我们感谢 Jochen Zeil, 保罗库珀和比尔吉德格雷纳分享他们的昆虫解剖学知识, 并展示了我们在这里描述的几种技术。我们感谢在雁努高级显微镜中心的有才华和支持性的工作人员和 MQU 的显微镜组。这项工作获得了澳大利亚研究理事会 (DE120100019、FT140100221、DP150101172) 的研究生奖学金和助学金的支持。

1. 试样制备
注意: 有必要首先了解复眼的相对位置和单眼, 并在头部。这可以通过获取头部背景色的图像来实现。为此, 我们建议处理样品的显微或使用 SEM 技术。下面是这两个过程所涉及的步骤。
<ol><li>
标本采集
	<ol>
<li>将标本直接收集到70% 乙醇中。尽可能收集不同的种姓。</li>
		<li>标记带有时间、日期和地点的标本以及任何其他相关观察 (例如, 在觅食时收集, 交配聚集, 树枝内嵌套,等等)</li>
		<li>收集足够多的标本, 在每次治疗中有多个复制。</li>
	</ol>
</li>
	<li>
显微和 Z 堆叠
	<ol>
<li>空气干燥的标本, 并安装在三角形点卡, 使用水溶性胶水, 然后在昆虫引脚。有关详细信息, 请参见参考18。</li>
		<li>图像使用高放大显微镜与 Z 步进电机和彩色相机。</li>
		<li>使用扩散器对标本进行均匀照明。</li>
		<li>在不同的焦点平面上捕获图像, 并以无损的文件格式 (如 tiff) 保存图像, 并使用商业可用的软件将其集中在堆栈上。</li>
	</ol>
</li>
	<li>
扫描电子显微 注: 用乙醇彻底清洁所有工具和工作表面, 以避免灰尘和其他微粒污染试样。<ol>
<li>在酒精中的脱水样品隔夜和空气干燥的培养皿。</li>
		<li>用锋利的刀片将头部与身体的其余部分分开。</li>
		<li>使用导电碳带或标签将头部安装在所需的视角 (如, 背朝上)。将碳带切成薄片, 并将其折叠以支撑头部胶囊。</li>
		<li>使用溅射涂布机将黄金应用于试样表面2分钟, 在20毫安的旋转阶段。时间和电流可能需要根据仪器调整。</li>
		<li>用新鲜碳带或标签将样品转移到新的铝存根上。 注: 未涂布的碳将提供一个黑色的背景, 但转移标本可能会损害金涂层。</li>
		<li>用解剖显微镜检查样品的方位是否正确, 并根据需要调整一双细钳或类似的工具。注意不要刮伤金镀层;尽可能少的处理。</li>
		<li>将试样加载到扫描电镜存根架上, 记下存根和试样相对的位置。 注: 一些中小型公司配备了舞台摄像头, 但许多不是, 它可能很难找到高放大率的小标本。</li>
		<li>图像的标本。使用低加速电压, 以避免充电和小孔径的良好景深。 注意: 在与专门使用特定仪器的技术人员协商时, 设置最优化。</li>
	</ol>
</li>
</ol>2. 量化小平面数和直径
<ol><li>
角膜复制品
	<ol>
<li>使用乙醇保存的蚂蚁或为此目的安装的脚 (步骤 1.1.1)。</li>
		<li>在昆虫针上或橡皮泥上安装动物。如果头部相对较大, 其余的身体部分可以被移除。</li>
		<li>使用昆虫针或细牙签拾起一小滴快速干燥无色指甲油并且迅速地传播它在眼睛。确保针脚不会划伤眼睛。指甲油应该覆盖整个眼睛和一些周围的头部胶囊。 注意: 重要的是指甲油的厚度相对均匀的眼睛。</li>
		<li>把指甲油放在室温下。</li>
		<li>一旦它完全设置, 使用一个细的昆虫别针轻轻地抬起副本从头部胶囊周围的眼睛。</li>
		<li>使用一副干净的镊子来举起复制品, 抓住覆盖头部而不是眼睛的部分。</li>
		<li>确保眼睛方向的意识: 前、后、背和腹区域。</li>
		<li>将复制副本放在玻璃幻灯片上。使用刀片修剪复制件, 小心地删除多余的材料周围的眼睛。用一根针或一副钳子来防止复制品移动。</li>
		<li>如果眼睛是非常凸的, 使用剃刀刀片, 使3-4 良好的部分径向切口周围的边缘, 以帮助 ' 拼合 ' 的副本。如果眼睛是相对平坦的, 就没有必要做这样的切口。</li>
		<li>将封面轻轻地放在眼睛副本上, 以确保复制副本的方向是已知的。不要施加压力, 因为这样可以消除指甲油上的角膜印象。</li>
		<li>密封的片使用很少指甲油在四个角落。如果指甲油在盖板和玻片之间流动, 就会损坏复制品。</li>
		<li>在复合显微镜上成像幻灯片。 注意: 如果只有一些方面是焦点, 那么这表明眼睛副本是不够扁平。丢弃并从步骤2.1.3 重新开始。</li>
		<li>将图像导入到自由可用的程序中, 如 ImageJ/斐济, 可以测量每个复眼的复眼数和大小。 注意: 此方法也可用于准备单眼的副本。因为它是一个单一的镜头, 我们建议保持所有的单眼在一个副本。</li>
	</ol>
</li>
</ol>3. 分析眼睛的结构
注意: 在大多数情况下, 研究眼睛的解剖需要两种互补的 LM 和 TEM 技术。初始处理阶段需要 LM 和 TEM 的相似技术。区别从剖切阶段起。处理样品需要使用危险化学品, 必须小心处理, 并负责任地丢弃。使用个人防护设备, 在通风柜中工作, 始终阅读安全数据表 (SDS), 并在启动前进行风险评估。
<ol><li>解剖
	<ol>
<li>麻醉试样通过冷却或暴露于气体 CO2。<ol>
<li>CO2麻醉非常快 (一般低于1分钟), 应注意避免过度曝光, 因为这可能导致标本死亡。如果使用干冰颗粒 (固态 CO2) 避免与标本直接接触, 因为这会导致冷灼伤。</li>
			<li>冷麻醉较慢;4° c 是充足的, 并且更冷的温度不推荐。为该物种建立适当的冷却时间。大或耐寒蚂蚁如公牛蚂蚁可能需要 > 10 分钟才能完全固定, 而较小的物种可能只需要1-2 分钟. 过度冷却会杀死标本 (避免与冰直接接触)。标本最好是存放在小, 泡沫塞, 塑料容器和放置在一个冰箱, 他们可以观察, 而不是在一个电动冰箱或冰柜。</li>
		</ol>
</li>
		<li>将标本放置在培养皿上, 并在解剖显微镜下进行调整。快速工作是重要的, 以保持麻醉和避免退化的组织一旦作出切口 (这可能发生在几秒钟内)。</li>
		<li>用镊子取下触角。如果与蛰虫一起工作, 最好先切除胃, 以免被蛰伤。</li>
		<li>用锋利的刀片除去嘴部;钳可以用来保存标本。切开眼睛的前部 (大标本) 或尽可能靠近眼睛 (小标本), 而不拉扯大脑, 因为这可能会撕裂视网膜。</li>
		<li>准备打开头部胶囊角度的标本, 使第一个切口是指着;这可能是在解剖显微镜下进行, 同时持有标本在钳或在视觉控制, 而持有标本之间的拇指和前指。</li>
		<li>通过头部进行横切口切除头部的腹部;部分腹侧眼可移除大种, 以改善固定和浸润。此时头部仍应附着在身体上。</li>
		<li>通过在复眼后方进行冠状切口, 从身体中切断头部囊。</li>
		<li>将经解剖的头胶囊与复合眼放在冰冷定影液中: 2.5% 戊二醛和2% 甲醛磷酸盐缓冲剂 (pH 7.2-7.5)。 注意: 定影液具有腐蚀性和毒性;穿戴适当的保护和工作在通风罩。 注意: 重要的是要迅速的工作, 以阻止神经组织变性。解剖应完成在2分钟或更少 (有效的解剖可能需要一些实践)。 注意: 如果眼睛需要适应明亮或黑暗的条件, 那么首先将动物暴露在需要的光照条件下几个小时。在各自的光照条件下进行解剖。解剖可以在红色的灯光下进行, 以模拟黑暗。</li>
	</ol></li>
	<li>试样加工
	<ol>
<li>将样品保存在室温下, 在一个轨道振动筛上运动, 2 h. 大型标本可能需要更长的固定时间。</li>
		<li>取下定影液, 并妥善处理。在室温磷酸缓冲液中洗涤样品 (3 次, 每瓶5分钟)。 注: 磷酸缓冲液包括8克氯化钠, 0.2 克氯化钾, 1.44克 Na2HPO4, 0.244 g 在蒸馏 H2O (pH 4) 的1升2中7.2。</li>
		<li>卸下磷酸盐缓冲并添加 2% OsO4.将试样罐放在通风罩上1-2 小时的振动筛上。这是一个 post-fixation 的步骤来修复脂肪, 也为 TEM 提供对比。 注: 锇固定时间受标本大小影响;作为粗略的经验法则, 每 1 mm3计算 1 h 固定。</li>
		<li>除去锇溶液并适当地处理它。将样品在室温缓冲液中洗涤 (3 次, 每次5分钟) 放在振动筛上。</li>
		<li>将试样放在乙醇或丙酮浓度增加的情况下脱水;例如, 50、70、80和95% 分别为10分钟和最后 100% (2 次, 每15分钟)。将试样放在溶液变化的振动筛上。 注: 如有必要, 标本可贮存于70% 乙醇过夜。</li>
		<li>排出乙醇, 加入100% 丙酮。在振动筛上留20分钟 (如果在丙酮中脱水, 请跳过这一步)。用新鲜丙酮代替, 再留出20分钟。</li>
		<li>用树脂将以下丙酮的比率渗透到组织中: 2:1 (3 小时)、1:1 (隔夜)、1:2 (4 h) 和纯树脂 (隔夜)。每一个步骤都在通风柜内的振动筛上留下标本, 并将容器盖在最后两步。 注: 树脂太粘, 不能排干, 所以在每一步都必须将试样移到新的一次性容器中。</li>
		<li>准备块以装载示例。块可以是定制的切割丙烯酸玻璃成小矩形块 (1.5 x 0.5 x 0.3 厘米)。块也可以通过浇注环氧树脂 (有许多商业套件可用) 到一个硅模具, 然后治愈它在12-14 小时的烤箱在60° c。 注意: 未 (液体) 树脂是致癌的, 应回到烤箱, 直到完全硬化。</li>
		<li>将块垂直放置在模具中。小心地从液体树脂中抽取样品, 允许多余的树脂流失, 并将试样放在块的顶部。少量的额外的树脂可以用来把标本绑在木块上。</li>
		<li>标记该块。打印纸张标签并将其嵌入块中或将其附加到块面上。</li>
		<li>在60° c 的温度下, 将模具与预埋块放在烤箱中12-14 小时。</li>
		<li>将标本块存放在一个干净的信封里。这样可以在几个月到几年内以这种方式存储。</li>
		<li>将使用过的容器、脏手套和其他受污染的设备放在油烟罩内, 使丙酮完全蒸发 (最低12小时)。</li>
		<li>在丢弃一次性设备或刮除镊子等其他物品的树脂之前, 在烤箱中固化树脂。</li>
	</ol></li>
	<li>切片
	<ol>
<li>观察解剖显微镜下的块, 确保标本定位适合于切片平面。</li>
		<li>如果方向是不适当的, 使用珠宝商的锯切出标本和重新使用集树脂和新鲜树脂的碎片, 以重新标本。头部也可分成两半, 分开两只眼睛。在进行前再次固化树脂。</li>
		<li>在可拆卸切片卡盘上安装块。取出卡盘并放置在支架上。</li>
		<li>在解剖显微镜下用刀片修剪树脂块。 注意: 当卡盘安装在切片臂上时, 不要这样做, 因为它可以摇晃手臂并损坏轴承。</li>
		<li>将卡盘重新装入切片臂上, 并对试样进行角度。</li>
		<li>在适当的角度安装刀架 (0 °为玻璃刀, 请参阅制造商关于金刚石刀具的说明)。 注: 玻璃刀可以便宜地用玻璃刀具制造, 但必须定期更换, 因为他们失去了他们的优势;高品质的金刚石刀可以购买, 但昂贵, 需要特别照顾, 不适合初学者。</li>
		<li>用装有过滤器的注射器 (0.45 µm 孔大小), 用蒸馏水填充刀船。</li>
		<li>把船填满, 直到水位到达刀的边缘;半月板可能是凸在其他地区的船。</li>
		<li>把船排出, 直到半月板非常轻微凹, 但仍然达到刀的边缘。水的水平可以在任何一点上调整, 但总是远离刀的边缘。</li>
		<li>小心地把刀对准标本, 并将木块与刀对齐。这是最好的完成缓慢, 定期检查靠近的刀通过目镜和从侧面。 注: 如仪器不同, 请检查仪器手册中的具体说明。</li>
		<li>设置切片的截面厚度。选择正确的厚度将取决于样本大小, 感兴趣的地区和使用的刀的种类。</li>
		<li>如果使用玻璃刀选择更高的设置 (例如, 4 µm), 如果在到达感兴趣的区域之前必须将大量材料切掉。如果使用的是金刚石刀或样本非常小, 1-2 µm 可能更合适。</li>
		<li>开始 "切割" (启动切片轮) 时, 刀是接近, 但尚未切割到标本, 以执行最后一部分的方法。部分应该开始出现在几个轮换;如果没有, 停下来, 非常小心地把刀子拉近一点。</li>
		<li>当接近感兴趣的区域时, 调整剖面收集厚度 (1 µm 薄节)。</li>
		<li>使用睫毛工具收集任何部分。 注: 睫毛工具, 可以用睫毛安装在薄棒指甲油。</li>
		<li>如果必须移除大量的材料, 则可以通过移除该刀并将其与水一起冲洗, 从而使这些部分堆积并移除成批. 如果使用一把玻璃刀, 这可能是一个适当的时间来改变到一个新的部分的刀或一刀。</li>
		<li>使用巴斯德吸管或理想情况下, 在幻灯片上放置一系列的蒸馏水小水滴, 过滤器配备注射器。</li>
		<li>小心地将收集到的部分放在睫毛上的水滴上。</li>
		<li>收集像这样的部分, 直到它是适当的检查切片的深度。 注意: 虽然它可能是乏味的, 它总是最好经常检查。</li>
		<li>将幻灯片放在板设置为60° c 的位置。允许所有的水蒸发和部分坚持幻灯片。</li>
		<li>以甲苯胺蓝为 10-60 s 的染料切片 (染色时间会随切片厚度而变化)。用装有过滤器的注射器 (如上所述) 分配染料。</li>
		<li>在幻灯片的一端放置一滴染料, 并在不触及或刮掉部分的情况下, 用针头的侧面展开。将幻灯片放置在板上, 以近似10-20s。</li>
		<li>用蒸馏水在洗瓶中喷洒, 然后把它放在热板上晾干。</li>
		<li>检查复合显微镜下的图像。</li>
		<li>重复, 直到到达感兴趣的区域。</li>
		<li>对于超薄切片: 设置40-60 纳米之间的切割厚度以收集 TEM 剖面。</li>
		<li>切割约3-5 节和检查厚度使用干涉颜色图。剖面应反射浅灰色时, 从一个角度看。 注: 氯仿烟雾可 pipetted 超过部分, 以放松任何折痕。这是最相关的经验丰富的用户, 初学者不必担心。太多的氯仿释放太靠近剖面可能损坏部分。</li>
		<li>拿起一个 Formvar 涂层, 铜, 槽格与钳与 Formvar 侧拿起。小心不要刺穿 Formvar 涂层。</li>
		<li>将栅格浸入上, 远离剖面, 然后将栅格与剖面下的表面平行。如有必要, 用睫毛在网格上引导切片。</li>
		<li>用滤纸小心翼翼地绕过钳子的尖端, 把水夹在胳膊之间。如果不这样做, 水紧张可以拉的网格之间的武器或使它坚持一侧。这可能导致污染或机械损伤。</li>
		<li>通过将栅格上在滤纸上, 小心地将多余的水从网格中移走。</li>
		<li>将网格放置在网格托架中。</li>
		<li>重复, 直到收集了足够的部分。</li>
		<li>半薄剖面可以直接成像任何复合显微镜配备相机。浸油可以直接放在剖面上。幻灯片应存储在幻灯片框中, 以防止变色。</li>
	</ol></li>
	<li>透射电镜对比超薄切片 注意: 以下步骤应在覆盖范围内进行, 因为染料是光, CO2敏感。此外, EM 染料使用重金属产生对比, 因此是有害物质。处理这些污渍时必须小心。<ol>
<li>用铝箔盖上几个大的培养皿来挡住光线。根据以下步骤进行操作。部分发现它们允许工作空间, 但尽快把盖子放回。</li>
		<li>切一块蜡膜, 小心地放置五滴6% 饱和铀乙酸在它使用巴斯德吸管。<ol>
<li>为制备溶液, 在蒸馏水中混合2克铀乙酸100毫升50% 甲醇, 并在使用前对溶液进行过滤。无法存储该解决方案, 每次19时都应使其新鲜。</li>
		</ol>
</li>
		<li>小心地拿起镊子的 TEM 网格和平衡上的染料滴与部分侧向下。离开25分钟。</li>
		<li>通过快速浸出蒸馏水, 分别冲洗网格;通过四不同的蒸馏水瓶取得进展。</li>
		<li>在一张新鲜的胶片上放五滴柠檬酸铅。在染料液滴周围安排一些氢氧化钠颗粒 (这吸收大气 CO2防止碳酸铅的沉淀)。<ol>
<li>制备柠檬酸铅溶液, 用蒸馏水煮沸0.5 小时. 让水冷却, 并在一个密封的容器, 添加0.3 克柠檬酸铅到100毫升的双蒸馏水。加入1毫升10米氢氧化钠, 密封容器紧紧, 并动摇, 直到溶解19。</li>
		</ol>
</li>
		<li>将栅格放在染料滴上, 如4.3 所述, 并盖上。染色5分钟。</li>
		<li>在蒸馏水中冲洗, 将网格浸入蒸馏水中20次。三罐蒸馏水的进展。</li>
		<li>用滤纸吸收多余的水, 使网格在格子箱中晾干。</li>
		<li>低加速电压下的透射电镜图像。</li>
	</ol></li>
</ol>

<ol>
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作者声明没有相互竞争的利益。

上面概述的一系列方法允许对蚂蚁和其他昆虫的光学系统进行有效的调查。这些技术告诉我们的理解采样分辨率, 光学灵敏度, 和潜在的偏振敏感性的眼睛正在研究。这些知识为他们的视觉能力的生理和行为调查提供了重要的基础。此外, 虽然这里详述的方法侧重于蚂蚁视觉系统, 但这些技术可以用于其他昆虫, 尽管在协议中稍加修改 (例如, 增加了固定的持续时间和在较厚的组织中渗透).稍加...

对于大多数动物来说, 视觉是一种重要的感官形态。在定位目标、建立和遵守路线以及获取罗盘信息1、2的背景下, 视觉尤为重要。昆虫检测视觉信息使用一对复眼, 在某些情况下, 一个到三背放置简单的眼睛称为单眼3,4,5。
蚂蚁的眼睛特别感兴趣, 因为蚂蚁的多样性非常多样, 它们在物种中保存了一些关键的特性。尽管解剖、大小和生态学有巨大的变化, 但绝大多数物种都是群居的, 生活在殖民地;因此, 不同物种在中心位置和资源之间来回导航方面面临着类似的视觉挑战。在蚂蚁之间, 同样的基本眼睛 bauplan 可以观察到的动物范围从 0.5-26 毫米的体长, 从完全白天到严格夜间物种, 从缓慢的步行地下到跳跃的视觉捕食者6,7, 8、9、10。所有这些在生态学和行为上的惊人的差异引起了相同的基本眼睛结构的无数置换, 以适应不同的环境、生活方式和身体尺寸1112。因此, 研究蚂蚁的视觉生态学为确定的调查者提供了一个真正的宝库的可能性。
理解昆虫的视觉系统对于了解它们的行为能力是至关重要的。这是显而易见的综合研究, 很好地结合了解剖学与生态学和行为的成功在几个昆虫组 (例如, 参考13,14,15,16,17). 虽然蚂蚁导航和蚂蚁行为在一般的领域已经相当成功, 但很少有人把重点放在一些选定物种之外的蚂蚁视觉上。在这里, 我们将详细阐述调查蚂蚁眼设计的技术。当我们将重点放在蚂蚁, 这些技术可以应用, 稍加修改, 其他昆虫, 太。

<table>
	<tbody>
		<tr>
			<td>Ant</td>
			<td>Myrmecia midas</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica M205 FA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sputter coater</td>
			<td>Pro Sci Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica MZ6</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insect Pin</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Colourless nail polish</td>
			<td>Non branded: from any cosmetic store</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foreceps</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slip</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compound microscope</td>
			<td>Leica DM5000 B</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformalydehyde</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>di-Sodium Hydrogen phosphate (Na2HPO4)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium di-Hydrogen Phosphate (KH2PO4)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Araldite Epoxy Resin</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidie Blue</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>Riechert HK120</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

techniques for investigating the anatomy of the ant visual system

本文概述了一套光学显微镜技术 (LM) 和电子显微镜 (EM), 可用于研究昆虫的内外眼解剖。这包括传统的组织学技术优化的工作在蚂蚁的眼睛, 并适应工作与其他技术, 如透射电子显微镜 (TEM) 和扫描电子显微镜 (SEM)。这些技术虽然非常有用, 但对于新手技术人员来说是很困难的, 因此本文将重点放在对不同标本的故障排除和优化上。我们提供了整个标本 (照片显微镜和 SEM) 成像技术的信息, 并讨论了它们的优缺点。我们强调了用于确定整个眼睛的透镜直径的技术, 并讨论了改进的新方法。最后, 我们讨论了制备样品的 LM 和 TEM, 切片, 染色和成像这些样品的技术。我们讨论在准备样本时可能遇到的障碍, 以及如何在它们周围进行最佳导航。

这里描述的方法能够详细研究蚂蚁的简单和复眼。使用 Z 堆栈显微技术, 可以对头部的背景色进行成像, 从而获得视觉系统布局的概览 (图 1)。这是很好的准备解剖和确定所需的剖切角度。这项技术对于测量头部宽度、眼睛长度和 ocellar 透镜直径也很有用。SEM 成像也提供了详细的概述图像, 但另外允许获得高放大率和高分辨率的图像。对眼睛感兴趣的?...

Watch this Scientific Journal Video about Techniques for Investigating the Anatomy of the Ant Visual System at JoVE.com

Techniques for Investigating the Anatomy of the Ant Visual System

本文概述了一套光和电子显微镜技术, 研究昆虫的内外眼解剖。这些技术包括针对 ant 眼睛的工作而优化的几种传统方法, 详细的疑难解答, 以及针对不同样本和感兴趣区域的优化建议。

蚂蚁视觉系统解剖学研究方法

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye ...

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13.1K 次观看.  Australian National University. 本文概述了一套光和电子显微镜技术, 研究昆虫的内外眼解剖。这些技术包括针对 ant 眼睛的工作而优化的几种传统方法, 详细的疑难解答, 以及针对不同样本和感兴趣区域的优化建议。

视频: Techniques for Investigating the Anatomy of the Ant Visual System

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced 1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production 3 and developed systems for both transient and stable transformation 4. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.
The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 &deg;C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

We have developed a methodology for performing crosses in Setaria viridis (S. viridis). The method involves pruning the panicle prior to a hot water treatment to kill viable pollen. Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.

方法中进行杂交<em&gt;狗尾草</em&gt;，新模型体系的草

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail ...

科研

环境科学

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

优化负染:通过电子显微镜检查小和不对称蛋白结构的高通量协议

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

经验、基因和计算技术阐明了杀菌剂危害蜜蜂健康的机制

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence the oxidizing capacity of the atmosphere through interactions with ozone and hydroxyl radicals. The main sink of NOx is the formation and deposition of nitric acid, a component of acid rain and a bioavailable nutrient. NOx is emitted from a mixture of natural and anthropogenic sources, which vary in space and time. The collocation of multiple sources and the short lifetime of NOx make it challenging to quantitatively constrain the influence of different emission sources and their impacts on the environment. Nitrogen isotopes of NOx have been suggested to vary amongst different sources, representing a potentially powerful tool to understand the sources and transport of NOx. However, previous methods of collecting atmospheric NOx integrate over long (week to month) time spans and are not validated for the efficient collection of NOx in relevant, diverse field conditions. We report on a new, highly efficient field-based system that collects atmospheric NOx for isotope analysis at a time resolution between 30 min and 2 hr. This method collects gaseous NOx in solution as nitrate with 100% efficiency under a variety of conditions. Protocols are presented for collecting air in urban settings under both stationary and mobile conditions. We detail the advantages and limitations of the method and demonstrate its application in the field. Data from several deployments are shown to 1) evaluate field-based collection efficiency by comparisons with in situ NOx concentration measurements, 2) test the stability of stored solutions before processing, 3) quantify in situ reproducibility in a variety of urban settings, and 4) demonstrate the range of N isotopes of NOx detected in ambient urban air and on heavily traveled roadways.

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

自动化，高清晰度移动采集系统对NO的氮同位素分析<sub系列&gt; X</sub

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

从塑料颗粒中提取有机氯农药和塑料类型分析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables allow quantification of the sources and rates of mortality in insect populations and contribute to understanding, predicting and manipulating population dynamics in agroecosystems. Methods for conducting and analyzing cohort-based life tables in the field for an insect with sessile immature life stages are presented.

用虱、烟粉虱在棉花中开发无梗昆虫生命表的方法作为模型系统

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

一氧化氮气体防治害虫实验室熏蒸程序

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

利用水下立体视频工具量化鱼类密度的新方法研究进展

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes.
One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

从刺客臭虫和其他 Heteropteran 昆虫身上采集毒液毒素

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

就地石蜡镶嵌成人珊瑚样品的杂交技术

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...