Name: drosophila preparation and longitudinal imaging of heart function in vivo using optical coherence microscopy (ocm)
Uploaded: 2016-12-12T12:30:00.000+00:00
Duration: 10 min 13 s
Description: Watch this Scientific Journal Video about Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM at JoVE.com

This work was supported by the Lehigh University Start-Up Fund, the NIH (R00EB010071 to C.Z., R15EB019704 to C.Z. and A.L., R03AR063271 to A.L., and R01AG014713 and R01MH060009 to R.E.T.), the NSF (1455613 to C.Z. and A.L.), the Cure Alzheimer's Fund (to R.E.T.), and the Massachusetts General Hospital (Executive Committee on Research Award to A.L.). M.C. and Y.M. was supported by the National Key Basic Research Program of China (973 Program) under Grant No. 2014CB340404.

1. 果蝇 16的光学成像OCM系统的研制<ol><li>选择一个分光计和高速行扫描摄像机，可提供至少80帧/秒的帧速率，以便该OCM系统将能够解决果蝇的心跳。 </li><li>使用宽带光源，以确保2微米轴向分辨率识别果蝇的心脏结构。 </li><li>使用10X的目标，以获得较高的横向分辨率。 </li><li>使用45°杆镜子反射参考臂的光束，并产生一个环形样品臂光束扩展聚焦深度的标本。 </li><li>开发一个定制的计算机程序，以控制该OCM系统并执行测量。 </li></ol> 2. 果蝇文化<ol><li> 标飞食品制作 <ol><li>放〜5毫升瞬间果蝇式成聚苯乙烯小瓶管纸槽的援助。 </li><li>倒〜8毫升水入公式正确饱和的食物。 </li><li>添加不同的添加剂的标准飞食品不同的实验。当全反式 -retinal（ATR）食品备战光遗传学实验起搏35，用吸管抽取100毫米ATR和溶入〜8毫升水进入食品1毫米的ATR浓度。该解决方案混合均匀后，将溶液倒入公式，并充分搅拌均匀。 </li><li>用于在果蝇研究肥胖相关心脏功能障碍用15ml水在杯子和热制备高脂肪的饮食，10,15组合〜10毫升式用于在微波炉30秒。把一些有机特级初榨椰子油在另一个杯子加热它在微波炉90秒。 </li><li>提取7.5毫升椰子油和制得的式混合足以使椰子油的重量/体积比为食品〜30/100，和第烯提取物约2ml混合食物，并把它到管的底部。 </li><li>等待1分钟，直到介质彻底饱和。仔细紧凑食品与一个平坦的表面，以优化果蝇的生活条件。添加6 - 酵母的8粒向制备的配方，并用棉的集群堵塞管。 </li></ol></li><li> 果蝇十字架和文化 <ol><li>采取与标准的准备飞食品管，并取出堵塞棉花。小心转移成蝇（男性和女性）的管中，并立即用棉塞的管。检查棉花，以确保有棉花与管壁，防止蝇从管逸出之间没有间隙。 </li><li>保持在孵化器的果蝇在25℃下进行杂交育种。大多数的基因被激活和细胞蛋白质在25℃下36-39合成。 </li><li>就拿出来管从孵化后8手转移一个dult飞出管在年龄相仿的实验控制，以获得鸡蛋。 </li><li>继续在25℃，这对于果蝇开发的8.5天40,41发展期的标准温度在孵化器中培养卵。 注:温度会影响发育时期（卵到成虫）和各种基因的表达水平。 </li></ol></li></ol> 3.执行与OCM光学成像<ol><li> 飞山为幼虫光学成像 注: 果蝇舱口22蛋- 24小时，在25℃至第一龄幼虫（L1）。接连24小时的第二龄幼虫出现。最大的幼虫形态是第三龄幼虫，这后约24小时换毛。在幼虫结构特征可用于区分其不同发育阶段。第一龄和第二龄之间的口器的大小是不同的。口第一龄幼虫的钩子都非常小，看起来像两个双很小的黑点，而第二龄幼虫的口钩较大，结构清晰。的气孔，通常用于识别第二龄期和第三龄。二龄幼虫有杵状前气孔，同时，第三龄，前气孔是支。深橙色圈将开始出现在第三龄幼虫后气门的一角。 <ol><li>应用一片双面胶带的到一个干净的显微镜载玻片。驱逐胶带下的气泡，以避免在成像期间由气泡引起的反射。 </li><li>采取与在幼虫期培养的果蝇出从培养箱管中的一个。 </li><li>确定幼虫在媒体，从媒体用软毛刷放于干净的纸巾将其删除。取下粘在幼虫任何食物用湿软刷和烘干的组织。 </li><li>移动cleaneð飞至组织宽视场显微镜的物镜下。 </li><li>调整显微镜的焦点，找到飞一个明确的说法。通过用显微镜其结构特征确定幼虫的右侧发育阶段。 </li><li>用软毛刷飞的位置。确保身体直朝上以用于安装在由背侧载玻片制备背侧。执行显微镜下这一步。 </li><li>确保幼虫是安装在磁带上之前完全干燥。否则，幼虫不会粘附到磁带上。 </li><li>被定位飞背侧粘到与中等压力的载玻片双面胶带。需要注意的是压力太大可能会杀死苍蝇和过少的力量会导致成像过程中飞行运动。 </li></ol></li><li> 在幼虫阶段（L2和L3）与OCM 果蝇的光学成像 注:心脏管的广泛管腔可FOUND位于在幼虫阶段A5之间的段至A8（ 图1）。横向OCM M模式图像（2D +时间）在所述心脏管的A7段获取的每个幼虫以促进收缩和舒张的分析。 <ol><li>放在一起向上面对物镜下方的背侧的y横向的OCM系统的可调节的试料台的安装幼虫。在样品台一个小孔是必要的放置幼虫，以避免其与阶段平面接触。 </li><li>调整样品台移动水果的心脏管飞摄像光束的焦平面。要轻松找到A7段，发现心脏管与实时截OCM图像在图像采集软件后部区域。然后移动台向前直到A7段是可见的。 </li><li>图像采集软件至100％B扫描的A扫描（帧），100 B扫描的设置参数，并且scanneř电压以覆盖〜0.28毫米在x横向，并在y横向方向0 V.点击软件的"开始"按钮通过用暗布阻断样品光束路径获取背景扣除背景噪声的数据。 注:在100帧的3可以用于背景减除。 </li><li>数据采集​​软件128每B扫描的A扫描，4096 B扫描，并且扫描仪电压的设定参数以覆盖〜0.28毫米在x横向，和0 V在y横向方向。点击软件的"开始"按钮在覆盖0.28点¯x0.57毫米2为约30秒的区域来获取整个飞心脏管的A7段横向M模式图像。 </li><li>在数据保存过程中使用深色的布，以避免飞心脏的长时间暴露在成像光阻断成像光束。 </li><li>重复测量5次获得心脏乐趣可靠的测量ction。 </li><li>图像采集软件400每B扫描的A扫描，800 B扫描，并且扫描仪电压的设定参数以覆盖〜1.7毫米在x横向，和〜4毫米的y横向方向。移动台在两个方向，以确保整个果蝇可以成像。点击软件中的"开始"按钮，获得一个数据集，以获得3维果蝇的图像。注:3D飞行结构可以用阿米拉3D软件渲染</li><li>用湿柔软的刷子沾测量飞行，轻轻地从载玻片上删除。移动它转化为不断发展的独立管。在接下来的发展阶段标注管纵向研究。 </li></ol></li><li> 图像在果蝇蛹的阶段 注:所有果蝇取出从PD1到PD5成像。如在图1b中的幼虫概略所示，宽腔保持在A5至第的A8段Ë心脏管，直到PD1。从PD2，锥形腔开始A1之间发展到A4段。为了获得一致的图像和促进心脏分析，从该A7段在PD1得到横M模式图像，并且从A1段PD2后，如标示于图1b。 <ol><li> 果蝇的图像在PD1 注: 果蝇将有一个白puparium短时间窗口（0 - 1小时）PD1中。这个时间窗口是理想的，因为透明度高导致更高的光线穿透的OCM成像早期蛹进行光学成像。 <ol><li>作为果蝇在管壁上发现，当他们变得蛹，用湿软刷除去从各个管的蛹成像在PD1，并用刷子清洗蛹如果有粘在身体的食物。 </li><li>安装在一个小的玻璃载玻片果蝇直接与湿刷和保持背侧朝上（ 图1a </str翁&gt;）。确保载玻片是小到足以回管一旦在此阶段的成像完成。 </li><li>从蝇体侧面除去过量的水。 </li><li>戴上OCM系统的样品台载玻片上，保持在顶部的果蝇。发现飞心脏利用在幼虫测量描述的相同策略的A7段清晰的实时图像。 </li><li>设置数据采集软件相同的参数在3.2节和图像在A7段心跳收购横向M模式和3D图像。 </li><li>成像后，使用镊子与蛹载玻片放回管的连续培养。 </li></ol></li><li> 果蝇的图像在PD2到PD5阶段 注:由于样品变为在蛹期越来越不透明，成像系统的穿透深度将减小。 <ol><li>使用镊子小心地将玻璃小号立德安装有在PD2从管用于成像的飞行。在PD2，试样壳变得微黄并与PD1（ 图1）的身体变得不太透明。 </li><li>把滑上该OCM系统的试料台。 </li><li>调整样品台到飞移动到OCM系统的成象光束的焦平面。找到具有实时剖OCM图像的心脏管的前端。移动〜50微米回来后方向找到心脏管的A1段。 注意:此时心脏发育（PD2）的，锥形腔将很小，可能无法跳动。 </li><li>收集来自A1段使用相同的方法与以前的发育阶段横M模式的数据集，以及3D数据。 </li><li>把滑回管仔细进行连续培养。 注:在PD3，在外壳试样的颜色比在PD2阶段暗。在PD4阶段，黑色条纹CAN为样品的外壳内观察。有些苍蝇会发展到这一阶段第二天的成年人，而其他人将演变成PD5。在PD5阶段，黑色条纹甚至在果蝇更为明显看到。这些苍蝇将成为第二天的成年人。 </li></ol></li></ol></li><li> 图像在果蝇成虫期 注:在成年阶段，雌性和雄蝇可以通过身体的大小和下腹部的颜色来区分。雌成虫有较大的尺寸，而男性更小，深色的小腹。 <ol><li>从孵化器采取管出来的时候，果蝇发育为成年，并转移成人飞到〜45毫升空瓶。 </li><li>蘸棒的吸收端（约1厘米长，〜3毫米直径）插入麻醉，把棒到小瓶，并用棉的集群堵塞管保持麻醉结束正下方插入棉花和ANESthetize飞3分钟。麻醉的持续时间取决于飞的大小，并且可以2.5 3.5分钟之间，以改变（例如:2.5 3雄性分钟，女性或3.5分钟）。 </li><li>准备用一块双面胶带的载玻片。 </li><li>移动飞麻醉到载玻片与背侧朝上使用软毛刷。 </li><li>单独使用镊子翅膀，并坚持在显微镜下磁带上的机翼固定飞和暴露心脏区域成像。 </li><li>图像从蝇心脏的A1段飞（ 图1）。在实验结束时，飞可能牺牲。 </li></ol></li></ol> 4.影像学分析16 <ol><li>开发Matlab的方案，以与所述图像采集软件将图像文件中收集的2D和3D的二进制文件转换。 </li><li>使用ImageJ的，以确定在横向M型图像的心脏管区域和魔杖算法来创造心脏部位的EA面具每个横向M模式图像。段的掩蔽区域，并使用一个峰寻找算法来识别的收缩和舒张的位置。计算从横M模式图像的时间依赖的心脏直径变化。 </li><li>基于所获取的时间相关的心脏的直径，计算出心脏参数如心率，心搏周期（CAP），舒张末期内径（EDD），收缩末期内径（ESD），舒张末期面积（EDA），并结束收缩期面积（欧空局）。计算短轴缩短率（FS）与<img alt="式（1）" src="/files/ftp_upload/55002/55002eq1.jpg" /></li><li>使用ImageJ的分析3D图像OCM可视化动态心脏的结构性发展。 </li></ol>

The authors declare no conflicts of interests related to the current study.

果蝇的心跳加快，大约在幼虫和成虫期400 BPM最大的人力资源，需要很高的成像速度来解决心脏diastoles和心脏收缩（根据经验不低于80帧/秒）。由于小心脏腔室的尺寸和微米尺度心脏壁的厚度（5 - 10微米），高空间分辨率（大于2μm更好）所需的解决心脏管结构。在这项研究中，高分辨率和超高速OCM系统的开发，其中，使用了用600线/毫米透射光栅的分光计和2,048像素行扫描摄像机。 20千赫?...

在小动物心脏的纵向研究有助于了解人类的各种心脑血管疾病，如基因相关的先天性心脏缺陷1,2。在过去的几十年中，各种动物模型，例如小鼠3,4-，爪蟾5,6-，斑马鱼7,8，禽流9，和果蝇10-16，已被用来进行人体心脏的开发有关的研究。小鼠模型已被广泛用来研究正常和异常心脏发育，并且由于其相似与人的心脏3,4-心脏缺陷的表型。非洲爪蟾胚胎心脏发育的研究中尤其有用，因为它易于处理和部分透明5,6。胚胎和斑马鱼模型的早期幼虫的透明度，使得心脏发育7,8容易光学观测。禽流模型是心脏发育研究的共同课题因为...E中的心脏可以去除蛋壳和禽流感的心给人类9形态相似后，很容易地访问。 果蝇模型具有一些独特的功能，这使得它非常适合进行心脏的纵向研究。首先， 果蝇的心脏管〜200微米背表面之下，这对于心脏的光纤接入和观察提供了方便。此外，许多分子机制和遗传途径是果蝇和脊椎动物之间保守。人类疾病基因的75％以上的同源基因在果蝇 ，这些都使得它广泛应用于转基因研究11,13被发现了。此外，它具有生命周期短，维护成本低，并已被广泛用作发育生物学研究14-16标本模型。 以前的报告中所述的协议监测果蝇的心脏功能，如他artbeat。然而，被要求17,18解剖程序。光学成像提供了可视化的动物心脏发育的有效方法，因为它的非侵入性的性质。不同的光学成像方法都在进行动物心脏研究中得到应用，如双光子显微镜19，共焦显微镜20,21，光片镜22和光学相干断层扫描（OCT）16,23-26。相比较而言，OCT能够提供在小动物的心大成像深度不使用造影剂，同时保持了高分辨率和超高成像的速度，这是用于成像活体动物重要的。此外，开发一个OCT系统的低成本已经普及这种技术的样品光学成像。 OCT已经被成功地用于果蝇的纵向研究。使用OCT，心脏形态和功能成像已进行研究心脏的结构中，func基因的国的角色，并在突变体模型心血管缺陷心脏发育过程中的机制。例如，年龄依赖性心功能下降是证实了下调血管紧张素转换酶相关（ACER）在果蝇基因与华侨城27基因相关的心肌病表型是使用OCT进行28-33 果蝇证实。研究采用OCT还揭示了人类基因SOX5在34果蝇的心脏功能的作用。与华侨城相比，OCM使用一个客观的具有更高的数值孔径，以提供更好的横向分辨率。在过去，所造成的沉默同源物人体生理基因dCry / DCLOCK的心脏功能障碍已被使用定制OCM系统15,16，以及高脂饮食对果蝇心肌病的效果，了解肥胖诱导人研究心脏疾病。 15 在这里，日Ë实验方案总结为果蝇在二龄（L2），三龄（L3），蛹1天（PD1），蛹天2（PD2），蛹第3天心脏形态和功能变化的纵向研究（PD3） ，蛹天4（PD4），蛹5天（PD5）和成人使用OCM以促进人类有关的先天性心脏病的研究（ 图1）。心脏功能参数，如HR和CAP物进行定量分析在不同发育阶段以显示心脏发育的功能。

<table><tbody><tr><td>Custom OCM imaging system</td><td>Developed in our lab</td><td></td><td></td></tr><tr><td>my Temp Mini Digital Incubator</td><td>Benchmark</td><td>H2200-HC</td><td></td></tr><tr><td>Cover glass</td><td>AmScope</td><td>200PCS</td><td></td></tr><tr><td>Cotton Ball</td><td>RITE AID</td><td></td><td></td></tr><tr><td>Instant Drosophila Formula</td><td>CAROLINA</td><td>formula 4-24</td><td></td></tr><tr><td>Yeast</td><td>ActiveDry</td><td></td><td></td></tr><tr><td>Microscope</td><td>SONY</td><td>WILD M420</td><td></td></tr><tr><td>Brush</td><td>Loew-Cornell</td><td>245B</td><td>being used to move specimens</td></tr><tr><td>Labview software</td><td>National Instruments</td><td></td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td></td><td></td></tr><tr><td>Matlab</td><td>Mathworks</td><td></td><td></td></tr><tr><td>Tweezer</td><td>Wiha</td><td>AA SA</td><td>to fix the fruit fly wings</td></tr><tr><td>FlyNap</td><td>Carolina Biological Supply Company</td><td>4,224,898</td><td></td></tr><tr><td>Scotch Permanent Double Sided Tape, 3 M</td><td>Scotch</td><td></td><td></td></tr><tr><td>Pipette</td><td>Fisherbrand</td><td>MU18837</td><td></td></tr><tr><td>Organic Extra Coconut Oil</td><td>Spring Valley</td><td>13183</td><td></td></tr><tr><td>Microscope Slide</td><td>CapitolBrand</td><td>M3504-E</td><td></td></tr><tr><td>&lt;i&gt;Drosophila&lt;/i&gt; Vials</td><td>SEOH</td><td>8401SS</td><td></td></tr><tr><td>All-trans-retinal</td><td>Sigma-Aldrich Co.</td><td>R2500</td><td></td></tr></tbody></table>

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.

纵向心脏成像用的是果蝇与24B-GAL4 / +在室温下用OCM应变进行。测量在L2，L3进行的，并在从PD1 8小时间隔PD4，和成人每天1（AD1）跟踪变态处理（ 表1）。幼虫，早期蛹，晚蛹和成虫蝇被安装在中所见图1A的载玻片上。的心脏对幼虫和成虫蝇的段特征在图1B中示意性表示被示出。 在这一发展的研究中，4096架是在32秒，我们的定制OCM系统来跟踪?...

Watch this Scientific Journal Video about Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM at JoVE.com

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 ...

drosophila-preparation-longitudinal-imaging-heart-function-vivo-using

Research

JoVE Journal

Biology

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

10.8K 次观看.  Lehigh University. 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.

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

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

科研

生物工程

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

单果蝇 Ommatidium解剖和影像

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

神经科学

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

可视化心脏跳动果蝇</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

生物学

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

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 ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

在幼虫的监测心脏功能果蝇生理研究

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Novel And Efficient Method for Drosophila Heart Fluorescence Staining with Cryosectioning

Drosophila heart models are widely employed in studying cardiac aging and modeling&#160;human cardiac diseases. However, the dissection of Drosophila hearts before imaging is a meticulous time-intensive process that requires advanced training and motor skills. To address these challenges, we present an innovative protocol that utilizes cryosectioning for the fluorescence imaging of Drosophila heart tissue. The protocol has been demonstrated in imaging the adult Drosophila heart but could be adapted for developmental stages. The method enhances both the efficiency and accessibility of fluorescence staining while preserving the integrity of the tissue. This protocol simplifies the process without compromising the quality of imaging, thereby reducing the dependency on technicians with highly developed training and motor skills. Specifically, we replace complex techniques, such as capillary vacuum suction, with more straightforward methods like tissue embedding. This approach allows for the visualization of cardiac structures with greater ease and reproducibility. We demonstrate the utility of this protocol by effectively detecting key cardiac markers and&#160;achieving high-resolution fluorescence and immunostaining imaging that unveils intricate details of heart morphology and cellular organization. This method provides a robust and accessible tool for researchers exploring Drosophila cardiac biology, facilitating detailed analyses of heart development, function, and disease models.

The Drosophila heart sectioning and fluorescence imaging protocol simplifies studying heart structure and pathologies. This approach involves straightforward sectioning, staining, and imaging, bypassing the technical expertise needed for traditional dissection. It enhances accessibility, making Drosophila a more widely usable model for cardiac-related research within the broader scientific community.

使用冷冻切片进行 果蝇 心脏荧光染色的新型高效方法

Drosophila heart models are widely employed in studying cardiac aging and modeling&#160;human cardiac diseases. However, the dissection of Drosophila ...

遗传学

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

发展和成人的制备果蝇大脑和视网膜实时成像

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function

Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular organization and genomic investigations to behavioral studies. Due to the accumulated scientific knowledge, in recent years, Drosophila was brought to the field of modeling human diseases, including heart disorders. The presented work describes the experimental system for monitoring and manipulating the heart function in the context of a whole live organism using red light (617 nm) and without invasive procedures. Control over the heart was achieved using optogenetic tools. Optogenetics combines the expression of light-sensitive transgenic opsins and their optical activation to regulate the biological tissue of interest. In this work, a custom integrated optical coherence tomography (OCT) imaging and optogenetic stimulation system was used to visualize and modulate the functioning D. melanogaster heart at the 3rd instar larval and early pupal developmental stages. The UAS/GAL4 dual genetic system was employed to express halorhodopsin (eNpHR2.0) and red-shifted channelrhodopsin (ReaChR), specifically in the fly heart. Details on preparing D. melanogaster for live OCT imaging and optogenetic pacing are provided. A lab-developed integration software processed the imaging data to create visual presentations and quantitative characteristics of Drosophila heart function. The results demonstrate the feasibility of initiating cardiac arrest and bradycardia caused by eNpHR2.0 activation and performing heart pacing upon ReaChR activation.

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function

The present protocol describes the generation of Drosophila melanogaster expressing eNpHR2.0 or ReaChR opsins in the heart for OCT imaging and optogenetic heart pacing. Detailed instructions for Drosophila OCT imaging and heart beating modulation, including the simulation of restorable heart arrest, bradycardia, and tachycardia in live animals at different developmental stages, are reported.

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）

摘要

探索更多视频

此视频中的章节

在小鼠心脏的动作电位和钙瞬变的光学测绘

单果蝇 Ommatidium解剖和影像

可视化心脏跳动果蝇

半自动小红桃光学心跳分析

为介观荧光体层摄影术在体内成像的发展果蝇

在幼虫的监测心脏功能果蝇生理研究

使用冷冻切片进行果蝇心脏荧光染色的新型高效方法

发展和成人的制备果蝇大脑和视网膜实时成像

GFP标记的蛋白质在实时成像

开发用于心脏功能成像和光遗传学控制的黑腹果蝇模型

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）

摘要

探索更多视频

此视频中的章节

在小鼠心脏的动作电位和钙瞬变的光学测绘

单果蝇 Ommatidium解剖和影像

可视化心脏跳动果蝇

半自动小红桃光学心跳分析

为介观荧光体层摄影术在体内成像的发展果蝇

在幼虫的监测心脏功能果蝇生理研究

使用冷冻切片进行 果蝇 心脏荧光染色的新型高效方法

发展和成人的制备果蝇大脑和视网膜实时成像

GFP标记的蛋白质在实时成像

开发用于心脏功能成像和光遗传学控制的 黑腹果蝇 模型

使用冷冻切片进行果蝇心脏荧光染色的新型高效方法

开发用于心脏功能成像和光遗传学控制的黑腹果蝇模型