样本收集
<ol><li>
水库水样收集:第一个案例研究（Exp I;低天然原位捕食者和猎物丰度系统）<ol>
<li>以适当的深度从所需位置收集水样。在运送到实验室过程中，将样品保存在温度控制的冷却器中，在原位温度下填充（避免温度冲击;应注意，原生动物的吸收率取决于温度）。 注:我们的取样是在中富营养峡谷形的切莫夫水库（南波希米亚，体积34.5 x 106 m3，最大深度43米，平均保留时间100天，二分）。取样地点位于30米深的大坝附近。2-l Friedinger 取样器采集了深度为 0.5 m 的混合样品。</li>
		<li>尽快继续第 1.2 节。</li>
	</ol>
</li>
	<li>
从食肉性尿反射植物的陷阱中收集液体:第二个案例研究（Exp II;具有高捕食者和猎物丰度、极小样本体积的系统）<ol>
<li>轻轻摇动水下的植物，从栽培容器中取出，放在吸水材料上吸收多余的水。根据植物的坚固性和陷阱的大小，选择8-10个植物个体。</li>
		<li>通过计算承载陷印器官的叶节点，将每个拍摄划分为大致相等的部分。每个拍摄部分将起到收集代表年轻、中年和旧陷阱的混合样本。</li>
		<li>将薄玻璃毛细管和 Eppendorf 小瓶连接到蠕动泵，用于收集样品。继续将毛细管尖端插入陷阱开口。使用真空泵，从每个疏水阀中吸出所有液体，直到为每个疏水阀年龄类别收集 900 ± 100 μL 的疏水阀液。</li>
		<li>使用池陷井液的200 μL的三联体子样品进行原石放牧实验。立即处理这些，详见第 3 节。保留样品的剩余 ±300 μL，用于对流体中微生物成分进行所有其他分析，详情如下（第 2 节）。</li>
		<li>立即转到第 2 节。</li>
	</ol>
</li>
</ol>2. 采集的样品的固定
<ol><li>Exp I 和 II:将水/陷阱液子样本固定在细菌枚举（第 4 节;分别为约 20 mL 和 0.3 mL）中，使用甲醛至少 1 小时，以获得 2% 的最终体积:每个样品的体积浓度。 注意:在烟罩中专门处理甲醛，并在操作样品时随时戴上手套。</li>
</ol>3. 样品过滤
<ol><li>样品稀释（Exp I）:储层水样无需稀释。（Exp II）:用无颗粒MQ水稀释疏水液样品10x-100x，在通过荧光显微镜计数之前，在过滤表面实现目标微生物的适当分布。</li>
	<li>使用过滤漏斗（直径 25 mm）过滤储液罐水的 1-2 mL （Exp I） 或疏水阀液子样品的 10-30 μL （Exp II），以便细菌计数到黑色 0.2 μm 孔径过滤器上。</li>
	<li>用DAPI（4'，6-二酰胺-2'-苯林多尔二盐，0.2%最终浓度）染色过滤器4分钟。 注意:避免皮肤和工作表面污染，并戴上手套。</li>
	<li>将装有浓缩微生物的过滤器放在显微镜幻灯片上的一滴浸入油（用于荧光显微镜）。将另一个油滴放在滤清器的中心，并盖上盖玻片，确保机油均匀分布。</li>
	<li>此时，最好立即处理样品，或者将其储存在冷冻室（-20°C）中数周至数月，直至进一步分析。</li>
</ol>4. 在过滤器上统计细菌数量
<ol><li>将幻灯片放在荧光显微镜下（过滤器设置与荧光DAPI相对应）。将 10 x 10 计数网格放入其中一个眼部。将幻灯片移动到随机位置。</li>
	<li>量化计数网格区域（放大倍数 1000x 下的细菌细胞（蓝色荧光）。在计数中，包括跨越计数网格左边缘和上边缘的单元格，同时排除位于右边缘和下边缘的单元格。</li>
	<li>移动到另一个随机位置，并在至少 10-15 个计数网格上重复枚举，总计 500 个单元格计数。</li>
	<li>根据对一个网格面积与过滤器总有效过滤面积的比率的了解，为给定显微镜和放大倍率建立转换系数。然后，将计数的细胞总数除以计数的网格数，每个网格产生平均细菌数。</li>
	<li>将后一个参数乘以建立的转换系数，并标准化样品每 mL 的结果数（取决于过滤的样本体积），以获得每 mL 的总细菌丰度。</li>
</ol>5. 确定原丰度
<ol><li>用谷醛（最终浓度为 1%，更适合含有叶绿素的颗粒在固定后数天到数周）固定水样（Exp I）或疏水液 （Exp II） 子样品，或使用在下面指定的二硫盐脱色技术。 注:这两种保存技术都防止从原药学家的食物中摄取的物分8。</li>
	<li>关于脱色剂脱色技术，将100μL/1 μL的Lugol溶液加入20mL/200 μL的水/陷阱流体亚样品（分别为Exp I/Exp II）。</li>
	<li>紧接着加入0.5 mL/50 μL的硼酸盐缓冲形式素，然后加入20μL/2μL的3%硫磺酸钠（分别为Exp I/Exp II）。 注:硫磺酸钠使Lugol的黄色变色，从而能够在荧光显微镜8下观察细胞。</li>
	<li>将已知样品体积（取决于目标原体数量）过滤到 1 μm 孔径黑色聚碳酸酯过滤器上。</li>
	<li>估计孕激素物种的数量是通过使用计数网格在放大倍数 600 倍下计算至少 200 个细胞（见上文）。</li>
	<li>将过滤漏斗中真空下的样品体积降低至约 2 mL。然后，释放压力下，加入DAPI氟铬（4'，6-二酰胺-2-苯二氯盐13，0.2%最终浓度）2分钟。</li>
</ol>6. 确定浮游生物样本中西利亚特动物的社区结构
注:淡水栖息地的西栖菌群落高度多样化，14、15、16、18，其微观测定具有挑战性。将纤毛组分为功能公会10、14、16、17，可以更详细地分析作为中上层细菌的不同纤细组。
<ol><li>通过结合以下两方面来评估纤毛社区结构:<ol>
<li>荧光显微镜中DAPI染色的样品（用不同尺寸和形态的宏观和微核的明亮荧光对纤细细胞进行局部化）与荧光标记细菌的吸收相结合（FLB8;详情，见下文），跟踪西利特以细菌为食的能力。</li>
		<li>在选定情况下进行现场抽样观察17，19。有关用于将纤毛分类分组到不同分类类别中的上述方法和标准的更多详细信息，请参阅以前的出版物16、17。 注:引用的研究表明，在硅酸中，来自锡科特里奇亚（属哈尔特里亚和佩拉戈哈尔特里亚）和奥利戈特里西亚（即里莫斯特龙比姆spp.）的杂食物种是在绝大多数淡水栖息地的浮游生物10，17，18。</li>
	</ol>
</li>
</ol>7. 估计西利特放牧率
<ol><li>根据示踪剂平均数量的变化（即每只毛草与孵育时间（5-15分钟）相关的FLB8]和添加的FLB的示踪量计算菌的取药率，最多占细菌总数的5%-15%。</li>
	<li>为了比较不同物种的接受率，将摄入率标准化为每小时每毛质动物的细菌数量，并根据实际孵育时间和添加的示踪剂FLB的比例进行计算。 注:FLB方法应用的一般方案，用于估计自然样本中细胞或物种特定和散装细菌率，如图1所示。</li>
	<li>从本地细菌菌株制备FLB到淡水环境8<ol>
<li>选择适当的大小（平均细胞体积 + MCV）和细菌的形态，以便它们有效地模仿被调查的水生系统中浮游杆菌/细菌细胞的典型大小。 注:对于Exp I，使用了从林诺比坦和多核细胞属的研究位置分离的菌株混合物（即，湖泊和池塘中典型的、高度丰富的浮游杆菌）20 。有关菌株形态和大小的详细信息，请参阅以前的出版物3、17、18、21。</li>
		<li>在早期固定阶段，通过离心（5，000 x g）从培养中收获细菌细胞15分钟，并在与所选位置的典型MCV细菌对应的混合物中以数值比例混合它们， 产生MCV + SD。</li>
		<li>将颗粒悬浮在10 mL的磷酸盐缓冲盐水中（PBS;pH = 9）。</li>
		<li>在磷酸盐盐缓冲液中向细胞悬浮液中加入2mg的黄绿色荧光染料5-（4，6-二氯三聚二苯-2-yl）氨基氟辛（DTAF，与蛋白质结合），并在60°C水浴中孵育2小时。</li>
		<li>孵育后，将细胞向下离心机，分解DTAF溶液，用PBS清洗和离心机3x。</li>
		<li>最后洗涤后，在PPi-盐碱缓冲液的20 mL中重新悬浮细胞。</li>
		<li>将FLB悬浮液和移液器1.5 mL等分液涡到2 mL低温瓶中，然后在PPi-盐碱缓冲液中保持冷冻（在-20°C下），直到使用。</li>
		<li>通过 0.2 μm 聚碳酸酯过滤器预过滤 PPi-盐碱缓冲液，用于下一步。</li>
		<li>要确定FLB浓度，将一个小等分（通常为20-40μL）转移到2 mL的无颗粒PPi-盐碱缓冲液，在30W下声波几次2s爆发，并过滤到0.2μm聚碳酸酯黑色过滤器上，通过荧光进行枚举在 DTAF （448 nm/520-540 nm） 的光学滤镜设置下进行显微镜（1，000 倍放大率）。</li>
	</ol></li>
	<li>用于估计西利茨细菌的跟踪器技术<ol>
<li>在天然木板栖息地进行放牧实验时，将300 mL样品放入经过良好冲洗的1 L瓶中，并在原位温度下孵育15分钟（让原生体从处理冲击中恢复）。</li>
		<li>添加FLB示踪剂，以占细菌总数的5%-15%，根据季节和水温增加量。 注: 在纤毛酸盐物种特定摄取率中，有非常广泛的、依赖于季节的光谱，跨越几个数量级（即，从 101-104细菌西利酸盐-1每小时）10、16 17，18，22，23，24。</li>
		<li>在具有高接受率的西利酸盐发生期间（通常在夏季），还运行平行孵育，FLB添加量非常低，仅占细菌总数的2%-4%，以避免由示踪剂FLB过度加载西利酸盐（参见图2中的示例 。</li>
		<li>用FLB孵育西利酸盐/浮游生物样品5-15分钟。</li>
		<li>有两种机会的样品固定，以防止从食物的食样剂的摄入材料的吸收8。通过添加 1% 谷醛（最终浓度更适合含叶绿素颗粒的样品，如藻类）来终止孵育。或者，将100μL/10 μL的Lugol溶液放入20 mL/200 μL的水/陷阱流体亚样品中，然后立即加入0.5 mL/10 μL的硼酸盐缓冲形式素，然后加入200 μL/2 μL的3%硫硫酸钠（Exp I/Exp II）。</li>
	</ol></li>
	<li>加入固定剂后，让样品在黑暗中在4°C下至少休息1小时，以确保完全保存纤毛细胞。</li>
	<li>从4-30 mL/10-30 μL（Exp I/Exp II;体积取决于西柳石丰度）和DAPI染色（最终浓度0.2%wt/vol;详情见上文步骤3.2）采集天然浮游生物子样本。</li>
	<li>通过 1 μm 黑色过滤器，并通过荧光显微镜检查，以计数纤毛（600 倍放大率），并列举摄念的FLB示踪剂数量（大部分以 1000 倍放大率），如前几篇出版物2、17 中详述的.保存后7天内检查样品。</li>
	<li>要估计原生动物/物种特异性放牧的总数量，请将所有西毛动物的平均接受率，或仅按原地丰度检测到的西利酸盐物种进行繁殖。</li>
	<li>从切莫夫水库的原位数据计算每单元的取水率的例子如下:<ol>
<li>假设细菌浓度为 3.55 x 106细菌/mL，添加示踪剂 FLB 为 0.25 x 106 FLB/mL，得出总细菌颗粒（天然细菌 + FLB = 100% 的猎物颗粒）的总和 3.8 x 106细菌/mL可用于天然样品中的噬菌体原生。 注:因此添加的FLB示踪剂代表细菌颗粒总量的6.58%（项目为0.25/0.038）。在5分钟的孵育中，每个哈尔特里亚sp的平均FLB为6.2FLB。</li>
		<li>要使每小时的摄入正常化，请使用以下计算:（6.2 x 12）/（6.58/100） = 每哈尔特里亚细胞/小时 1131 细菌。 注:有关在可变水温下检测到的Halteria sp.的单个摄取率分布的更多示例、添加的示踪剂FLB的量（百分比）以及FLB的不同孵育时间，请参见图3。</li>
	</ol></li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+DAPI+for+identifying+and+counting+aquatic+microflora.">The use of DAPI for identifying and counting aquatic microflora.</a> Limnology and Oceanography. 25, 943-948 (1980).</li><li>Foissner, W., Berger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+user-friendly+guide+to+the+ciliates+(Protozoa,+Ciliophora)+commonly+used+by+hydrobiologists+as+bioindicators+in+rivers,+lakes,+and+waste+waters,+with+notes+on+their+ecology.">A user-friendly guide to the ciliates (Protozoa, Ciliophora) commonly used by hydrobiologists as bioindicators in rivers, lakes, and waste waters, with notes on their ecology.</a> Freshwater Biology. 35, 375-482 (1996).</li><li>Foissner, W., Berger, H., Schaumburg, J. <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+ecology+of+limnetic+plankton+ciliates.">Identification and ecology of limnetic plankton ciliates.</a> Informationsberichte des Bayer Landesamtes f&#252;r Wasserwirtschaft Heft. , 3-99 (1999).</li><li>&#352;imek, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliate+grazing+on+picoplankton+in+a+eutrophic+reservoir+during+the+summer+phytoplankton+maximum:+a+study+at+the+species+and+community+level.">Ciliate grazing on picoplankton in a eutrophic reservoir during the summer phytoplankton maximum: a study at the species and community level.</a> Limnology &amp; Oceanography. 40, 1077-1090 (1995).</li><li>Skibbe, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+quantitative+protargol+stain+for+ciliates+and+other+planktonic+protists.">An improved quantitative protargol stain for ciliates and other planktonic protists.</a> Archiv f&#252;r. Hydrobiolgie. 130, 339-347 (1994).</li><li>Macek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+rates+of+dominant+planktonic+ciliates+in+two+freshwater+bodies+of+different+trophic+degree.">Growth rates of dominant planktonic ciliates in two freshwater bodies of different trophic degree.</a> Journal of Plankton Research. 18, 463-481 (1996).</li><li>Šimek, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+food+webs+in+hypertrophic+fishponds:+omnivorous+ciliate+taxa+are+major+protistan+bacterivores.">Microbial food webs in hypertrophic fishponds: omnivorous ciliate taxa are major protistan bacterivores.</a> Limnology &amp; Oceanography. , (2019).</li><li>Jezbera, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+freshwater+bacterioplankton+groups:+Contrasting+trends+in+distribution+of+Limnohabitans+and+Polynucleobacter+lineages+along+a+pH+gradient+of+72+habitats.">Major freshwater bacterioplankton groups: Contrasting trends in distribution of Limnohabitans and Polynucleobacter lineages along a pH gradient of 72 habitats.</a> FEMS Microbiology Ecology. 81, 467-479 (2012).</li><li>Kasalick&#253;, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diversity+of+the+Limnohabitans+genus,+an+important+group+of+freshwater+bacterioplankton,+by+characterization+of+35+isolated+strains.">The diversity of the Limnohabitans genus, an important group of freshwater bacterioplankton, by characterization of 35 isolated strains.</a> PLoS One. 8, 58209 (2013).</li><li>Stabell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliate+bacterivory+in+epilimnetic+waters.">Ciliate bacterivory in epilimnetic waters.</a> Aquatic Microbial Ecology. 10, 265-272 (1996).</li><li>Zingel, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliates+are+the+dominant+grazers+on+pico-+and+nanoplankton+in+a+shallow,+naturally+highly+eutrophic+lake.">Ciliates are the dominant grazers on pico- and nanoplankton in a shallow, naturally highly eutrophic lake.</a> Microbial Ecology. 53, 134-142 (2007).</li><li>Bickel, S. L., Tang, K. W., Grossart, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliate+epibionts+associated+with+crustacean+zooplankton+in+german+lakes:+distribution,+motility,+and+bacterivory.">Ciliate epibionts associated with crustacean zooplankton in german lakes: distribution, motility, and bacterivory.</a> Frontiers in Microbiology. 3 (243), (2012).</li><li>Sirov&#225;, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hunters+or+gardeners?+Linking+community+structure+and+function+of+trap-associated+microbes+to+the+nutrient+acquisition+strategy+of+a+carnivorous+plant.">Hunters or gardeners? Linking community structure and function of trap-associated microbes to the nutrient acquisition strategy of a carnivorous plant.</a> Microbiome. 6, 225 (2018).</li><li>&#352;imek, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+traits+of+a+zoochlorellae-bearing+Tetrahymena+sp.+(Ciliophora)+living+in+traps+of+the+carnivorous+aquatic+plant+Utricularia+reflexa.">Ecological traits of a zoochlorellae-bearing Tetrahymena sp. (Ciliophora) living in traps of the carnivorous aquatic plant Utricularia reflexa.</a> Journal of Eukaryotic Microbiology. 64, 336-348 (2017).</li><li>Pitsch, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+Tetrahymena+utriculariae+n.+sp.+(Ciliophora,+Oligohymenophorea)+with+its+endosymbiotic+algae+(Micractinium+sp.),+living+in+the+feeding+traps+of+a+carnivorous+aquatic+plant.">The green Tetrahymena utriculariae n. sp. (Ciliophora, Oligohymenophorea) with its endosymbiotic algae (Micractinium sp.), living in the feeding traps of a carnivorous aquatic plant.</a> Journal of Eukaryotic Microbiology. 64, 322-335 (2017).</li><li>Nielsen, J. M., Clare, E. L., Hayden, B., Brett, M. T., Kratina, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet+tracing+in+ecology:+Method+comparison+and+selection.">Diet tracing in ecology: Method comparison and selection.</a> Methods in Ecology and Evaluation. 9, 278-291 (2018).</li><li>Beisner, B. E., Grossart, H. P., Gasol, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+methods+for+estimating+phago-mixotrophy+in+nanophytoplankton.">A guide to methods for estimating phago-mixotrophy in nanophytoplankton.</a> Journal of Plankton Research. , 1-13 (2019).</li><li>Dolan, J. D., &#352;imek, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+ingested+matter+in+Strombidium+sulcatum,+a+marine+ciliate+(Oligotrichida).">Processing of ingested matter in Strombidium sulcatum, a marine ciliate (Oligotrichida).</a> Limnology and Oceanography. 42, 393-397 (1997).</li><li>Massana, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grazing+rates+and+functional+diversity+of+uncultured+heterotrophic+flagellates.">Grazing rates and functional diversity of uncultured heterotrophic flagellates.</a> The ISME Journal. 3, 588-596 (2009).</li><li>Gruj&#269;i&#263;, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptophyta+as+major+freshwater+bacterivores+in+experiments+with+manipulated+bacterial+prey.">Cryptophyta as major freshwater bacterivores in experiments with manipulated bacterial prey.</a> The ISME Journal. 12, 1668-1681 (2018).</li></ol>

作者没有什么可透露的。

在水生系统中破译营养相互作用总是具有挑战性的28，特别是在纳米浮游生物秤涉及原发动物和他们的猎物，细菌。在营养摄入途径和定量方面，由于生物相互作用的复杂性高，在高营养水平下成功使用的方法的应用的可能性较小。例如，这些方法包括稳定的同位素标记方法。该协议显示了使用荧光显微镜和荧光标记细菌作为示踪剂来跟踪和半定量/估计碳流动的优点（细菌猎物:...

异养原核生物是水生系统中的关键生物成分，在浮游生物生物量1、2、3中占很大比例。控制其丰度、多样性和活性的因素对于理解其在生物地球化学循环中的作用（即有机碳和其他营养物质的命运以及从原核生物到更高的营养水平的能量流动）至关重要。原生动物放牧是这些重要因素之一。异营养纳米旗和纤毛的细菌对原核生物丰度、群落功能、结构、多样性，甚至细胞形态和特定细菌群的生长速率施加了强大的自上而下的控制。 5，6.在一些系统中，原发学家是细菌死亡的主要原因6，7。
用于评估原生动物细菌的标准方法，已经使用了一段时间，涉及使用荧光标记细菌（FLB）作为猎物类似物和荧光显微镜。细胞特异性的吸收率可以通过在选定的时间程8中量化前列腺食物中标记的猎物颗粒的数量来确定。这种方法有几个优点。追踪器被添加到自然样品与自然捕食者和猎物组合。孵育前有最少的样品操作，添加的FLB示踪剂对样品的改变最小，孵育时间较短，以确保在接近原位条件下获得良好结果。或者，在细菌原虫或浮游动物数量少的环境中（例如近海海洋系统），通过流动细胞测定，可长期（12-24小时）在样品中添加少量FLB的消失率（2%-3%示踪剂）孵化实验。然后，在起点和终点的FLB数量（集成所有细菌的影响）通过流动细胞测定法进行量化（详情请参阅以前的出版物9）。然而，这种参数只代表不能直接归因于任何特定的前列腺动物和浮游动物食草动物群体或物种的总细菌率。
总体而言，准确量化水生环境中的古生物物种或形态特定细菌死亡率可能具有挑战性。一些原生动物是选择性的放牧者，添加的FLB示踪剂的大小和细胞形状可能会扭曲猎物摄入的自然速率10，11。此外，前列腺活性和代谢高度温敏12;因此，需要针对每种样品类型仔细操纵添加的FLB示踪剂量（不仅基于细菌的自然丰度、大小和形态以及细菌的流行类型，还基于温度）。大多数研究侧重于散装牧草放牧活动;然而，特定原物种的细菌通常具有较高的信息价值，并且可能更可取。在这种情况下，需要对样本中存在的原科物种的分类学知识，并了解它们的行为。因此，需要大量的时间和劳动力，才能获得针对特定物种的细菌感染率的健全结果，该比率可归因于特定的前列腺组或物种。
尽管存在这些困难，这种方法仍然是目前可用于在自然环境中评估前列腺细菌的最合适工具。这里介绍了一种在水生微生物生态学研究中使用FLB作为示踪剂的综合性、易于遵循的方法。该方法的所有问题方面都得到了说明，并描述了改进的工作流程，其中有两个来自对比环境的实验以及对比的西利特物种作为示例。
第一个案例研究是在捷克共和国的中生代什莫夫水库的表观环境中进行的，结果表明，与大多数地表淡水体相媲美的食草和细菌丰度（参见第5、7）。第二个案例研究是在水生食肉植物Utricularia反射的陷阱内高度特殊的环境中进行的，这种反射中含有极高数量的两种放牧混合营养性纤酸（四海梅纳眼）和细菌细胞。显示了这两种样本中细胞特异性放牧率和细菌常备库的计算。然后讨论了对结果的一系列生态解释，最后提出了可能的后续研究实例。

<table><tbody><tr><td>0.2-&amp;micro;m pore-size filters&amp;nbsp;</td><td>SPI supplies, https://www.2spi.com/</td><td>B0225-MB</td><td>Black, polycarbonate track etch membrane filters, diameter approprite for filtering apparatus used</td></tr><tr><td>5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF)</td><td>Any brand</td><td></td><td></td></tr><tr><td>Automatic pipettes with adjustable volumes&amp;nbsp;</td><td>Any brand, various sizes</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>22 000 x g</td><td></td><td></td></tr><tr><td>Cryovials</td><td>Any brand, 2 mL size</td><td></td><td></td></tr><tr><td>DAPI (4&amp;acute;,6-Diamidino-2&amp;acute;-phenylindole dihydrochloride)</td><td>Any brand&amp;nbsp;</td><td></td><td>1 mg ml&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Epiflorescence microscope</td><td>Magnification from 400 x up to 1000 x</td><td></td><td></td></tr><tr><td></td><td>Filters appropriate for viewing in the DAPI and DTAF range</td><td></td><td></td></tr><tr><td></td><td>Counting grid in one of the oculars</td><td></td><td></td></tr><tr><td>Filtering apparatus</td><td>Usually with a diameter of 25 mm&amp;nbsp;</td><td></td><td></td></tr><tr><td>Formaldehyde</td><td>A brand for microscopy</td><td></td><td></td></tr><tr><td>Glutaraldehyde</td><td>A brand for microscopy</td><td></td><td></td></tr><tr><td>Immersion oil for microscopy</td><td>Specific oil with low fluorescence</td><td></td><td></td></tr><tr><td>Lugol&amp;acute;s solution</td><td>Any brand or see comment</td><td></td><td>Make an alkaline Lugol&#039; solution as follows: Solution 1 - dissolve&amp;nbsp; 10 g of potassium iodide in 20 ml in MQ water, then add 5 g of iodine. Solution 2 - add 5 g of sodium acetate&amp;nbsp; to 50 ml of MQ water. Add the solution 2 to the solution 1 and thoroughly mix</td></tr><tr><td>Methanol stabilized formalin</td><td>Any brand available for microscopy purposes</td><td></td><td></td></tr><tr><td>Microscope slides and cover slips</td><td>Any brand produced for microscopy purposes&amp;nbsp;</td><td></td><td></td></tr><tr><td>MQ water for diluting samples</td><td>Any brand&lt;br/&gt; &amp;nbsp;</td><td></td><td></td></tr><tr><td>Phosphate-buffered saline (PBS; pH = 9)</td><td>Any brand</td><td></td><td>0.05 M Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;-NaCl solution, adjusted to pH 9</td></tr><tr><td>PP&lt;sub&gt;i&lt;/sub&gt;-saline buffer</td><td>Any brand</td><td></td><td>0.02 M Na&lt;sub&gt;4&lt;/sub&gt;P&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;-NaCl solution. Add 0.53 g Na&lt;sub&gt;4&lt;/sub&gt;P&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt; to 100 ml of MQ water plus 0.85 g NaCl&amp;nbsp;</td></tr><tr><td>Sampling device&amp;nbsp;</td><td>Appropriate for obtaining representative sample&amp;nbsp;</td><td></td><td>e.g. Friedinger sampler for lake plankton</td></tr><tr><td>Sodium thiosulfate solution</td><td>Any brand</td><td></td><td>3% solution is used in the protocol</td></tr><tr><td>Sonicator</td><td>Any brand</td><td></td><td>30 W</td></tr><tr><td>Vortex</td><td>Any brand allowing&amp;nbsp; thorough mixing of the solutes and samples</td><td></td><td></td></tr><tr><td>Water bath</td><td>Any brand allowing temperature to be maintained at 60 &amp;deg;C</td><td></td><td></td></tr></tbody></table>

fluorescently labeled bacteria as a tracer to reveal novel pathways of organic carbon flow in aquatic ecosystems

阐明营养相互作用，如掠夺及其影响，是生态学许多研究者经常的任务。微生物群落的研究有许多局限性，确定捕食者、猎物和捕食率通常很困难。这里介绍的是一种基于添加荧光标记的猎物作为示踪剂的优化方法，它允许可靠地定量水生食肉真核生物的放牧率，并估计营养转移到更高的营养水平。

例如，实验是在切莫夫水库（南波希米亚，CZ），这是一个自然地点，较低的自然原位捕食者和猎物丰度。报告具有代表性的数据为杂食性香菜物种哈尔特里亚格兰尼拉，这是一个丰富和高效的肉食虫颗粒（<2 μm）颗粒10，16，17，18 ， 22.图...

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Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

这里介绍的是一种基于单细胞的表氟显微镜技术的协议，用于以高精度和分类分辨率量化水生捕食真核生物的放牧率。

荧光标记细菌作为追踪器揭示水生生态系统中有机碳流动的新途径

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

这种方法意义重大，因为它使我们能够以非常高的分辨率可视化和估计从原核生物到微生物食物网中更高营养水平的碳和能量流动。这是目前唯一可用于确定哪些原生物种原位使用细菌的技术，同时在单细胞水平上量化这个过程的速率。由于细菌是唯一重要的微生物，将溶解的有机碳改变为颗粒有机碳，因此，参与者的知识和发生这种物质的比率构成了了解水生生态系统的基础。我们强烈建议研究人员在进行放牧实验之前熟悉他们的研究系统。这涉及到光和荧光的显微镜检查样品，并了解主要分类存在和他们的季节性动态。演示程序将是吉特卡 · 耶兹贝罗夫

fluorescently-labeled-bacteria-as-tracer-to-reveal-novel-pathways

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7.0K 次观看.  Biology Centre CAS. 这种方法意义重大，因为它使我们能够以非常高的分辨率可视化和估计从原核生物到微生物食物网中更高营养水平的碳和能量流动。这是目前唯一可用于确定哪些原生物种原位使用细菌的技术，同时在单细胞水平上量化这个过程的速率。由于细菌是唯一重要的微生物，将溶解的有机碳改变为颗粒有机碳，因此，参与者的知识和发生这种物质的比率构成了了解水生生态系统的?...

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Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

在表面定植微生物种群监测空间分异

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

科研

遗传学

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

全细胞Bioreporter方法​​评估交通有机污染物的生物利用度和水饱和系统

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

环境科学

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

利用荧光蛋白可视化和定量<em&gt;衣原体</em&gt;液泡生长动态的活细胞

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

免疫与感染

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

可视化单分子络合物在体内使用高级荧光显微

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

生物学

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

溴脱氧尿嘧啶核苷（BrdU）标记和文化独立的鉴定溶解有机随后的荧光激活细胞分选碳有辱人格的浮游细菌

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

单细胞物种间细菌相互作用的动力学可视化

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

通过荧光报告基因定量进行全器官病原体追踪

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

作者聚焦:揭示细菌对组织中抗生素和免疫系统的反应

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

微生物感染的高分辨率三维全器官断层扫描

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

通过电穿孔内在和生物分子荧光观察生活中的微生物

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

荧光标记细菌作为追踪器揭示水生生态系统中有机碳流动的新途径

副本

摘要

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在表面定植微生物种群监测空间分异

全细胞Bioreporter方法评估交通有机污染物的生物利用度和水饱和系统

利用荧光蛋白可视化和定量

可视化单分子络合物在体内使用高级荧光显微

溴脱氧尿嘧啶核苷（BrdU）标记和文化独立的鉴定溶解有机随后的荧光激活细胞分选碳有辱人格的浮游细菌

单细胞物种间细菌相互作用的动力学可视化

微生物感染的高分辨率三维全器官断层扫描

微生物感染的高分辨率三维全器官断层扫描

微生物感染的高分辨率三维全器官断层扫描

通过电穿孔内在和生物分子荧光观察生活中的微生物

荧光标记细菌作为追踪器揭示水生生态系统中有机碳流动的新途径

副本

摘要

探索更多视频

此视频中的章节

在表面定植微生物种群监测空间分异

全细胞Bioreporter方法​​评估交通有机污染物的生物利用度和水饱和系统

利用荧光蛋白可视化和定量

可视化单分子络合物在体内使用高级荧光显微

溴脱氧尿嘧啶核苷（BrdU）标记和文化独立的鉴定溶解有机随后的荧光激活细胞分选碳有辱人格的浮游细菌

单细胞物种间细菌相互作用的动力学可视化

微生物感染的高分辨率三维全器官断层扫描

微生物感染的高分辨率三维全器官断层扫描

微生物感染的高分辨率三维全器官断层扫描

通过电穿孔内在和生物分子荧光观察生活中的微生物

全细胞Bioreporter方法评估交通有机污染物的生物利用度和水饱和系统