该研究由国家农业和食品技术研究所（RFP2015-00015-00和RTA2017-00003-00）资助;Gobierno de Aragón——欧洲社会基金，欧洲联盟（Grupo Consolidado A12-17R）和Jente de Extremadura——Fondo Europeo de desarrollo Regional （FEDER），Regional de Investigación（IB16181），Grupo de Investigación（AGA001，GR18196）。B.I. Guerrero得到了墨西哥国家科学与技术委员会（CONACYT，471839）的奖学金支持。

1. 田间手工授粉
<ol><li>
花粉提取
	<ol>
<li>为了获得花粉，在阶段D24收集花蕾，根据BBCH量表25，26上的第57阶段。 注意:与其他 李 子相比，日本李子需要更多的花蕾，因为它们的花药产生的花粉较少。</li>
		<li>使用塑料网（2 mm x 2 mm孔径）取出花药，并将其在室温下放在纸上24小时，直到花药开裂。</li>
		<li>通过细网（0.26 mm x 0.26 mm孔径）筛选花粉颗粒，并将它们保存在10 mL玻璃管中，盖子在4°C直至使用。</li>
	</ol>
</li>
	<li>
阉割花的授粉
	<ol>
<li>当10%-20%的花朵开放时，选择并标记几个分支。去除开放的花朵和年轻的花蕾，只留下阶段D24的花蕾，根据BBCH等级25，26的第57阶段。</li>
		<li>每次使用指甲或镊子去除800至1，000朵花蕾的花瓣，萼片和雄蕊。</li>
		<li>阉割后24小时，用精细的画笔帮助手授粉雌蕊。一些分支含有一半的雌蕊花粉，花粉为同一品种，另一半含有来自其他栽培品种的相容花粉作为对照。注意不要用其他品种的花粉颗粒污染指尖或画笔。</li>
		<li>记录每周的花朵和发育中的果实计数，以表征果实掉落模式并量化每次授粉处理中的最终果实。</li>
	</ol>
</li>
	<li>
田间补充授粉
	<ol>
<li>在第一朵花开前几天，将选定的树木围在0.8毫米网状笼中，以避免授粉昆虫的到来。</li>
		<li>当10%-20%的花朵开放时，每次授粉处理选择并标记几个分支，每次处理留下1，000-1，500朵花。</li>
		<li>第二天，当花朵开放时，在画笔的帮助下，用相应的花粉（来自同一品种的花粉用于自花授粉，来自其他兼容品种的花粉作为交叉授粉对照）授粉。</li>
		<li>每隔一天授粉，直到所有花朵都开开。</li>
		<li>记录从花开到收获的每周花卉和发育果实的数量，以表征果实掉落模式并量化每次授粉处理中的最终果实集。</li>
	</ol>
</li>
</ol>2. 实验室中的手工授粉
<ol><li>根据BBCH等级25，26的第57阶段，在D阶段24收集50-100朵花。</li>
	<li>在实验室中，每次处理阉割30朵花（自花授粉和异花授粉）。 注意:阉割应小心进行，以避免对雌蕊造成任何损害。</li>
	<li>在水下对每个花梗的基部进行新鲜切割，然后将其放在一块湿花店泡沫上（每次授粉处理一块泡沫）。</li>
	<li>24小时后，使用精细的画笔用先前收集的花粉手工授粉每个雌蕊（见第1.1节）。用来自同一品种的花粉授粉一组雌蕊，另一组用来自兼容品种的花粉作为对照。</li>
	<li>在室温下授粉后72小时离开授粉的雌蕊。花卉泡沫应不断用水润湿。</li>
	<li>将雌蕊固定在乙醇/乙酸（3:1）的固定溶液中，在4°C下至少24小时。 用75%乙醇代替固定剂。样品可以在4°C下保存在该溶液中，直到使用27。 注:确保样品完全浸没在溶液中。</li>
</ol>3. 微观观察
<ol><li>
体外花粉萌发的评估
	<ol>
<li>为了制备花粉发芽培养基，将25克蔗糖溶解在250毫升蒸馏水中，然后加入0.075克硝酸钙[Ca（NO 3）2]和0.075克硼酸（H3BO3）。</li>
		<li>向溶液中加入2克琼脂并混合直至完全溶解28。</li>
		<li>为了对培养基进行灭菌，请在120°C下高压灭菌20分钟。冷却培养基，在固化之前，将每个无菌培养皿（55 mm x 12 mm）3 mL分配到无菌层流罩中。介质固化后，将包裹在铝箔中的培养皿保存在4°C直至使用。</li>
		<li>将先前用作花粉供体的每个品种的花粉在两个培养皿中对照授粉，并在25°C下孵育24小时。 注意:接种的培养基可以在使用后立即用显微镜观察或储存在-20°C直至使用。为此，应在显微镜观察前24小时将培养皿从冰箱更换为冰箱。</li>
		<li>为了观察花粉颗粒，准备1%（v / v）苯胺蓝溶液，使胼胼体染色。首先，通过将7.97g K3PO4溶解在1，000 mL蒸馏水中来制备0.1 N磷酸钾三元酸钾（K3PO4） 溶液。为了阐述1%（v / v）苯胺蓝溶液，将1mL苯胺蓝溶解在100mL 0.1 N K3PO4中。</li>
		<li>向每个培养皿板加入2-3滴苯胺蓝溶液，并在5分钟后使用兴奋剂过滤器BP340-390和屏障过滤器LP425在紫外落射荧光显微镜下观察。将活的和不可存活的花粉粒计算在每盘的三块田中，每块田含有100-200粒花粉粒，在每个品种的两个培养皿中。</li>
	</ol>
</li>
	<li>
花粉管生长
	<ol>
<li>用蒸馏水冲洗固定的雌蕊三次（每个1小时，总共3小时），并在4°C下转移到5%（w / v）亚硫酸钠（Na2SO3）24小时。为了制备该溶液，将5克亚硫酸钠溶解在100毫升蒸馏水中。</li>
		<li>将雌蕊在120°C下在5%（w / v）亚硫酸钠中高压灭菌8分钟以软化组织。</li>
		<li>南瓜软化雌蕊在载玻片上的盖玻片下滴1%（v / v）苯胺蓝溶液以染色胼脖。</li>
		<li>使用励磁剂过滤器BP340-390和屏障过滤器LP425在显微镜下观察花粉管沿样式的生长。</li>
	</ol>
</li>
</ol>4. 确定不相容关系
<ol><li>从叶子中提取DNA<ol>
<li>为了提取DNA，请在田间收集每个品种的3-4片嫩叶，最好在春季。 注意:DNA也可以从成熟叶中提取，但幼叶中的DNA含有较少的酚类化合物。</li>
		<li>使用商业试剂盒分离DNA，并遵循提供的实验方案试剂盒（见 材料表）。</li>
		<li>在紫外可见分光光度计中定量DNA浓度并评估每个样品在260nm处的DNA质量。将DNA浓度调节至10 ng / μL。</li>
	</ol>
</li>
	<li>片段扩增的 PCR 条件<ol>
<li>标记 0.2 mL PCR 管和盖。</li>
		<li>根据 表1 制备PCR试剂，并让它们在冰上解冻。</li>
		<li>根据反应次数加上10%的过量，在1.5mL微管中设置每对引物的预混液体积，考虑每次反应的体积为16μL。按照 表1 中的顺序加入试剂并充分混合。</li>
		<li>将16μL预混液等分到每个含有4μLDNA模板或4μL灭菌蒸馏水的0.2mL PCR管中作为阴性对照（C-）。使用已知基因型的品种的DNA作为阳性对照。轻轻混合，用盖子关闭反应管，并以2，000×g 离心30秒，以收集反应管底部的整个体积。</li>
		<li>将反应管置于热循环器中，并使用以下温度曲线设置PCR程序:在94°C下初始步骤3分钟，在94°C下循环35次1分钟，在56°C下循环1分钟，在72°C下3分钟，最后一步在72°C下7分钟20。</li>
	</ol>
</li>
	<li>电泳和片段大小的估计<ol>
<li>要制备1.7%（w / v）琼脂糖迷你凝胶，将0.68g琼脂糖和40mL 1x TBE缓冲液溶解到100 mL锥形瓶中。通过以30秒的间隔在600 W的微波炉中加热溶液来熔化溶液，以避免沸腾。</li>
		<li>让溶液留在工作台上冷却，然后加入3.5μL核酸染色溶液。</li>
		<li>将凝胶托盘放入铸造支架中，然后将选定的梳子放入凝胶模具中。确保每个PCR产物和DNA分子量标准品有足够的孔。</li>
		<li>将琼脂糖溶液倒入凝胶模具中，使其冷却直至聚合。取出聚合凝胶的梳子。将凝胶置于含有足够1x TBE缓冲液的水平电泳系统中以覆盖凝胶表面。</li>
		<li>用2μL的DNA分子量分子量标准品（1 kb DNA分子量标准品）加载第一个和最后一个孔。将PCR的每种产物的3μL加载到其他孔中。关闭腔室，打开电源，然后将凝胶在100 V下运行30分钟。</li>
		<li>使用凝胶记录系统在紫外光下观察凝胶。使用DNA分子量分子量标准品确定扩增片段的大小，并将其与阳性对照进行比较，以鉴定相应的等位基因。</li>
	</ol>
</li>
</ol>5. 监测花期
<ol><li>监测不同品种不同树种在不同年份开花时的物候。确定从第一朵（约5%）到最后一朵开花（约95%）的开花期的长度。当至少50%的花朵处于F24阶段时，根据BBCH等级25，26的第65阶段，则考虑盛开。</li>
	<li>为了比较相互兼容的品种的开花日期并确定每年开花时间的开花时间，请用几年的数据制定开花时间日历。</li>
</ol>

作者没有什么可透露的。

本文描述的日本李子品种授粉要求的方法要求通过在田间或实验室中控制授粉来确定每个品种的自（不）相容性，并随后用荧光显微镜观察花粉管的生长。不相容关系是通过分子基因分型表征 S-等位基因建立的。最后，通过监测物候学进行授粉媒介的选择，以检测每年开花时重合的品种。
由于大量授粉要求未知的新品种，并且其中大多数是自相容的，因此在日本新的?...

日本李子（Prunus salicina Lindl.）原产于中国1.在19世纪，这种作物从日本引入美国，在那里它与其他北美二倍体李子2杂交。在20世纪，其中一些杂交种被传播到世界各地的温带地区。如今，术语"日本李子"是指广泛的种间杂交种，这些杂交种来自原始水杨假单胞菌与多达15个其他二倍体李子属之间的杂交种。
与蔷薇科的其他物种一样，日本李子表现出配子体自不相容性（GSI），其由包含多个等位基因的单个高度多态性的S位点控制6。S-位点包含两个基因，它们编码在雌蕊中表达的核糖核酸酶（S-RNase）和在花粉粒7中表达的F-box蛋白（SFB）。在自不相容反应中，当花粉粒（单倍体）中表达的S-等位基因与雌蕊（二倍体）中表达的两个中的一个相同时，由于S-RNase8的作用使花粉管RNA降解，跨样式的花粉管的生长被阻止。由于该过程阻止了胚珠中雌性配子体的受精，因此GSI促进了品种之间的异交。
虽然一些日本李子品种是自相容的，但目前种植的大多数品种都是自相容的，需要来自互相容捐赠者的花粉来施肥3。在杏仁9、杏10、11、12和甜樱桃13等李属的核果品种中，可以通过不同的方法确定品种的授粉需求。自（不）相容性可以通过在田间自花授粉和随后监测坐果来确定，或者通过在实验室的受控条件下进行半体内自花授粉和在显微镜下观察花粉管来确定14，15，16，17，18.品种间的不相容关系可以通过在田间或实验室使用潜在传粉品种的花粉进行异花授粉来确定，并通过PCR分析鉴定每个品种的S-等位基因14，15，16，19，20，21，22.在甜樱桃或杏仁等物种中，也可以通过鉴定与自相容性相关的特定S等位基因来评估自身（in）相容性，如甜樱桃13中的S4'或杏仁23中的Sf。 
来自主要生产国的几个李子育种计划正在释放一些新的品种2，14，其中许多具有未知的授粉要求。本文介绍了日本李子型杂交种授粉需求测定方法。自（不）相容性由田间和实验室的自花授粉决定，然后在荧光显微镜下观察花粉管。授粉品种的选择结合了通过PCR分析鉴定S基因型和监测田间开花时间。

<table>
	<tbody>
		<tr>
			<td>Acetic Acid Glacial</td>
			<td>Panreac</td>
			<td>131008.1611</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>iNtRON Biotechnology</td>
			<td>25999</td>
			<td></td>
		</tr>
		<tr>
			<td>Aniline blue</td>
			<td>Difco</td>
			<td>8504-88</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric Acid (H3BO4)</td>
			<td>Panreac</td>
			<td>131015.1210</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Nitrate 4-hydrate (Ca(NO3)2&#183;4H2O)</td>
			<td>Panreac</td>
			<td>131231.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverglass</td>
			<td>Deltalab</td>
			<td>D102460</td>
			<td>24 mm x 60 mm</td>
		</tr>
		<tr>
			<td>Digital Camera</td>
			<td>Imaging Developmet Systems</td>
			<td>UI-1490SE</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Camera Software Suite</td>
			<td>Imaging Developmet Systems</td>
			<td>4.93.0.</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Oligos</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Mix, 10 mM each</td>
			<td>ThermoSischer Scientific</td>
			<td>R0193</td>
			<td></td>
		</tr>
		<tr>
			<td>DreamTaq Green DNA polymerase</td>
			<td>ThermoFisher Scientific</td>
			<td>EP0713</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96&#176;</td>
			<td>VWR-Chemicals</td>
			<td>83804.360</td>
			<td></td>
		</tr>
		<tr>
			<td>1Kb DNA Ladder (U.S. Patent No. 4.403.036) (500pb-12Kb)</td>
			<td>Invitrogen</td>
			<td>15615-016</td>
			<td>Size: 250&#181;g; Conc: 1.0 &#181;g/&#181;l</td>
		</tr>
		<tr>
			<td>Gel Documentation System</td>
			<td>Bio-Rad</td>
			<td>1708195</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand Counter</td>
			<td>Tamaco</td>
			<td>TM-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Lab Software</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>Image Analyse System for Gel Documentation System</td>
		</tr>
		<tr>
			<td>MetaPhor Agarose</td>
			<td>Lonza</td>
			<td>50180</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge 5415 R</td>
			<td>Eppendorf</td>
			<td>Z605212</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope with UV epiflurescence</td>
			<td>Leica</td>
			<td>DM2500</td>
			<td>Exciter filter BP340-390, Barrier filter LP425</td>
		</tr>
		<tr>
			<td>Microslides</td>
			<td>Deltalab</td>
			<td>D100004</td>
			<td>26 mm x 76 mm</td>
		</tr>
		<tr>
			<td>Mini Electrophoresis System</td>
			<td>Fisherbrand</td>
			<td>14955170</td>
			<td></td>
		</tr>
		<tr>
			<td>Minicentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>15334204</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 1000 Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Deltalab</td>
			<td>200201</td>
			<td>55 mm x 14 mm</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Tribasic (K3PO4&#183;1.5H2O)</td>
			<td>Panreac</td>
			<td>141513</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer forward &#39;Pru C2&#39;</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer forward Pru T2&#39;</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer reverse &#39;PCER&#39;</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RedSafe Nucleic Acid Staining Solution</td>
			<td>iNtRON Biotechnology</td>
			<td>21141</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharose</td>
			<td>Panreac</td>
			<td>131621.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium sulphite anhydrous (Na2SO3)</td>
			<td>Panreac</td>
			<td>131717.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Speedtools plant DNA extraction Kit</td>
			<td>Biotools</td>
			<td>21272</td>
			<td></td>
		</tr>
		<tr>
			<td>TBE Buffer (10X)</td>
			<td>Panreac</td>
			<td>A0972,5000PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler T100</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer comfort</td>
			<td>Eppendorf</td>
			<td>T1317</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Autoclave Presoclave II</td>
			<td>JP Selecta</td>
			<td>4001725</td>
			<td></td>
		</tr>
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			<td>Vortex</td>
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establishing pollination requirements in japanese plum by phenological monitoring, hand pollinations, fluorescence microscopy and molecular genotyping

通常种植的日本李子品种是种间杂交种，来源于原始 水杨 李与其他 李 子物种之间的杂交。大多数杂交种表现出配子体自不相容性，其由包含多个等位基因的单个高度多态性的 S-位点控制。大多数栽培的杂交种是自不相容的，需要来自相容供体的花粉来施肥。由于大量新品种的授粉需求未知，在日本李子中建立授粉要求变得越来越重要。本文介绍了日本李子型杂交种授粉需求测定方法。自（不）相容性是通过在田间和实验室中手工授粉来确定的，然后用荧光显微镜监测花粉管伸长率，以及监测果实在田间的成熟度。通过将PCR分析对 S基因型的鉴定与田间开花时间监测相结合，对授粉品种的选择进行评估。了解品种的授粉要求有助于选择品种用于新果园的设计，并允许早期发现与已建立果园授粉不足相关的生产力问题。

每个日本李子花蕾包含一个花序，有1-3朵花。与其他核果物种一样，每朵花由四个漩涡组成:腕骨，雄蕊，花瓣和萼片，它们在花的底部融合形成一个杯子。花的结构比其他核果小，雌蕊短而脆弱，被含有少量花粉颗粒的雄蕊包围。在盛开时，每个花序的花朵在短茎上分开，显示白色花瓣在花开前几天形成一个被绿色萼片包围的气球（D期，57 BBCH）（图1A）。花在花开时完全?...

Watch this Scientific Journal Video about Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping at JoVE

Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping

本文介绍了一种测定日本李子型杂交种授粉需求的方法，该方法将田间和实验室授粉以及荧光显微镜下花粉管的观察与通过PCR鉴定 S基因型和监测开花以选择授粉者相结合。

通过物候监测、手授粉、荧光显微镜和分子基因分型确定日本李子的授粉要求

The Japanese plum cultivars commonly grown are interspecific hybrids derived from crosses between the original Prunus salicina with other Prunus species. ...

establishing-pollination-requirements-japanese-plum-phenological

Research

JoVE Journal

Developmental Biology

3.0K 次观看.  Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA). 本文介绍了一种测定日本李子型杂交种授粉需求的方法，该方法将田间和实验室授粉以及荧光显微镜下花粉管的观察与通过PCR鉴定 S基因型和监测开花以选择授粉者相结合。

视频: Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

科研

发育生物学

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 &#176;C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

日本鹌鹑胚胎的卵黄囊膜主内胚上皮细胞培养

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

光片为主的生活的荧光显微镜或固定和染色<em&gt;赤拟谷盗</em&gt;胚胎

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye. Subsequent cell tracking and quantification of the integrated fluorescence intensity over time results in an exponential decay curve for each tracked cell, allowing for determining protein degradation rates in single cells by curve fitting. This method provides an estimate for the heterogeneity of half-lives in a population of cultured cells, which cannot easily be assessed by other methods. The approach presented here is applicable to any type of cultured adherent cells expressing a protein of interest fused to a SNAP-tag. Here we use mouse embryonic stem (ES) cells grown on E-cadherin-coated cell culture plates to illustrate how single cell degradation rates of proteins with a broad range of half-lives can be determined.

This protocol describes a method to determine protein half-lives in single living adherent cells, using pulse labeling and fluorescence time-lapse imaging of SNAP-tag fusion proteins.

粘附细胞培养的时间推移荧光显微镜对蛋白质降解率的单细胞定量研究

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.