作者感谢翻译基因组小组成员和凯瑟琳·汉森的反馈和对该协议的改进。这个项目是与研究赠款 SAF2015-64500-R, 其中包括欧洲区域发展基金, 授予 R. A 由 Ministerio de Economia y Competitividad。

1. 固定和树脂嵌入
<ol><li>麻醉与二氧化碳 (CO2) 或通过低温使用冰块的苍蝇。使用解剖显微镜 (用低放大率观察整个苍蝇) 和剪刀, 以消除腿部, 翅膀, 头部和腹部的末端部分, 以促进固定剂的穿透。为了避免胸部的变形, 需要在这一步骤中小心地处理尸体。 注: 每个基因型至少有12只苍蝇, 以确保在手术结束时有足够数量的经过适当处理的个体。</li>
	<li>将尸体转移到冰上的1.5 毫升管上 (每管最多放置6个人), 其中含有200µL 溶液1。 注: 屠体不能在解决方案1中保留超过20分钟。<ol>
<li>对于解决方案 1, 混合组件在这些体积比例: ¼4% 多聚甲醛, ¼8% 戊二醛, ¼0.2 米Na2HPO 4 和¼ 0.2 MNaH2PO4。</li>
	</ol>
</li>
	<li>加入200µL 溶液 2, 在冰上孵育30分钟。<ol>
<li>解决方案 2: 在 1:1 (v/v) 比例中混合解决方案1和 OsO4 。 警告: OsO4非常有毒。使用适当的安全和处置措施在通风罩中操作。</li>
	</ol>
</li>
	<li>除去所有的液体。加入200µL 溶液 2, 在冰上孵育1-2 小时。</li>
	<li>去除溶液, 通过分级乙醇系列脱水样品: 一次在 30%, 50%, 70%, 90%, 和两次在绝对乙醇;每人5分钟。添加1毫升的乙醇来覆盖样品。 注: 一旦将组织放置在90% 乙醇中, 随后的步骤可以在室温下进行。</li>
	<li>在丙烯氧化物中孵化两次样品, 每次孵化10分钟。 注意: 氧化丙烯是有毒的, 用在通风罩里。</li>
	<li>用 1:1 (v/v) 的环氧树脂和环氧丙烷混合物取代丙烯氧化物, 在室温下孵化一夜。 注意: 树脂在液体状态下是极其有毒的。该树脂可作为一套四组件 (a、B、C 和 D) 提供。<ol>
<li>对于环氧树脂, 在一次性烧杯中加入54克树脂 (a)、44.5 克硬化剂 (B)、2.5 克加速器 (C) 和10克增塑剂 (D)。将所有树脂元件组合在通风罩中。将树脂贮存在整除数-20 摄氏度 (长达6月) 或-80 摄氏度 (数年)。</li>
		<li>为了去除树脂, 烘烤它在70°c 为 24 h, 因为固体树脂不是毒物。根据适当的国际程序, 丢弃重金属组中以前的所有解决方案, 因为解决方案可以包含 OsO4 的跟踪.
</li>
	</ol>
</li>
	<li>用100% 树脂取代以前的混合物, 并孵育样品4小时。</li>
	<li>将壳体转移到含有新树脂的塑料模具 (模具中每个井中的一个胴体)。使用削尖的木棒或针转移和定向在水井中的尸体, 并让树脂聚合一夜之间在巴斯德烤箱在摄氏70摄氏度。 注: 为了确保后修剪和切片的胴体的适当方向, 尸体应该躺在他们的长边, 与前部的胴体非常接近井的边缘。</li>
</ol>2. 砌块的修整和切片
<ol><li>从模具中取出聚合树脂块。使用剃刀刀片修剪树脂, 形成一个梯形形状周围的胴体, 并给予适当的方向在切片。开始切片所有的尸体后, 横向缝合, 以获得可比的结果。 注意: 该节必须垂直于 DLMs 的前后轴, 才能正确修剪示例 (图 3)。</li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56179/56179fig3.jpg"> 图 3.Dorsoventral 部分的树脂嵌入成人胸膛的强直营养不良模型苍蝇.在 (A) 中, 样品的修剪不正确, 因为图像的右背部分的部分不是完全横向的肌肉。因此, 右上角的三块肌肉束似乎比左侧的大。这个错误会导致对肌肉区域的高估。(B) 部分从正确修剪的树脂块。刻度条: 200 µm。
<ol><li>切割 1.5-µm 厚的部分与 ultramicrotome, 并转移到水下降到糊幻灯片的部分。<ol>
<li>从 mesothorax 段获取所有样本的部分, 以获得可比较的结果。</li>
	</ol>
</li>
	<li>将包含节的幻灯片放在加热块上70-80 摄氏度, 直到 H2O 蒸发。此时, 可以使用 OsO4对比度 (图 4A) 来观察示例。</li>
</ol>3. 染色 IFM 部分
<ol><li>盖部分与足够的甲苯胺蓝色大约三十年代在热化块。<ol>
<li>甲苯胺蓝溶液, 溶解1% 甲苯胺蓝和1% 硼砂在 H2O。</li>
	</ol>
</li>
	<li>用 H2进行冲洗, 并将幻灯片放在暖盘上以蒸发水。如果需要增强对比度 (图 4B), 请重复使用甲苯胺蓝染色。</li>
</ol><img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56179/56179fig4.jpg"> 图 4.Dorsoventral 部分树脂嵌入成人胸膛不同对比度染色.两个面板都显示了 DM1 模型苍蝇的图像, 与 Abp1 一样, 如图 2D所示。(a) 带有 OsO4对比度的节的图像。(B) 染色甲苯胺蓝的部分, 可以更好地可视化肌肉束。刻度条: 200 µm。
4. 安装 IFM 部分
<ol><li>用加热块干燥幻灯片, 直到 H2O 蒸发。</li>
	<li>将1或2滴的安装介质放在切片上, 然后放在盖玻片上。 注意: 在通风罩中执行这一步骤, 因为安装介质是有毒的。</li>
</ol>5. 图像的采集和肌肉面积的量化
<ol><li>以低放大率的图像, 使整个 DLM 肌肉集中。我们通常使用10X。<ol>
<li>统计分析每飞至少需要5个序列图像, 每实验组分析至少5只苍蝇。</li>
	</ol>
</li>
	<li>选择包含整个肌肉的图像 (图 5A)。检查该部分的轴或方向, 并丢弃不适当的方向的样本, 这将扭曲结果 (比较图 3/3B, 以区分严重和良好的部分, 分别)。</li>
	<li>使用 ImageJ 软件选择仅包含 DLMs (图 5A/5B) 的区域 (以像素为单位)。为了比较不同的实验组, 要使用的参考区域总是以最大的肌肉飞行。在要比较的不同基因型的所有图像中, 选择包含参照区域 (图 5B) 的相等尺寸 DLMs 的区域。</li>
	<li>Binarize 图像-J 命令:进程/二进制/二进制并删除与感兴趣的肌肉不对应的区域或像素 (图 5C)。<ol>
<li>如果 artefactual 黑点出现在肌肉以外的区域, 请使用命令绘图工具将其删除, 然后选择橡皮擦符号。</li>
	</ol>
</li>
</ol><img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56179/56179fig5.jpg"> 图 5.肌肉面积测定的图像分析程序.(A) 强直营养不良症1型的横断面与含有 DLM 的矩形一起飞行胸部。(B) 选定区域仅有间接飞行肌肉和肠道 (*)。(C) 二值化图像。黑色区域对应于兴趣的肌肉。肠道已被清除, 以准确定量的肌肉面积。刻度条: 200 µm。
<ol start="5"><li>将与肌肉组织 (黑色像素) 对应的像素百分比量化为命令:分析/测量并选择区域分数选项。这个百分比的面积是估计的 DLM 肌肉质量的每一个苍蝇。</li>
</ol>

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没有宣布利益冲突。

已证明,果蝇是研究人类神经肌肉疾病的有用模型7,10,11, 包括强直白斑, 其特点是外观肌肉萎缩。这里提出的协议是一个有用的工具, 以量化的肌肉退化造成的发病或进展的某一特定疾病的苍蝇模型。例如, 通过对不同年龄的苍蝇进行这种分析, 也可以追踪和量化肌肉纤维的变性。
该协议中有关键步骤。为了避免组织的退化, 必须尽量减少在固定之前解剖苍蝇所需的时间。为了使固定液在肌肉中的渗透 (图 6), 必须清除苍蝇的头部、翅膀和腿, 以确保解决方案的内部飞行。重要的是要获得的横向部分, 其中的肌肉的利益是完全可见的。量化的样本的二值化是至关重要的, 因此请注意, 选定的像素 (黑色像素) 对应于感兴趣的组织, 并选择整个感兴趣的区域。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56179/56179fig6.jpg"> 图 6。成年的果蝇胸腔, 在肌肉 (*) 中, 固定剂的穿透力不足, 这会在样品加工过程中导致组织的退化和变形。刻度条: 200 µm。
此协议中使用的试剂之一是 OsO4, 它可以非常好地保存大多数结构, 特别是在细胞学级别, 同时提供与图像5的对比度。但是, 该方法的一个重要缺点是 OsO4的毒性, 必须始终使用在通风罩中, 并且需要安全的处理和处理。该协议不仅允许精确定量的肌肉组织由于固定和嵌入程序, 它也可以用于其他应用, 如电子显微镜 (EM) 准备。然而, 由于所产生的组织学切片不能用于随后的化验, 如免疫组化, 因此它受到固有的限制。
最后, 这种方法也被优化, 以检测小的肌肉区域差异, 从而允许可靠的鉴定的修饰语在基因或化学屏幕8,9。然而, 由于肌肉面积增加并不一定意味着更好的肌肉功能, 横断面肌肉面积的确定应该与功能化验, 如飞行化验12理想地相关。

果蝇的胸部包含两种不同种类的飞行肌肉, 它们在功能上、生理上和解剖学上截然不同。这些肌肉是: 间接飞行肌肉 (IFM), 由 dorso 纵向 (DLM) 和 dorso 腹 (DVM) 肌肉 (图 1) 和同步飞行控制肌肉1,2组成。这些肌肉共同产生飞行所需的高机械动力。IFMs 的大小、分布和 rostro 尾部配置允许对横向切片3 (图 2A) 进行简单的定位。因此, 我们选择了这些肌肉来研究果蝇的肌肉萎缩症。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56179/56179fig1.jpg"> 图1。图的胸部的果蝇显示间接飞行肌肉 (IFMs) 安排。(左) 代表侧面视图, (右) 表示胸部的横断面。IFMs 由 Dorso 纵向 (DLM) 肌肉 (红色) 和 Dorso 腹 (DVM) 肌肉 (绿色) 组成。
组织结构的保存和对 dorso-腹侧轴方向的控制是确保正确评估肌肉横截面积的关键。为了保持肌肉结构, 我们使用了从汤姆林森et . 修改的固定混合物。4 。此外, 由于肌肉是内部组织, 因此, 由于固定混合物无法穿透靶组织, 因此,果蝇外骨骼的抗渗性是一个问题。为了规避这个问题, 我们删除了苍蝇头, 腿, 翅膀和最后两个部分的腹部创造孔, 使固定合剂进入。作为固定协议的一部分, 我们包括使用锇毒气 (OsO4)5进行治疗, 因为它能够修复脂肪, 包括甘油三酯, 因此被广泛使用。OsO4保留大多数结构非常好, 特别是在细胞学级别, 同时提供与图像的对比度。固定后,果蝇胸膛嵌入在树脂中进行横向半薄切片 (1.5 µm)。为改善对比, 组织可以另外染色甲苯胺蓝。完全胸膛的图像是在10X 和肌肉区域被量化的 binarizing 图像 (等维度) 和量化百分比的像素对应的肌肉组织 (黑色像素) 的总和, 与 ImageJ 软件。
在本议定书中介绍的 OsO4和戊二醛溶液浓度的增加, 对组织制备和固定混合物的修改, 使肌肉组织得以独特的保存。这是因为该协议避免了组织的退化和变形, 使样品的后分析更可靠, 即使在高度萎缩的情况下与神经肌肉退行性疾病, 如强直营养不良 (DM) 有关。在更常见的形式, DM 类型 1, 这种罕见的遗传紊乱是由扩大 CUG 重复在强直营养不良蛋白激酶(DMPK) 转录。突变 DMPK rna 聚集形成 ribonuclear 病灶, 它能封存 Muscleblind 核 RNA 结合蛋白 (MBNL1-3;果蝇)6中的 Muscleblind。我们通过在肌球蛋白重链启动子 (Mhc-Gal4) 下表达 250 CTG 重复, 生成了一种强直营养不良的果蝇模型。模型苍蝇是不能飞的, 典型的 "上升的翅膀" 表型和严重的肌肉萎缩, 在他们的 IFMs (图 2B)。以前在我们实验室进行的研究表明, IFMs 的肌肉面积的测定是一种可靠的方法来量化不同化学或遗传修饰剂的肌肉萎缩的影响, 这些模型苍蝇7。举例来说, 在飞行中表达 250 CTG 重复的异构体 C 在肌肉中的过度表达, 实现了肌肉区域的抢救, 因为螯合被封存的损耗是 DM1 发病机制8 (图 2C) 的触发因素。肌肉区域也获救后喂养 DM 模型苍蝇与 Abp1, 一个 hexapeptide 与证明 anti-DM1 活动9 (图 2D)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56179/56179fig2.jpg"> 图2。树脂嵌入成人胸膛 dorsoventral 段的量化。(A D) 间接飞行肌肉的果蝇与指定的相关基因型。(A) 控制苍蝇 (yw)。(B) 在肌肉中250非编码 CTG 重复的表达 (无人参与-ctg (250) x) 导致 DLMs 中肌肉面积的减少与控制苍蝇相比. (C) 这种肌肉萎缩表型是通过过度表达 Muscleblind (MblC) (无人飞机-ctg (250) x 无人) 和 (D) 喂养模型苍蝇与 hexapeptide Abp1 (无人飞机-ctg (250) x Abp1)。在所有图像中, 背侧位于顶部。转基因是用肌球蛋白重链启动子 (Mhc)-Gal4 驱动肌肉。(E) 相对于对照蝇的肌肉面积百分比进行量化, 证实差异很大。直方图显示的意思是 S.E.M.p< 0.01 和 * p < 0.05 (Student´s t 测试)。刻度条: 200 µm.<a href="//cloudflare.jove.com/files/ftp_upload/56179/56179fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
本文所报道的方法将对研究肌肉发育、维持和衰老、疾病病理学和药物检测的研究者们感兴趣, 因为它提供了有关肌肉组织对内源和外在因素的反应的可靠信息。

<table><tbody><tr><td>Image-J software</td><td>National Institutes of Health</td><td></td><td>https://imagej.nih.gov/ij/</td></tr><tr><td>Ultramicrotome</td><td>Leica</td><td>Leica UC6</td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td>Leica MZ6</td><td>Bright field technique.</td></tr><tr><td>Razor blades</td><td>Electron Microscopy Sciences</td><td>71970</td><td>Several alternative providers exist.</td></tr><tr><td>Scissors</td><td>World Precision World</td><td>14003</td><td>Several alternative providers exist.</td></tr><tr><td>Embedding molds</td><td>Electron Microscopy Sciences</td><td>70900</td><td>Several alternative providers exist.</td></tr><tr><td>Glutaraldehyde</td><td>Fluka (Sigma)</td><td>49624</td><td>Toxic.</td></tr><tr><td>OsO&lt;sub&gt;4&lt;/sub&gt;</td><td>Polyscience</td><td>0972A</td><td>Extremely toxic.</td></tr><tr><td>Propylene oxide</td><td>Sigma Aldrich</td><td>82320-250ML</td><td>Extremely toxic.</td></tr><tr><td>resin (Durcupan)</td><td>Sigma Aldrich</td><td>44611-44614</td><td>Carcinogenic when it is unpolymerized.</td></tr><tr><td>Toluidine blue</td><td>Panreac</td><td>251176</td><td>Toxic.</td></tr><tr><td>Mountant Medium (DPX)</td><td>Sigma Aldrich</td><td>44581</td><td>Dangerous.</td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148-500G</td><td>Harmful.</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Panreac</td><td>122507</td><td>0.2 M dilution.</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Panreac</td><td>121677</td><td>0.2 M dilution.</td></tr><tr><td>Borax</td><td>Panreac</td><td>3052</td><td>Toxic.</td></tr></tbody></table>

optical cross-sectional muscle area determination of drosophila melanogaster adult indirect flight muscles

肌肉大量的浪费, 称为肌肉萎缩, 是一个常见的表型在果蝇模型的神经肌肉疾病。我们使用的间接飞行肌肉 (IFMs) 的苍蝇, 特别是 dorso-纵肌 (DLM), 作为实验对象, 以衡量的萎缩表型所带来的不同的遗传原因。在该协议中, 我们描述了如何将飞胸肌嵌入半薄切片, 如何获得良好的肌肉和周围组织的对比, 以及如何处理光学显微镜图像的半自动获取可量化的数据和分析.我们描述方法管道的三具体应用。首先, 我们展示了如何将该方法用于量化强直营养不良蝇模型中的肌肉退化;第二, 肌肉横截面积的测量可以帮助识别促进或防止肌肉萎缩和/或肌肉退化的基因;第三, 该协议可用于确定候选化合物是否能够显著改变由致病突变或环境触发器诱发的特定萎缩表型.

为了量化 MblC 或 Abp1 的过度表达是否对强直营养不良蝇模型的萎缩表型有任何影响, 我们重点关注 DLMs, 这是 IFMs 的一部分 (图 1)。我们确定, 模型苍蝇, 这表明250非编码 CTG 重复整个肌肉组织驱动的肌球蛋白重链启动子 (Mhc)-Gal4, 有一个50% 的减少肌肉面积相比, 控制苍蝇。相反, MblC 和扩张的 CTG 的共同表达与同一驱动程序重复, 强烈抑制这种表型和 crossectional 肌肉区是70% 的控制 (正常) 苍蝇。营养培养基中 Abp1 hexapeptide 的管理同样抑制了萎缩性表型, 而采用该化合物的模型苍蝇则显示了大约60% 的正常肌肉面积 (而非50% 是典型的 DM1 蝇;图 2E)。对于这些样品的量化, 我们使用了染甲苯胺蓝的切片来增强对比度 (图 4和5)。
 
我们注意到, 在程序中, 一些技术故障可能会严重干扰最终量化。一个常见的是, 树脂块没有正确修剪, 这可能 misorient 样品和导致斜切片的 DLMs。因此, 一些 DLM 肌肉可能看起来更大的一侧相比, 对侧一个 (见例如图 3), 其中引入了一个重大错误的最终量化。另一个可能发生的错误是, 在肌肉组织 (图 6) 中, 固定剂的穿透力不足, 这看起来是退化和变形的, 使肌肉区域的精确量化成为不可能。

Watch this Scientific Journal Video about Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles at JoVE.com

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

果蝇成人间接飞行肌的光学横截肌面积测定

我们报告的方法, 以量化肌肉面积, 这是一个间接的方法来确定肌肉质量在果蝇成人。我们通过分析强直营养不良症的果蝇模型中的间接飞行肌肉来证明我们的方法的应用。

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...

optical-cross-sectional-muscle-area-determination-drosophila

Nanyang Technological University

Research

JoVE Journal

Biology

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

8.9K 次观看.  University of Valencia. 我们报告的方法, 以量化肌肉面积, 这是一个间接的方法来确定肌肉质量在果蝇成人。我们通过分析强直营养不良症的果蝇模型中的间接飞行肌肉来证明我们的方法的应用。

视频: Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

解剖果蝇黑色素组飞行肌肉为奥米克斯方法

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and ...

科研

遗传学

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

在体内果蝇蛹肌肌腱形态的影像学研究

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

发育生物学

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

为介观荧光体层摄影术在体内成像的发展果蝇</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

生物学

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

果蝇胚胎肌肉收缩的实时成像和分析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

果蝇中单个 mRNA 分子的检测和定量

Studying Muscle Transcriptional Dynamics at Single-molecule Scales in Drosophila

Skeletal muscles are large syncytia made up of many bundled myofibers that produce forces and enable body motion. Drosophila is a classical model to study muscle biology. The combination of both Drosophila genetics and advanced omics approaches led to the identification of key conserved molecules that regulate muscle morphogenesis and regeneration. However, the transcriptional dynamics of these molecules and the spatial distribution of their messenger RNA within the syncytia cannot be assessed by conventional methods. Here we optimized an existing single-molecule RNA fluorescence in situ hybridization (smFISH) method to enable the detection and quantification of individual mRNA molecules within adult flight muscles and their muscle stem cells. As a proof of concept, we have analyzed the mRNA expression and distribution of two evolutionary conserved transcription factors, Mef2 and Zfh1/Zeb. We show that this method can efficiently detect and quantify single mRNA molecules for both transcripts in the muscle precursor cells, adult muscles, and muscle stem cells.

作者聚焦：研究果蝇肌肉组织中的 mRNA 空间分布 

Drosophila is a well-established model for studying key molecules that regulate myogenesis. However, current methods are insufficient to determine mRNA transcriptional dynamics and spatial distribution within syncytia. To address this limitation, we optimized an RNA fluorescence in situ hybridization method allowing the detection and quantification of mRNAs at a single-molecule scale.

研究果蝇单分子尺度的肌肉转录动力学

Skeletal muscles are large syncytia made up of many bundled myofibers that produce forces and enable body motion. Drosophila is a classical model to study ...

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The well characterized larval neuromuscular junction is often used for such studies. However, rapid larval development followed by muscle histolysis and nervous system remodeling during metamorphosis makes this model problematic for the study of slow age-dependent degenerative changes like those occurring in amyotrophic lateral sclerosis. Alternatively, adult flies live for 90 days and the adult leg can be used to study motor neuron changes over the course of adult lifespan using in vivo fluorescent imaging through the cuticle. Here, we describe a leg dissection technique coupled with immunocytochemistry, which allows for the study of molecular changes at the neuromuscular junction of identified adult leg motor neurons. These techniques can be coupled with a myriad of antibodies labeling both pre- and post-synaptic structures. Together these procedures allow a more complete characterization of slow age-dependent changes in adult flies and can be applied across multiple motor neuron disease models.

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

We describe a dissection technique that preserves the architecture of the neuromuscular junction and enables a detailed immunocytochemical study of motor neurons in the adult Drosophila leg.

果蝇成腿的解剖和免疫组织化学，以检测已识别运动神经元的神经肌肉接头处的变化

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...

神经科学

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

果蝇成人间接飞行肌的光学横截肌面积测定

摘要

探索更多视频

此视频中的章节

解剖果蝇黑色素组飞行肌肉为奥米克斯方法

在体内果蝇蛹肌肌腱形态的影像学研究

为介观荧光体层摄影术在体内成像的发展果蝇

果蝇胚胎肌肉收缩的实时成像和分析

石蜡包埋及冷冻的部分果蝇成人肌肉

研究果蝇单分子尺度的肌肉转录动力学

研究果蝇单分子尺度的肌肉转录动力学

研究果蝇单分子尺度的肌肉转录动力学

果蝇成腿的解剖和免疫组织化学，以检测已识别运动神经元的神经肌肉接头处的变化

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）