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 border="0" cellpadding="0" cellspacing="0" width="515">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Custom OCM imaging system</td>
			<td style="width:136px;">Developed in our lab</td>
			<td style="width:95px;"></td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">my Temp Mini Digital Incubator</td>
			<td style="width:136px;">Benchmark</td>
			<td style="width:95px;">H2200-HC</td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Cover glass</td>
			<td style="width:136px;">AmScope</td>
			<td style="width:95px;">200PCS</td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Cotton Ball</td>
			<td style="width:136px;">RITE AID</td>
			<td style="width:95px;"></td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Instant Drosophila Formula</td>
			<td style="width:136px;">CAROLINA</td>
			<td style="width:95px;">formula 4-24</td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Yeast</td>
			<td style="width:136px;">ActiveDry</td>
			<td style="width:95px;"></td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Microscope</td>
			<td style="width:136px;">SONY</td>
			<td style="width:95px;">WILD M420</td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Brush</td>
			<td style="width:136px;">Loew-Cornell</td>
			<td style="width:95px;">245B</td>
			<td style="width:117px;">being used to move specimens</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Labview software</td>
			<td style="width:136px;">National Instruments</td>
			<td style="width:95px;"></td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Image J</td>
			<td style="width:136px;">National Institutes of Health</td>
			<td style="width:95px;"></td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Matlab</td>
			<td style="width:136px;">Mathworks</td>
			<td style="width:95px;"></td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Tweezer</td>
			<td style="width:136px;">Wiha</td>
			<td style="width:95px;">AA SA</td>
			<td style="width:117px;">to fix the fruit fly wings</td>
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			<td style="width:95px;">4,224,898</td>
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			<td height="21" style="height:21px;width:168px;">Pipette</td>
			<td style="width:136px;">Fisherbrand</td>
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			<td height="42" style="height:42px;width:168px;">Organic Extra Coconut Oil</td>
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			<td height="21" style="height:21px;width:168px;">Drosophila Vials</td>
			<td style="width:136px;">SEOH</td>
			<td style="width:95px;">8401SS</td>
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</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

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

适当的照顾和清洁的显微镜

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

科研

生物学

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

相衬，微分干涉对比（DIC）显微镜

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

生于正常化荧光整个心脏成像的光学投影层析成像

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

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  ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

制备果蝇 S2细胞，光学显微镜

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

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）

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