我们感谢Colby Carpenter和Spencer Mathias在3D打印设计方面的帮助。我们感谢艾伦·克林格（Ellen Klinger）协助制作摄影人物，并感谢乔纳森·B·科赫（Jonathan B. Koch）协助进行修订。资金由美国农业部 - ARS授粉昆虫生物学，管理和系统学研究部门提供。

1. 打印花粉陷阱结构
<ol><li>下载大黄蜂筑巢的巢箱的相应STL文件（例如，Biobest或Koppert风格的蜂巢，https://www.ars.usda.gov/pacific-west-area/logan-ut/pollinating-insect-biology-management-systematics-research/docs/pollen-traps/）。这些文件向公众开放，最终用户可以免费下载和修改。</li>
	<li>在打印机程序中打开 STL 文件。按照打印机制造商的说明构建四个陷印组件。 注：打印疏水阀体约 3 小时，打印集水盆约 2 小时，滤镜和陷印盒闭合插入物各 30 分钟。疏水阀主体尺寸为 6 厘米 x 3.8 厘米 x 7 厘米。</li>
</ol>2. 花粉捕集器组件
<ol><li>取下印有疏水阀体和集水池的支撑结构，包括疏水阀体的筛分结构（图1）。</li>
	<li>使用安装在手钻中的 3/16 英寸（0.476 厘米）钻头清除穿过花粉过滤器凸起边缘的任何塑料股，这些塑料股可能会阻碍蜜蜂通过过滤器孔。使用箱式切割剃须刀片和砂纸，将花粉过滤器平坦侧的任何凸起或凸起的边缘均匀。</li>
	<li>将花粉过滤器轻轻推过陷阱主体的一侧，将花粉过滤器放入疏水阀体中。过滤器只能以一种方式安装，因为疏水阀的左侧有一个更大的开口，以适应凸起的过滤器锥体的通过。<ol>
<li>如果侧缝太小，花粉过滤器无法顺利滑过，请使用剃须刀或其他可以一次去除一小部分塑料的工具，从疏水阀体的狭缝中刮出足够的塑料。确保花粉过滤器牢固地安装到位，花粉过滤器和捕集器体之间的间隙不超过2 mm。</li>
	</ol>
</li>
	<li>通过将捕集器主体底部的凸起边缘滑入集水盆顶部的凹槽中，将集水盆连接到捕集器主体上。集水池应直接位于疏水阀体的筛分区域下方（图 1A\u2012E）。</li>
	<li>通过切割或打磨塑料进行适当的修改，以便从疏水阀体上平滑放置和移除集水盆。集水池的放置会将花粉过滤器固定到位，在收集盆被移除之前，花粉过滤器无法移除，在集水盆就位时也无法插入花粉过滤器。 注意：如果将多个蜂箱彼此靠近放置，除了部署具有不同方向的蜂巢外，为每个蜂巢提供陷阱体，集水盆和花粉过滤器结构的独特颜色组合将有助于返回的工人找到巢穴。</li>
</ol>3. 大黄蜂蜂群准备
<ol><li>在部署菌落之前，停止花粉喂养24\u201248小时。这将导致工蚁用尽任何储存的花粉，并刺激它们离开巢穴寻找花粉。</li>
	<li>通过将疏水阀闭合插件插入过滤器槽中，防止蜜蜂在安装疏水阀时逃逸，准备用于安装蜂巢陷阱体。</li>
	<li>在红灯下工作以防止蜜蜂飞翔，将塑料巢箱从纸板外箱中取出。</li>
	<li>找到巢前方的塑料巢入口。根据巢供应商的不同，有两种入口样式：Koppert样式的盒子（步骤3.5）和Biobest风格的盒子（步骤3.6）。</li>
	<li>对于Koppert风格的蜂巢入口，通过拉动入口标签直到两个入口孔都打开，将花粉陷阱安装到巢穴入口处。<ol>
<li>将花粉捕集器的两根管子插入入口孔中，确保花粉捕集器的筛子在底部。轻轻向下推塑料入口卡舌，将花粉陷阱固定到位。</li>
	</ol>
</li>
	<li>要将花粉陷阱安装到Biobest式蜂巢入口处，请使用平头螺丝刀从巢箱中轻轻撬出塑料入口装置。将花粉陷阱插入巢穴入口孔中，直到花粉陷阱牢固地贴在巢穴上（图1E）。<ol>
<li>如果需要，使用胶带或速干胶将疏水阀固定在巢穴中，陷阱与巢箱接触。</li>
	</ol>
</li>
	<li>将塑料巢箱放回纸板箱。纸板可能需要切掉以容纳花粉陷阱。</li>
</ol>4. 巢穴的部署
<ol><li>将巢箱放入研究区域。提供覆盖物以防止降水和风锚定，因为这些会对收集的花粉的质量和数量产生不利影响。为放置在温室中的蜂箱提供足够的阳光覆盖，以减少过热。</li>
	<li>从陷阱体上取下陷阱封闭插件，让蜜蜂自由觅食，以便觅食者可以根据周围区域和巢穴的位置定位自己。在正常情况下，定向飞行时间应在24小时内完成。</li>
</ol>5. 花粉采集
<ol><li>要接合疏水阀，请将花粉过滤器滑入过滤器槽，确保其牢固到位。</li>
	<li>通过从正面将集水盆滑到疏水阀体上直到其完全关闭来安装集水盆。如果集水盆过度松动或从疏水阀体上脱落，请使用橡皮筋将其固定在疏水阀体上。</li>
	<li>观察蜜蜂在首次部署时进入和退出花粉陷阱，以确保花粉过滤器孔足够大以容纳蜜蜂。<ol>
<li>如果工人无法通过花粉过滤器，请取下过滤器，然后使用大于3/16英寸（0.476厘米）的钻头来增加孔的尺寸。按顺序进行，每次增加孔径1/32英寸（0.079厘米），因为太大的孔不会收集任何花粉。</li>
		<li>一旦蜜蜂能够通过过滤器，继续观察入口，以确保花粉在重新进入时被去除。 注意：蜜蜂在花粉过滤器孔中移动应该有些困难，尤其是它们通过的头几次。如果它们太容易通过，花粉可能不会从corbiculae中脱落。</li>
	</ol>
</li>
	<li>在指定的花粉收集期后，从捕集器体中滑动并取出集水盆。</li>
	<li>根据您的实验设计处理花粉负载。</li>
	<li>取下花粉过滤器，让工人自由觅食，直到下一个花粉采集期。在实验期间，陷阱体可能仍然附着在蜂巢上。 注意：只要研究人员希望从蜂群中收集花粉，花粉陷阱就可以参与。然而，在一周内部署花粉诱捕器超过24小时可能导致蜂巢中的育雏饥饿并延缓菌落发育。</li>
</ol>

<ol>
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作者没有什么可透露的。

从大黄蜂群入口收集花粉可以进行各种生态和农业研究。确定大黄蜂收集花粉的花卉来源为有助于殖民地整体饮食的植物多样性提供了有价值的信息和见解19。确定花粉来源对农业生产和野生土地生态系统服务的研究都有影响12，20。通过收集相对较大的样本大小的皮质花粉负载，研究人员可以确定大黄蜂是否在它们部署的目标作物<sup ...

大黄蜂（Bombus spp.）是大型健壮的昆虫，遍布世界温带，高山和北极地区1。它们对植物群落很重要，并为它们访问的农作物提供重要的授粉服务2.最近几个物种的丰度和分布的下降使它们作为传粉媒介的重要性成为公众意识的前沿3.研究人员已经确定了几个可能导致人口下降的压力因素，包括缺乏多样和丰富的花卉资源，大黄蜂以此为食4。确定大黄蜂从哪种植物物种中觅食，使研究人员和土地管理人员能够了解大黄蜂如何应对资源可用性，竞争和人为干扰的变化5，6。
调查大黄蜂花粉觅食偏好的研究通常由研究人员进行，研究人员捕捉在花朵处觅食的个体蜜蜂，然后从标本中去除皮质花粉负荷以进行进一步处理和鉴定7，8，9，10。虽然这种方法可以深入了解一个物种或大黄蜂物种的组合如何利用区域7中的资源，但它是时间密集型的，如果没有额外的分子分析来确定觅食蜂11的起源殖民地，就无法辨别蜂巢之间偏好的潜在差异。
对于一些觅食动力学的研究，希望在单个殖民地进行研究;然而，野生大黄蜂巢通常位于地下或地面，因此很难找到12。商业生产的大黄蜂蜂巢为研究人员提供了更多的访问和更好的实验控制，并且从工人身上去除花粉仍然主要通过捕获觅食者返回蜂巢并手动去除其Corbicular花粉负载13，14。从蜜蜂的鞘翅中手工去除花粉是时间密集型的，每小时的花粉产量很低，特别是在蜂巢入口处，返回花粉觅食者的比率可能很低。此外，手动清除蜜蜂的花粉会导致受到干扰的工人被蜇伤。
花粉陷阱已被用于实验性地去除蜜蜂的花粉数十年15;然而，一种从大黄蜂中去除花粉的被动方法尚未开发出来。开发一种机制以从返回的觅食者大黄蜂中去除花粉的主要障碍是大黄蜂群中存在的工蜂大小的巨大变化16。蜜蜂花粉陷阱之所以有效，很大程度上是因为蜜蜂工人的大小变化不大。此外，这些陷阱在安装后只需要轻微的操作，并且不需要牺牲蜜蜂17。这是使用屏幕或塑料表面实现的，当工人返回蜂巢时，这些屏幕或塑料表面会将花粉从工人的后腿上移开。这些诱捕器仅从返回的觅食者身上去除一部分花粉负荷，并且这些诱捕器的各种设计导致花粉收集效率不同。当花粉从蜂腿上取出时，它通过屏幕落入蜜蜂无法进入的收集盆中，因此研究人员可以在对蜂巢产生轻微干扰的情况下将其移除。
本研究的目的是调整用于从蜜蜂蜂巢中收集花粉的技术，并使用3D打印结构将其应用于大黄蜂巢，并在 Bombus huntii的菌落上测试陷阱设计。设计过程遵循的假设是，陷阱的生产成本应该低廉，适应各种大黄蜂物种，对蜜蜂造成的伤害或干扰最小，花粉去除率应超过手工收集的花粉。三维打印技术用途广泛，易于访问，并且是一种具有成本效益的工具，允许研究人员为特定目的复制和修改对象18。这里介绍的技术指示用户建立花粉陷阱，并将它们附着在市售的大黄蜂群落上。这些陷阱不是为野生殖民地设计的。这些陷阱被动地从携带大黄蜂的花粉的后腿上去除皮质花粉负荷，因为它们返回巢箱。

<table border="0" cellpadding="0" cellspacing="0" style="width:461px;" width="462">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:71px;">MakerBot Replicator+</td>
			<td style="width:87px;">MakerBot</td>
			<td style="width:87px;">Model PABH65</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:71px;">MakerBot Tough Material</td>
			<td style="width:87px;">PLA Filament</td>
			<td style="width:87px;"></td>
			<td>various colors</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:71px;">Nest Box</td>
			<td style="width:87px;">Biobest</td>
			<td style="width:87px;"></td>
			<td>Not sold publicly without bee purchase</td>
		</tr>
	</tbody>
</table>

a 3d printed pollen trap for bumble bee (bombus) hive entrances

为了验证大黄蜂觅食花粉的植物来源，必须收集个体以去除其皮质花粉负荷以进行分析。 传统上，这是通过在巢穴入口处或花朵上网捕觅食者来完成的，在冰上冷却蜜蜂，然后用镊子或刷子从corbiculae中去除花粉负载。 这种方法是时间和劳动密集型的，可能会改变正常的觅食行为，并可能导致执行任务的工人发生刺痛事件。 花粉陷阱，例如用于蜜蜂蜂箱的花粉陷阱，通过在通过巢穴入口处的屏幕时将工人腿部的皮质花粉负载移开来收集花粉。 陷阱可以用最少的劳动力从返回的觅食蜂中去除大量花粉，但迄今为止还没有这样的陷阱可用于大黄蜂群落。大黄蜂蜂群内的工蚁大小可能不同，使得入口的大小选择难以使这种机制适应商业饲养的大黄蜂蜂巢。 使用3D打印设计程序，我们创建了一个花粉陷阱，成功地从返回的大黄蜂觅食者的腿上移除了皮质花粉负荷。 这种方法大大减少了研究人员从返回蜂群的大黄蜂觅食者那里收集花粉所需的时间。我们介绍了设计，花粉去除效率测试的结果，并为调查人员建议了修改区域，以使陷阱适应各种大黄蜂物种或巢箱设计。

测试了八种不同的花粉过滤器设计，以确定它们在从返回的大黄蜂工人中去除皮质花粉负荷方面的功效和效率。所有设计都成功地从返回的觅食者身上消除了至少一个皮质花粉负荷。然而，有些被发现会减慢工人离开或进入蜂巢的速度，或者无法去除花粉负荷（表1）。在温室中生长的Phacelia tanacetifolia上的4个实验室饲养的B. huntii Greene菌落上依次测试了带有各种过滤器的?...

Watch this Scientific Journal Video about A 3D Printed Pollen Trap for Bumble Bee Bombus Hive Entrances at JoVE.com

A 3D Printed Pollen Trap for Bumble Bee Bombus Hive Entrances

我们提出了一种非致命和自动化的机制，从返回蜂巢的大黄蜂（Bombus）工人那里收集花粉。包括有关生产、准备、安装和使用设备的说明。通过使用3D打印对象，对设计的修改是及时，有效的，并允许快速周转进行测试。

用于大黄蜂（Bombus）蜂巢入口的3D打印花粉陷阱

To verify the plant sources from which bumble bees forage for pollen, individuals must be collected to remove their corbicular pollen loads for analysis.  ...

a-3d-printed-pollen-trap-for-bumble-bee-bombus-hive-entrances

Research

JoVE Journal

Biology

A 3D Printed Pollen Trap for Bumble Bee (Bombus) Hive Entrances

5.0K 次观看.  Utah State University. 我们提出了一种非致命和自动化的机制，从返回蜂巢的大黄蜂（Bombus）工人那里收集花粉。包括有关生产、准备、安装和使用设备的说明。通过使用3D打印对象，对设计的修改是及时，有效的，并允许快速周转进行测试。

视频: A 3D Printed Pollen Trap for Bumble Bee Bombus Hive Entrances

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

测量细菌细胞的抗弯刚度，使用光学陷阱

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

科研

生物学

Associated Chromosome Trap for Identifying Long-range DNA Interactions

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure. Each chromosome occupies a specific territory, that may change according to stage of development or cell cycle. Gene expression can occur in specialized transcriptional factories where chromatin segments may loop out from various chromosome territories, leading to co-localization of DNA segments which may exist on different chromosomes or far apart on the same chromosome. The Associated Chromosome Trap (ACT) assay provides an effective methodology to identify these long-range DNA associations in an unbiased fashion by extending and modifying the chromosome conformation capture technique. The ACT assay makes it possible for us to investigate mechanisms of transcriptional regulation in trans, and can help explain the relationship of nuclear architecture to gene expression in normal physiology and during disease states.

The associated chromosome trap (ACT) assay is a novel unbiased method for identifying long-range DNA interactions. The characterization of long range DNA interactions will allow us to determine the relationship of nuclear architecture to gene expression in both normal physiology and in diseased states.

确定远距离的DNA相互作用的相关染色体陷阱

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure.  Each chromosome occupies a specific territory, ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

对于在比较营养Beebread由非洲化和欧洲蜜蜂的方法和淋巴蛋白质滴度影响

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment methods. Here we describe methods to measure the quantities of adult bees, brood, and food resources by weighing hives and hive parts, by photographing frames, and by installing hives on scales and with internal sensors. Data from these periodic evaluations are then combined with running average and daily detrended data on hive weight and internal hive temperature. The resulting datasets have been used to detect colony-level effects of imidacloprid applied in a sugar syrup as low as 5 parts per billion. The methods are objective, require little training, and provide permanent records in the form of sensor output and photographs.

Sublethal doses of pesticides may affect colonies in ways that are difficult to detect using only periodic or visual methods. Methods to measure total adult bee mass, brood, and food resources by weighing hives and hive parts, photographing frames, and installing sensors, are provided. Data analysis is also addressed.

蜜蜂致死农药暴露对蜂群水平的影响

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment ...

Collection and Identification of Pollen from Honey Bee Colonies

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate pesticide exposure of bees via pollen. Described herein is an effective pollen-trapping method for collecting corbicular pollen from honey bees returning to their hives. This collection method results in large quantities of corbicular pollen that can be used for research purposes. Honey bees collect pollen from many plant species, but typically visit one species during each collection trip. Therefore, each corbicular pollen pellet predominantly represents one plant species, and each pollen pellet can be described by color. This allows the sorting of samples of corbicular pollen by color to segregate plant sources. Researchers can further classify corbicular pollen by analyzing the morphology of acetolyzed pollen grains for taxonomic identification. These methods are commonly used in studies related to pollinators such as pollination efficiency, pollinator foraging dynamics, diet quality, and diversity. Detailed methodologies are presented for collecting corbicular pollen using pollen traps, sorting pollen by color, and acetolyzing pollen grains. Also presented are results pertaining to the frequency of pellet colors and taxa of corbicular pollen collected from honey bees in five different cropping systems.

We describe methods for collecting corbicular pollen from honey bees as well as protocols for color sorting, acetolysis, and microscope slide preparation of pollen for taxonomic identification. In addition, we present pellet color and taxonomic diversity of corbicular pollen collected from five cropping systems using pollen traps.

蜜蜂群落花粉的采集和鉴定

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate ...

Assessing Agrochemical Risk to Mated Honey Bee Queens

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not accurately capture the effects of agrochemical exposure on honey bee queens. As the sole producer of fertilized eggs inside a honeybee colony, the queen is arguably the most important single member of a functioning colony unit. Therefore, understanding how agrochemicals affect queen health and productivity should be considered a critical aspect of pesticide risk assessment. Here, an adapted method is presented to expose honey bee queens and worker queen attendants to agrochemical stressors administered through a worker diet, followed by tracking egg production in the laboratory and assessing first instar eclosion using a specialized cage, referred to as a Queen Monitoring Cage. To illustrate the method&#39;s intended use, results of an experiment in which worker queen attendants were fed diet containing sublethal doses of imidacloprid and effects on queens were monitored are described.

This protocol was developed to enhance the understanding of how agrochemicals affect honey bee (Apis mellifera) reproduction by establishing methods to expose honey bee queens and their worker caretakers to agrochemicals in a controlled, laboratory setting and carefully monitoring their relevant responses.

评估交配蜜蜂女王的农用化学品风险

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not ...

An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of conditioned media remains time-consuming and far from physiological and pathological conditions. Although a few models of co-culture are commercially available, they remain restricted to specific assays and are mostly for two types of cells. 
Here, 3D-printed inserts are used that are compatible with numerous functional assays. The insert allows the separation of one well of a 6-well plate into four compartments. A wide range of combinations can be set. Moreover, windows are designed in each wall of the compartments so that potential intercellular communication between every compartment is possible in the culture medium in a volume-dependent manner. For example, paracrine intercellular communication can be studied between four cell types in monolayer, in 3D (spheroids), or by combining both. In addition, a mix of different cell types can be seeded in the same compartment in 2D or 3D (organoids) format. The absence of a bottom in the 3D-printed inserts allows the usual culture conditions on the plate, possible coating on the plate containing the insert, and direct visualization by optical microscopy. The multiple compartments provide the possibility to collect different cell types independently or to use, in each compartment, different reagents for RNA or protein extraction. In this study, a detailed methodology is provided to use the new 3D-printed insert as a co-culture system. To demonstrate several capacities of this flexible and simple model, previously published functional assays of cell communication were performed in the new 3D-printed inserts and were demonstrated to be reproducible. The 3D-printed inserts and the conventional cell culture using conditioned media led to similar results. In conclusion, the 3D-printed insert is a simple device that can be adapted to numerous models of co-cultures with adherent cell types.

In this paper, a newly designed 3D-printed insert is presented as a model of co-culture and validated through the study of the paracrine intercellular communication between endothelial cells and keratinocytes.

创新的 3D 打印插入物，旨在实现简单的 2D 和 3D 细胞培养

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of ...

一种测量拟南芥花粉水合作用曲线的改进方法

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

作者聚焦：拟南芥的高分辨率、单粒、体内花粉水合生物测定  

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

拟南芥的高分辨率、单粒、体内花粉水合生物测定

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...