这项研究得到了R35GM148160（C.C.）和美国国立卫生研究院（NIH）培训补助金T32 GM007270（R.C.S）的支持。

注意： 图1 显示了进行本研究所需的材料。
1. 实验的注意事项和准备
<ol><li>防止幼虫过度拥挤。 注意：外植体幼虫大脑的质量与解剖前幼虫的健康和质量直接相关。因过度拥挤而营养不良的幼虫通常会产生质量较低的大脑30。<ol><li>确保每个餐盖培养皿的幼虫不超过20-30只，以避免营养不良。这些示例如图 2 所示。</li>
	</ol></li>
	<li>使用前过滤并等分施耐德培养基。<ol><li>对于每次解剖，通过用1%牛生长血清（BGS）补充等分的施耐德昆虫培养基来制备新鲜的成像和解剖培养基。5 mL 的解剖和成像介质体积通常足以进行成像实验。</li>
		<li>使用前将补充的培养基加热至室温 （RT）。</li>
	</ol></li>
	<li>考虑要收集的短片的长度，并用它来考虑成像介质的补充、安装方法和显微镜的采集设置。 注意：在最佳条件下，仅补充BGS的大脑中的NB将强有力地分裂3小时以上。<ol><li>通过在成像介质中添加幼虫脂肪体组织来补充成像培养基，以支持在进行需要更长电影的实验时超过4小时的分裂。 注意：脂肪体分泌支持NB增殖的有丝分裂原31，来自10个幼虫的整个脂肪体足以支持4到5个大脑。此外，用膜结合载玻片成像的样品已被证明分裂超过10小时13,32，而用多孔载玻片成像的样品通常分裂的频率较低（未发表的观察结果）。</li>
		<li>或者，为较长的电影实现更复杂的成像介质，如前所述33.通过调整曝光时间、激光功率和采样频率来最大程度地减少光损伤，以获得最佳效果。</li>
	</ol></li>
</ol>2. 幼虫分期和收集（图2）
<ol><li>将1-5日龄的雌性处女苍蝇与1-7日龄的成年雄性苍蝇杂交，以产生具有所需基因型的后代。为了获得最佳产量，将 10-15 个女性处女与 5-10 个男性杂交。将这些苍蝇放入带有餐盖的苍蝇笼中（图2A-C），并在25°C下孵育。</li>
	<li>每天交换餐盖。这可以防止餐盖被幼虫过度拥挤，从而降低解剖大脑的质量。</li>
	<li>如果餐盖被幼虫明显覆盖（即>30），请将该餐盖分成两半，并用同样切成两半的新鲜餐盖代替一半。或者，更频繁地交换餐盖（即每 12 小时而不是每 24 小时）。餐帽过多的示例如图2E，F所示。</li>
	<li>将餐盖与幼虫在25°C孵育，直到幼虫达到所需年龄。</li>
</ol>3.幼虫脂肪体解剖（图3）
注意：本协议描述了使用3孔解剖皿的解剖。
<ol><li>将 ~400 μL 解剖和成像培养基移液到 3 孔解剖皿的每个孔中。</li>
	<li>用解剖钳轻轻握住十只 72-96 小时龄喂养良好的野生型幼虫，并将它们浸入和浸出最底部孔中的解剖溶液，直到所有食物颗粒都被洗掉。冲洗后，将干净的幼虫移到中间孔中。</li>
	<li>使用一组镊子，用嘴钩握住幼虫。用另一组镊子，破坏幼虫角质层的一侧。</li>
	<li>这种破裂会导致脂肪体从幼虫中溢出。脂肪体是灰白色和半透明的，将具有格子状结构（图3I）。肥胖的身体也倾向于粘在自己和解剖镊子上。一旦确定，从每个幼虫中收集尽可能多的脂肪体，并用镊子将其转移到带有 400 μL RT 解剖培养基的最顶层孔中。</li>
</ol>4.幼虫脑解剖（图3）
<ol><li>如上所述在解剖和成像介质中清洗实验幼虫，以清除它们的食物残渣。为获得最佳效果，请避免将未解剖的幼虫存放在解剖溶液中。这将导致幼虫“淹死”，并对解剖大脑的质量产生负面影响。</li>
	<li>使用一组镊子，用嘴钩握住幼虫。使用另一组镊子，从后侧轻轻切割/撕下大约 1/3 的幼虫（图 3A）。这将导致消化道，脂肪体，结缔组织和神经系统的元素从幼虫破裂的一侧“爆发”出来（图3B）。</li>
	<li>使用一组镊子，用嘴钩握住幼虫。用另一组镊子，轻轻地将角质层刷向口钩，同时用镊子握住口钩向内“推动”，直到整个幼虫翻过来。这种运动类似于将袜子“从内到外”转动（图3C，D）。</li>
	<li>将幼虫倒置，使中枢神经系统和其他组织朝外，同时仍与角质层相连。在此步骤中，找到中枢神经系统（CNS）以避免意外移除。使用镊子轻轻去除所有非CNS组织，仅留下附着在角质层上的CNS和大脑（图3E）。</li>
	<li>大脑将通过轴突连接 附着 在角质层上。使用显微解剖剪刀，切断这些轴突连接，将大脑从角质层释放出来。为此，首先轻轻切开脑叶下方（图3F）。重复腹侧神经索下的连接。 注意：如果没有显微切割剪刀，则可以使用镊子完成此步骤。使用镊子时要特别小心，以确保脑组织在从角质层取出时不会过度拉伸，因为机械应力会对大脑健康产生负面影响。</li>
	<li>将解剖的大脑转移到带有解剖和成像介质的孔中。对于超过3小时的成像实验，如上所述使用补充脂肪体的解剖和成像介质。分批解剖幼虫，以保持解剖时间在20分钟以下。</li>
</ol>5. 安装和成像（图4）
<ol><li>对于使用膜结合玻片34成像：<ol><li>在解剖盘的最后一口井中收集解剖的大脑和孤立的脂肪体。</li>
		<li>通过在载玻片背面放置透气膜来组装载玻片的一半，然后将分体环压入中心，将其固定到位（图4A-C）。</li>
		<li>使用 200 μL 微量移液器，将 ~130-140 μL 解剖和成像介质中的多达 10 个解剖大脑和尽可能多的脂肪体（见上文）转移到膜上。确保将培养基与样品沉积在透气膜的中心（图4D，E）。</li>
		<li>针对要成像的NB群体和正在使用的显微镜类型定向大脑（图4E）。将样品放置在尽可能靠近显微镜物镜的位置。例如，要对中央脑叶中的NB进行成像，请调整大脑的方向，使脑叶最接近目标（图4H）。</li>
		<li>一旦大脑定向，轻轻地将玻璃盖玻片放在膜上的溶液顶部。这将导致含有大脑和脂肪体的溶液扩散到整个膜上（图4F）。</li>
		<li>通过将实验室组织靠近盖玻片边缘来吸干过量的溶液。当大脑接触盖玻片而不被压扁时，可以达到最佳溶液量。如果在此步骤中需要重新定向，请小心地移动盖玻片以移动大脑。</li>
		<li>用画笔沿着盖玻片的边缘涂抹融化的凡士林来固定盖玻片。让果冻凝固（图4G）。</li>
	</ol></li>
	<li>对于使用多孔成像载玻片成像（图4）：<ol><li>将 400 μL 成像培养基添加到多孔载玻片的孔中（在此处进行的实验中，使用腔室 8 孔微型 [μ] 载玻片; 图4I）。将先前解剖的脂肪体转移到该孔中（见步骤3.4）。</li>
		<li>在孔中心附近的簇中沉积多达10个大脑（图4J）。</li>
		<li>如步骤5.1.4（图4K）中所述，针对要成像的NB群体和正在使用的显微镜类型定向大脑。排列样本，使它们彼此靠近。这将最大限度地减少样品台在样品之间必须移动的距离，从而减少采集过程中的样品漂移。</li>
		<li>一旦大脑在井中定向，让大脑沉淀2-5分钟。这增加了它们在运输/成像过程中的稳定性。在此期间准备显微镜以进行采集。</li>
		<li>用载玻片盖盖住载玻片μ载玻片，然后将其转移到显微镜上。以尽可能低的激光功率和曝光时间开始采集，以最大限度地减少光漂白。</li>
	</ol></li>
</ol>6. 数据处理和管理最佳实践
<ol><li>根据可用的分析软件根据需要处理数据。<ol><li>对于此处显示的示例，使用幻灯片软件将采集的数据保存为幻灯片图像文件 （.sld）。</li>
		<li>要使用 Imaris 文件转换器转换为 Imaris 的专有文件类型 （.ims），请在单独的窗口中打开 Imaris 文件转换器。单击并将.sld文件拖到Imaris文件转换器的“输入”部分。</li>
		<li>确定转换后文件的所需输出位置，然后单击“全部启动”。</li>
		<li>转换后，在Imaris软件中查看和注释数据。 注意：可以使用图像分析的替代方案代替Imaris，例如斐济（https://hpc.nih.gov/apps/Fiji.html），Aivia（https://www.aivia-software.com/），Volocity（https://www.volocity4d.com/）或其他。</li>
	</ol></li>
	<li>保留尽可能多的原始数据，以便妥善保存记录。例如，如果采集软件保存为一种文件格式，但转换为另一种格式进行分析，请保留采集的数据版本。</li>
	<li>对于数据分析，请尽可能多地记录有关样品和采集设置的详细信息。要保留的关键信息包括解剖幼虫的基因型、解剖前幼虫的年龄、饲养它们的餐盖状态、成像期间使用的激光功率、曝光时间、采集时间和时间分辨率。</li>
</ol>7. 细胞周期长度定量示例（图5）
注意：在本例中，对表达极性标记针（Pins：：EGFP16）和微管结合蛋白木星25 （樱桃：：木星13）的幼虫进行成像。随后的分析是使用Imaris软件进行的。
<ol><li>使用所选的图像分析软件打开电影。滚动浏览电影的长度以识别划分的 NB，并标记它们以供将来参考。通过其不同的有丝分裂纺锤体识别分裂的NB（图5C-E）。</li>
	<li>确定参考细胞周期阶段以确定细胞周期长度。在本例中，中期用作参考。</li>
	<li>手动确定连续中期之间的帧数，并将其转换为分钟或小时以确定完成一个细胞周期所需的时间。<ol><li>为此，请采用电影的时间分辨率并将其乘以中期之间的帧数。例如，如果电影的时间分辨率为每 5 分钟一帧，并且在第 13 帧和第 35 帧观察到中期，则这些中期之间的时间为 110 分钟（[35 − 13] × 5）。</li>
	</ol></li>
	<li>使用任何适当的软件绘制数据。此处显示的数据是使用PRISM软件绘制的。</li>
</ol>8. 细胞纺锤对准的量化示例（图5）
注意：在本例中，使用 Imaris 软件执行分析。
<ol><li>在 Imaris 或其他所选软件中打开电影文件。滚动浏览电影的长度以识别划分的 NB，并标记它们以供将来参考。</li>
	<li>使用顶端和基底中心体（用 m表示）确定纺锤体极形成的载体，如下所示： <img alt="Equation 1" src="/files/ftp_upload/65538/65538eq01.jpg" style="margin: auto;"> 其中 Ax、 Ay 和 Az 是顶端中心体的坐标， Bx、 By 和 Bz 是基底中心体的坐标。类似地，分裂向量的轴（用 n表示）由顶端Pins：：EGFP新月形和基底皮层的中点形成： <img alt="Equation 2" src="/files/ftp_upload/65538/65538eq02.jpg" style="margin: auto;"> 其中 Ax、 Ay 和 Az 是 Pins：：EGFP 新月形中点的坐标， Bx、 By 和 Bz 是基底皮层中点的坐标。</li>
	<li>确定向量 m 和 n 的大小： m的星等：<img alt="Equation 3" src="/files/ftp_upload/65538/65538eq03.jpg" style="margin: auto;"> n 的大小：<img alt="Equation 4" src="/files/ftp_upload/65538/65538eq04.jpg" style="margin: auto;"></li>
	<li>确定 m 和 n 的点积（用 k 表示）： <img alt="Equation 5" src="/files/ftp_upload/65538/65538eq05.jpg" style="margin: auto;"></li>
	<li>使用点积 k 和矢量幅度 m 和 n，确定矢量之间的角度： <img alt="Equation 6" src="/files/ftp_upload/65538/65538eq06.jpg" style="margin: auto;"></li>
	<li>在所选软件中绘制数据。此处显示的数据是在 Excel Microsoft 中准备的，并在 PRISM 中可视化。</li>
</ol>

来自 黑腹果蝇 第三龄幼虫的活脑外植体成像

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S., Otte, F., Cabernard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+polarity+regulates+biased+myosin+activity+and+dynamics+during+asymmetric+cell+division+via+Drosophila+rho+kinase+and+protein+kinase+N.">Cell polarity regulates biased myosin activity and dynamics during asymmetric cell division via Drosophila rho kinase and protein kinase N.</a> Developmental Cell. 42 (2), 143-155 (2017).</li><li>Montembault, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+efflux+promotes+cell+elongation+to+coordinate+chromosome+segregation+with+cell+cleavage.">Myosin efflux promotes cell elongation to coordinate chromosome segregation with cell cleavage.</a> Nature Communications. 8 (1), 326 (2017).</li><li>Roubinet, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporally+separated+cortical+flows+and+spindle+geometry+establish+physical+asymmetry+in+fly+neural+stem+cells.">Spatio-temporally separated cortical flows and spindle geometry establish physical asymmetry in fly neural stem cells.</a> Nature Communications. 8 (1), 1383 (2017).</li><li>Januschke, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrobin+controls+mother-daughter+centriole+asymmetry+in+Drosophila+neuroblasts.">Centrobin controls mother-daughter centriole asymmetry in Drosophila neuroblasts.</a> Nature Cell Biology. 15 (3), 241-248 (2013).</li><li>Januschke, J., Llamazares, S., Reina, J., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+neuroblasts+retain+the+daughter+centrosome.">Drosophila neuroblasts retain the daughter centrosome.</a> Nature Communications. 2 (1), 243 (2011).</li><li>Rebollo, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+unequal+centrosomes+drive+spindle+orientation+in+asymmetrically+dividing+Drosophila+neural+stem+cells.">Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells.</a> Developmental Cell. 12 (3), 467-474 (2007).</li><li>Januschke, J., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interphase+microtubule+aster+is+a+determinant+of+asymmetric+division+orientation+in+Drosophila+neuroblasts.">The interphase microtubule aster is a determinant of asymmetric division orientation in Drosophila neuroblasts.</a> The Journal of Cell Biology. 188 (5), 693-706 (2010).</li><li>Rusan, N. M., Peifer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+a+novel+centrosome+cycle+in+asymmetric+cell+division.">A role for a novel centrosome cycle in asymmetric cell division.</a> The Journal of Cell Biology. 177 (1), 13-20 (2007).</li><li>Lerit, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interphase+centrosome+organization+by+the+PLP-Cnn+scaffold+is+required+for+centrosome+function.">Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function.</a> Journal of Cell Biology. 210 (1), 79-97 (2015).</li><li>Gallaud, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+centriolar+localization+of+Polo+and+Centrobin+in+early+mitosis+primes+centrosome+asymmetry.">Dynamic centriolar localization of Polo and Centrobin in early mitosis primes centrosome asymmetry.</a> PLoS Biology. 18 (8), e3000762 (2020).</li><li>Ramdas Nair, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microcephaly-associated+protein+Wdr62/CG7337+is+required+to+maintain+centrosome+asymmetry+in+Drosophila+neuroblasts.">The microcephaly-associated protein Wdr62/CG7337 is required to maintain centrosome asymmetry in Drosophila neuroblasts.</a> Cell Reports. 14 (5), 1100-1113 (2016).</li><li>Singh, P., Nair, A. R., Cabernard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+centriolar+protein+Bld10/Cep135+is+required+to+establish+centrosome+asymmetry+in+Drosophila+neuroblasts.">The centriolar protein Bld10/Cep135 is required to establish centrosome asymmetry in Drosophila neuroblasts.</a> Current Biology. 24 (13), 1548-1555 (2014).</li><li>LaFoya, B., Prehoda, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consumption+of+a+polarized+membrane+reservoir+drives+asymmetric+membrane+expansion+during+the+unequal+divisions+of+neural+stem+cells.">Consumption of a polarized membrane reservoir drives asymmetric membrane expansion during the unequal divisions of neural stem cells.</a> Developmental Cell. 1534 (23), 00159 (2023).</li><li>Sunchu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+chromatin+retention+and+nuclear+envelopes+separate+chromosomes+in+fused+cells+in+vivo.">Asymmetric chromatin retention and nuclear envelopes separate chromosomes in fused cells in vivo.</a> Communications Biology. 5 (1), 953 (2022).</li><li>Oliveira, A. C., Rebelo, A. R., Homem, C. C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+animal+development:+How+hormones+and+metabolism+regulate+developmental+transitions+and+brain+formation.">Integrating animal development: How hormones and metabolism regulate developmental transitions and brain formation.</a> Developmental Biology. 475, 256-264 (2021).</li><li>Britton, J. S., Edgar, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+control+of+the+cell+cycle+in+Drosophila:+nutrition+activates+mitotic+and+endoreplicative+cells+by+distinct+mechanisms.">Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms.</a> Development. 125 (11), 2149-2158 (1998).</li><li>Lee, C. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Aurora-A+kinase+inhibits+neuroblast+self-renewal+by+regulating+aPKC/Numb+cortical+polarity+and+spindle+orientation.">Drosophila Aurora-A kinase inhibits neuroblast self-renewal by regulating aPKC/Numb cortical polarity and spindle orientation.</a> Genes &amp; Development. 20 (24), 3464-3474 (2006).</li><li>Homem, C. C. F., Reichardt, I., Berger, C., Lendl, T., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+live+cell+imaging+and+automated+4D+analysis+of+Drosophila+neuroblast+lineages.">Long-term live cell imaging and automated 4D analysis of Drosophila neuroblast lineages.</a> PLoS ONE. 8 (11), e79588 (2013).</li><li>Cabernard, C., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+neuroblast+lineages+within+intact+larval+brains+in+Drosophila.">Live imaging of neuroblast lineages within intact larval brains in Drosophila.</a> Cold Spring Harbor Protocols. 2013 (10), 970-977 (2013).</li><li>Karpova, N., Bobinnec, Y., Fouix, S., Huitorel, P., Debec, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jupiter,+a+new+Drosophila+protein+associated+with+microtubules.">Jupiter, a new Drosophila protein associated with microtubules.</a> Cell Motility and the Cytoskeleton. 63 (5), 301-312 (2006).</li><li>Loyer, N., Januschke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+last-born+daughter+cell+contributes+to+division+orientation+of+Drosophila+larval+neuroblasts.">The last-born daughter cell contributes to division orientation of Drosophila larval neuroblasts.</a> Nature Communications. 9 (1), 3745 (2018).</li><li>Bostock, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immobilization+technique+for+long-term+time-lapse+imaging+of+explanted+Drosophila+tissues.">An immobilization technique for long-term time-lapse imaging of explanted Drosophila tissues.</a> Frontiers in Cell and Developmental Biology. 8, 590094 (2020).</li></ol>

作者没有财务披露需要申报。

该协议概述了一种从 黑腹果蝇 幼虫对活外植体大脑进行成像的方法。这里描述的方案允许在正确的实验条件下观察外植体大脑12-20小时。必须特别考虑样品的制备和所需实验的设计。如上所述，决定解剖组织质量的最关键因素之一是幼虫的健康。为了达到尽可能高的质量，必须确保幼虫在采集前得到良好的喂养。不健康的幼虫最常见的原因是过度拥挤。为了解决这个问题，必须确保通过增...

不对称细胞分裂 （ACD） 是亚细胞成分（如 RNA、蛋白质和细胞器）在子细胞之间分配不均等的过程 1,2。这个过程在干细胞中很常见，干细胞经历ACD以产生具有不同发育命运的子细胞。果蝇NBs不对称地分裂以产生一个NB，保留其干性，以及一个神经节母细胞（GMC）。GMC经历进一步的分裂以产生分化的神经元或神经胶质细胞3。不对称分裂的NBs在三龄幼虫的发育大脑中很丰富，通过显微镜很容易观察到。在第三龄幼虫阶段，每个中枢脑叶中大约有100个NBs3,4,5,6。
不对称细胞分裂是一个高度动态的过程。活细胞成像方案已被用于测量和量化细胞极性7,8,9,10，纺锤体取向11,12,13，肌动肌蛋白皮层14,15,16,17,18，微管和中心体生物学19,20的动力学，21,22,23,24,25,26,27和膜10,28和染色质动力学29。ACD的定性和定量描述依赖于可靠的方法和协议来成像完整活体大脑中的NBs分割。以下协议概述了使用两种不同的安装方法制备，解剖和成像用于体内活细胞成像的第三龄幼虫大脑的方法。这些方法最适合对干细胞分裂以及其他脑细胞分裂的时空动力学感兴趣的研究人员，因为它们允许对细胞事件进行短期和长期观察。此外，这些技术对于该领域的新手来说很容易获得。我们证明了这种方法在表达荧光标记的微管和皮质融合蛋白的幼虫脑中的有效性和适应性。我们还讨论了分析方法和在其他研究中应用的注意事项。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m polyethersulfone (PES) Membrane</td>
			<td>Genesee</td>
			<td>25-231</td>
			<td>Vacuum-driven filters</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Genesee</td>
			<td>20-248</td>
			<td>granulated agar</td>
		</tr>
		<tr>
			<td>Analytical Computer</td>
			<td>Dell</td>
			<td>NA</td>
			<td>Intel Xeon Gold 5222 CPU with two 3.80 GHz processors running Windows 10 on a 64-bit operating system</td>
		</tr>
		<tr>
			<td>Bovine Growth Serum</td>
			<td>HyClone</td>
			<td>SH30541.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Chambered Imaging Slides</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Nikon</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom-machined metal slide</td>
			<td>NA</td>
			<td>NA</td>
			<td>See Cabernard and Doe 2013 (Ref. 34) for specifications</td>
		</tr>
		<tr>
			<td>Dissection Dishes</td>
			<td>Fisher Scientific</td>
			<td>5024343</td>
			<td>3-well porcelain micro spot plate</td>
		</tr>
		<tr>
			<td>Dissection Forceps</td>
			<td>World Precision Instruments</td>
			<td>Dumont #5</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Microscope</td>
			<td>Leica</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Scissors</td>
			<td>Fine Science Tools (FST)</td>
			<td>15003-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo collection cage</td>
			<td>Genesee</td>
			<td>59-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Flypad with access to CO2 to anesthetize adult flies</td>
			<td>Genesee</td>
			<td>59-172</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas-permeable membrane</td>
			<td>YSI</td>
			<td>98095</td>
			<td>Gas-permeable membrane</td>
		</tr>
		<tr>
			<td>Glass Cover Slides</td>
			<td>Electron Microscopy Sciences</td>
			<td>72204-03</td>
			<td># 1.5; 22 mm x 40 mm glass coverslips</td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Oxford Instruments</td>
			<td>NA</td>
			<td>Alternatives: Fiji, Volocity, Aivia</td>
		</tr>
		<tr>
			<td>Imaris File Converter</td>
			<td>Oxford Instruments</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant Yeast</td>
			<td>Saf-Instant</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Molasses</td>
			<td>Genesee</td>
			<td>62-117</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Greiner Bio-One</td>
			<td>628161</td>
			<td>60 mm x 15 mm Petri dish</td>
		</tr>
		<tr>
			<td>Petroleum Jelly</td>
			<td>Vaseline</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Schneider&#39;s Insect Medium with L-glutamine and sodium bicarbonate liquid</td>
			<td>Millipore Sigma</td>
			<td>S0146</td>
			<td></td>
		</tr>
		<tr>
			<td>SlideBook acquisition software</td>
			<td>3i</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum-Driven Filtration Unit with a 0.22 &#181;&#181;m PES membrane filter</td>
			<td>Genesee Scientific, GenClone</td>
			<td>25-231</td>
			<td></td>
		</tr>
	</tbody>
</table>

live-cell imaging of drosophila melanogaster third instar larval brains

果蝇 神经干细胞（神经母细胞，以下简称NBs）经历不对称分裂，再生自我更新的神经母细胞，同时还形成分化神经节母细胞（GMC），该母细胞将经历一次额外的分裂以产生两个神经元或神经胶质细胞。NBs的研究揭示了细胞极性，纺锤体取向，神经干细胞自我更新和分化的分子机制。这些不对称的细胞分裂很容易通过活细胞成像 观察到 ，使幼虫NB非常适合研究活组织中不对称细胞分裂的时空动力学。当在营养补充培养基中正确解剖和成像时，外植体大脑中的NBs强烈分裂12-20小时。前面描述的方法在技术上是困难的，对于那些刚进入该领域的人来说可能具有挑战性。在这里，描述了使用脂肪体补充剂制备、解剖、安装和成像活的三龄幼虫脑外植体的方案。还讨论了潜在的问题，并提供了如何使用这种技术的示例。

Asymmetric cell division (ACD) is the process by which subcellular components such as RNA, proteins, and organelles are partitioned unequally between daughter cells1,2. This process is commonly seen in stem cells, which undergo ACD to give rise to daughter cells with different developmental fates. Drosophila NBs divide asymmetrically to produce one NB, which retains its stemness, and one ganglion mother cell (GMC). The GMC undergoes further divisions to produce differentiating neurons or glia3. Asymmetrically dividing NBs are abundant in the developing brains of third-instar larvae, which are readily observed via microscopy. At the third instar larval stage, there are roughly 100 NBs present in each central brain lobe3,4,5,6.
Asymmetric cell division is a highly dynamic process. Live-cell imaging protocols have been used to measure and quantify the dynamics of cell polarity7,8,9,10, spindle orientation11,12,13, the dynamics of the actomyosin cortex14,15,16,17,18, microtubule and centrosome biology19,20,21,22,23,24,25,26,27, and membrane10,28 and chromatin dynamics29. Qualitative and quantitative descriptions of ACD rely on robust methods and protocols to image dividing NBs in intact living brains. The following protocol outlines methods to prepare, dissect, and image third instar larval brains for live-cell imaging in vivo using two different mounting approaches. These methods are best suited for researchers interested in the spatiotemporal dynamics of stem cell divisions, as well as divisions in other brain cells, as they allow for short- and long-term observations of cellular events. Additionally, these techniques are readily accessible to newcomers to the field. We demonstrate the effectiveness and adaptability of this approach with larval brains expressing fluorescently tagged microtubule and cortical fusion proteins. We additionally discuss methods of analysis and considerations for application in other studies.

作者聚焦：研究不对称细胞分裂动力学：果蝇幼虫脑外植体实时成像的方案 

黑腹果蝇第三龄幼虫脑的活细胞成像

Our research focuses on studying the impact of asymmetric cell division or ACD, on stem cell proliferation and differentiation. We employ Drosophila neural stem cells or neuroblasts as a model to understand the mechanisms, regulations, and impacts of asymmetric cell division on development and neurogenesis. The recent developments in artificial intelligence have been making major strides in image quantification and analysis, which has significantly advanced the field of life cell imaging.Maintaining sample quality for a long-term movie is a long-standing challenge. While it's insightful to observe a sample for eight to 10 hours, logistical constraints, like the sample's imaging requirements and available imaging tools, make it difficult. Our protocol addresses this by demonstrating long-term imaging without impacting the sample's quality.Our technique could be easily adopted and applied to the field by newcomers. With some time and practice, one can feasibly generate short and long-term movies, which have the potential to yield insightful and meaningful data.

中枢脑叶NBs的解剖和成像表达Pins：：EGFP和樱桃：：木星 为了展示这一协议，表达UAS驱动的樱桃：：木星13和内源性标记的Pins：：EGFP16（w; worGal4，UAS-cherry：：jupiter/CyO;引脚：：EGFP / TM6B，Tb）使用多孔成像载玻片使用所述方案成像4小时（图5C，D）。其他数据取自表达UAS驱动的樱桃：：木星13的...

Watch this Scientific Journal Video about Investigating Asymmetric Cell Division Dynamics: A Protocol for Live-Imaging of Drosophila Larval Brain Explants at JoVE.com

在这里，我们讨论了一种工作流程，用于准备、解剖、安装和成像来自黑 腹果蝇 第三龄幼虫的活外植体大脑，以观察生理条件下的细胞和亚细胞动力学。

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...

我们的研究重点是研究不对称细胞分裂或ACD对干细胞增殖和分化的影响。我们使用果蝇神经干细胞或神经母细胞作为模型来了解不对称细胞分裂对发育和神经发生的机制、法规和影响。人工智能的最新发展在图像量化和分析方面取得了重大进展，这大大推动了生命细胞成像领域的发展。保持长期电影的采样质量是一项长期挑战。虽然观察样品 8 到 10 小时很有见地，但后勤限制，如样品的成像要求和可用的成像工具，使其变得困难。我们的实验方案通过在不影响样品质量的情况下展示长期成像来解决这个问题。我们的技术可以很容易地被新来者采用和应用于该领域。通过一些时间和实践，人们可以制作短期和长期电影，这些电影有可能产生有见地和有意义的数据。

live-cell-imaging-drosophila-melanogaster-third-instar-larval

Research

JoVE Journal

Developmental Biology

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

1.3K 次观看.  University of Washington. 我们的研究重点是研究不对称细胞分裂或ACD对干细胞增殖和分化的影响。我们使用果蝇神经干细胞或神经母细胞作为模型来了解不对称细胞分裂对发育和神经发生的机制、法规和影响。人工智能的最新发展在图像量化和分析方面取得了重大进展，这大大推动了生命细胞成像领域的发展。保持长期电影的采样质量是一项长期挑战。虽然观察样品 8 到 10 小时很有见地，但后...

视频: 作者聚焦：研究不对称细胞分裂动力学：果蝇幼虫脑外植体实时成像的方案 

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

科研

发育生物学

﻿Staging and Collecting Drosophila melanogaster Third Instar Larvae

Staging and Collecting Drosophila melanogaster Third Instar Larvae  

Begin by crossing one to five-day-old female virgin flies with one to seven-day-old adult male flies to produce progeny with the desired genotype. To do so, place the flies in a fly cage with a meal cap. Use 10 to 15 female virgins with five to 10 males to get optimal yield.Incubate the fly cage containing flies at 25 degrees Celsius. Change the meal cap daily to prevent overcrowding of larvae which reduces the quality of the dissected brains. If the meal cap has more than 30 larvae, split it into half and replace one portion with a fresh meal cap cut in half.Incubate the meal caps with larvae at 25 degrees Celsius until the larvae reached the desired age.

分期和收集黑 腹果蝇 第三龄幼虫

Fat Body Collection from Drosophila melanogaster Third Instar Larvae

Fat Body Collection from Drosophila melanogaster Third Instar Larvae  

Begin by pipetting around 400 microliters of dissection and imaging medium into each well of a three-well dissection dish. Under a microscope, wash 10, well-fed, wild-type larvae by gently holding them with dissection forceps and dipping them in and out of the dissection medium in the bottommost well to remove all food particulates. While continuing to work under the dissection microscope, transfer the clean larvae to the middle well.Hold the larva by the mouth hooks using one set of tweezers. With another set of tweezers, rupture one side of the larva's cuticle to spill out the fat bodies. Using forceps, collect as much fat body from each larva as possible and transfer it to the topmost well containing 400 microliters of dissection medium at room temperature.

黑腹果蝇第三龄幼虫的脂肪体收集

Dissection of Drosophila melanogaster Third Instar Larval Brain for Live-Cell Imaging

Dissection of Drosophila melanogaster Third Instar Larval Brain for Live-Cell Imaging  

Begin by pipetting around 400 microliters of dissection and imaging medium into each well of a three-well dissection dish. Working under a dissection microscope, wash the experimental larvae in dissection and imaging medium to remove food residues. While continuing to work under the dissection microscope, hold the larvae by the mouth hooks using one set of tweezers.With another set of tweezers, carefully cut off approximately 1/3 of the larva from the posterior side. Next, while holding the larva by the mouth hooks using one set of tweezers, use the other set of tweezers to brush the cuticle towards the mouth hooks gently. Simultaneously, push inward with the tweezers holding the mouth hooks until the entire larva is turned inside out.Invert the larva to expose the central nervous system and other tissues while maintaining their connection to the cuticle. Locate the central nervous system, or CNS, to avoid accidental removal. Gently remove non-CNS tissues using tweezers, keeping only the CNS and brain attached to the cuticle.To release the brain from the cuticle by severing the axonal connections, using microdissection scissors, cut gently beneath the brain lobes. Then, cut the connections under the ventral nerve cord. Dissect larvae in batches to keep the dissection time under 20 minutes.Transfer the dissected brain into a well containing the dissection and imaging medium. For imaging lasting over three hours, supplement the medium with fat bodies.

解剖黑腹果蝇第三龄幼虫脑进行活细胞成像

Mounting and Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brain

Mounting and Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brain  

To perform live cell imaging of the third instar larval brain, begin by collecting the dissected brains and the isolated fat bodies in the last well of a three well dissection dish containing the dissection and imaging medium. Place a gas permeable membrane on the back of a slide and press the split ring into the center. Using a 200 microliter micro pipette, transfer up to 10 dissected brains with maximum possible fat bodies and 130 to 140 microliters of the medium onto the center of the membrane.Orient the brains according to the neural stem cell population to be imaged and the microscope type, ensuring the samples are close to the microscope's objective. For example, to image neural stem cells in the central brain lobes, ensure the brain lobes are closest to the objective. Then gently place a glass cover slip over the solution on the membrane to spread the solution with the brain's and fat bodies throughout the membrane.Hold the laboratory tissue at the cover slip edge to blot excess solution until the brains touch the cover slip without being squashed. Next, immobilize the cover slip by applying molten petroleum jelly along its edges using a paintbrush and allow the jelly to solidify. Add 400 microliters of imaging medium to a well in a multi well micro slide.Transfer the previously dissected fat bodies to this well. Then, place up to 10 brains in a cluster near the center of the well. Orient the brains according to the neural stem cell population to be imaged and the microscope type.Allow the brains to settle in the well for two to five minutes. Cover the micro slide with the slide cover before transferring it to the microscope. Start the acquisition process using the lowest laser power and exposure time to minimize photo bleaching.Imaging of larval brains expressing UAS driven Cherry-Jupiter and androgynously tagged pins GFP using multi-well imaging slides and without fat body supplementation revealed that the pins formed a pronounced apical crescent in dividing neuroblasts to which the mitotic spindles consistently aligned during mitosis. In those samples, the cell cycle length increased with increasing imaging time. Samples that were imaged in a fat body supplemented medium on a membrane-bound slide did not show an increase in cell cycle length.Furthermore, neuroblasts with four divisions were observed on the 10 hour membrane-bound slide.