方法文章

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

DOI：

10.3791/55002

⸱

2016年12月12日

 ,  ,  ,  ,  ,  ,  ,  , 
1Bioengineering Program, Lehigh University, 2Center for Photonics and Nanoelectronics, Lehigh University, 3Department of Electrical and Computer Engineering, Lehigh University, 4State Key Laboratory of Software Engineering, Wuhan University, 5Genetics and Aging Research Unit, Department of Neurology, Massachusetts General Hospital and Harvard Medical School

本文内容

摘要

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这里描述了用于准备不同发育阶段的果蝇并使用定制的光学相干显微镜 （OCM） 系统对果蝇心跳进行纵向光学成像的实验方案。通过分析 OCM 图像中的心脏结构和功能参数，可以定量表征心脏形态和动力学变化。

摘要

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小动物心跳的纵向研究有助于了解心脏发育过程中的结构和功能变化。光学相干显微镜 （OCM） 已被证明能够以高空间分辨率和超高成像速度对小动物心脏进行成像。高图像对比度和无创特性使 OCM 成为进行纵向研究的理想选择，无需组织解剖或染色。果蝇因其大量直系同源人类疾病基因、分子机制和遗传途径与脊椎动物相似、生命周期短、培养成本低等优点，在心脏发育研究中被广泛用作模式生物。在这里，描述了在标本的整个生命周期中使用定制的 OCM 系统制备果蝇和心跳光学成像的实验方案。按照本报告中提供的步骤，可以获取横向 M 型和 3D OCM 图像，以对果蝇的心脏形态和功能进行纵向研究。还显示了面部和轴向截面 OCM 图像以及心率 （HR） 和心脏活动期 （CAP） 直方图，以分析果蝇过程中的心脏结构变化并量化心脏动力学，并结合用 M 模式图像构建的视频直观地追踪心脏活动。由于果蝇和脊椎动物之间的遗传相似性，对果蝇心脏形态和动力学的纵向研究有助于揭示人类心脏病的起源。这里的协议将提供一种有效的方法来进行广泛的研究，以了解人类心脏病的机制。

引言

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Longitudinal study of the heart in small animals contributes to understanding a variety of human related cardiovascular diseases, such as gene related congenital heart defects1,2. In the past decades, various animal models, such as mouse3,4, Xenopus5,6, zebrafish7,8, avian9, and Drosophila10-16, have been used to conduct the human heart-development related research. The mouse model has been widely used to study normal and abnormal cardiac development and cardiac defect phenotypes due to its similarities with the human heart3,4. The Xenopus embryo is especially useful in the study of heart development due to its easy handling and partial transparency5,6. The transparency of the embryo and early larva of the zebrafish model allows for easy optical observation of cardiac development7,8. The avian model is a common subject of developmental heart studies because the heart can be easily accessed after removing the eggshells and the morphological similarity of avian hearts to humans9. The Drosophila model has some unique features which make it ideal for performing longitudinal studies of the heart. First, the heart tube of Drosophila is ~ 200 µm below the dorsal surface, which provides convenience for optical access and observation of the heart. Additionally, many molecular mechanisms and genetic pathways are conserved between Drosophila and vertebrates. The orthologs of over 75% of human disease genes were found in Drosophila, which have made it widely used in transgenic studies11,13. Furthermore, it has a short life cycle and low maintenance costs, and has been commonly used as a specimen model for developmental biology research14-16.

Previous reports described the protocols for monitoring Drosophila cardiac functions such as the heartbeat. However, dissection procedures were required17,18. Optical imaging provides an effective way to visualize cardiac development in animals due to its non-invasive nature. Different optical imaging modalities have been applied in performing animal cardiac study, such as two-photon microscopy19, confocal microscopy20,21, light sheet microscopy22, and optical coherence tomography (OCT)16,23-26. Comparatively, OCT is capable of providing great imaging depth in small animal hearts without using contrast agents, while keeping a high resolution and an ultrahigh imaging speed, which are important for imaging live animals. Additionally, the low cost of developing an OCT system has popularized this technique for optical imaging of specimens. OCT has been successfully used for the longitudinal study of Drosophila. Using OCT, cardiac morphological and functional imaging has been performed to study the heart structures, the functional roles of genes, and the mechanisms of cardiovascular defects in mutant models during cardiac development. For example, age-dependent cardiac function decline was confirmed with down-regulated angiotensin-converting enzyme-related (ACER) gene in Drosophila with OCT27. Phenotyping of gene related cardiomyopathy was demonstrated in Drosophila using OCT28-33. Research using OCT also revealed the functional role of the human SOX5 gene in the heart of Drosophila34. Compared with OCT, OCM uses an objective with a higher numerical aperture to provide better transverse resolution. In the past, the heart dysfunction caused by silencing an ortholog human circadian gene dCry/dClock has been studied using a custom OCM system15,16, as well as the effect of high-fat-diet on cardiomyopathies in Drosophila to understand obesity induced human cardiac diseases.15

Here, the experimental protocol is summarized for longitudinal study of the cardiac morphological and functional changes in Drosophila at second instar (L2), third instar (L3), pupa day 1 (PD1), pupa day 2 (PD2), pupa day 3 (PD3), pupa day 4 (PD4), pupa day 5 (PD5), and adult (Figure 1) using OCM to facilitate study of human-related congenital cardiac diseases. Cardiac functional parameters, such as HR and CAP were quantitatively analyzed at different developmental stages to reveal the cardiac development features.

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方案

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1. Preparation of OCM System for Optical Imaging of Drosophila16

  1. Select a spectrometer and a high-speed line scan camera that provides a frame rate of at least 80 frame/sec so the OCM system will be able to resolve the heartbeat of Drosophila.
  2. Use a broadband light source to ensure the axial resolution of 2 µm to identify the heart structure of Drosophila.
  3. Use a 10X objective to obtain a high transverse resolution.
  4. Use a 45° rod mirror to reflect the reference arm light beam and to generate an annular sample arm light beam to extend the depth of focus in the specimens.
  5. Develop a custom computer program to control the OCM system and perform measurements.

2. Drosophila Culture

  1.  Standard Fly Food Preparation
    1. Put ~ 5 ml instant Drosophila formula into a polystyrene vial tube with the assistance of a paper chute.
    2. Pour ~ 8 ml water into the formula to properly saturate the food.
    3. Add different supplements to the standard fly food for different experiments. When preparing for all-trans-retinal (ATR) food for the optogenetic pacing experiment35, use a pipette to extract 100 mM ATR and dissolve into ~ 8 ml water to get the ATR concentration of 1 mM in food. After mixing the solution uniformly, pour the solution into the formula and stir it sufficiently.
    4. To prepare a high-fat-diet for studying obesity related heart dysfunctions in Drosophila,10,15 mix ~10 ml formula with 15 ml water in a cup and heat for 30 sec in a microwave oven. Put some organic extra virgin coconut oil in another cup and heat it for 90 sec in the microwave oven.
    5. Extract 7.5 ml coconut oil and mix with the prepared formula sufficiently to make the weight/volume ratio of coconut oil to food ~ 30/100, and then extract ~ 2 ml mixed food and put it to the bottom of a tube.
    6. Wait for 1 min until the medium is thoroughly saturated. Compact the food carefully with a flat surface to optimize the living conditions for the Drosophila. Add 6 - 8 grains of yeast to the prepared formula, and plug the tube with a cluster of cotton.
  2. Fruit Fly Crosses and Culture
    1. Take a tube with prepared standard fly food, and remove the plugged cotton. Carefully transfer the adult flies (male and female) to the tube, and plug the tube with cotton immediately. Check the cotton to make sure that there is no gap between the cotton and the tube wall to prevent flies from escaping from the tube.
    2. Keep the fruit flies in the incubator at 25 °C for cross breeding. Most of the genes are active and the cellular proteins are synthesized at 25 °C36-39.
    3. Take the tube out from the incubator after 8 hand transfer the adult flies out of the tube to obtain the eggs at similar age for experimental control.
    4. Continue culturing the eggs in the incubator at 25 °C, which is the standard temperature for Drosophila development with the development period of 8.5 days40,41.
      NOTE: Temperature influences the developmental period (egg to adult) and expression level of various genes.

3. Performing Optical Imaging with OCM

  1. Mount Fly Larva for Optical Imaging
    NOTE: The egg of Drosophila hatches in 22 - 24 hr at 25 °C to the first instar larva (L1). The second instar larva emerges after another 24 hr. The largest larval form is the third instar larva, which molts after about 24 hr. Structural characteristics in larva can be used to distinguish their different developmental stages. The size of the mouthparts between the first instar and the second instar is different. The mouth hooks of the first instar larva are very small and look like two pairs of tiny black spots, while the mouth hooks of the second instar larva are larger and the structure is clearer. The spiracles are usually used to identify the second instar and the third instar. The second instar larva has clubbed anterior spiracles, while, for the third instar, the anterior spiracles are branched. A dark orange ring will begin to appear at the tip of the posterior spiracles in the third instar larva.
    1. Apply a piece of double sided tape to a clean microscope glass slide. Expel the air bubbles under the tape to avoid the reflections caused by air bubbles during imaging.
    2. Take one of the tubes with the cultured flies out from the incubator at the larval stage.
    3. Identify the larva in the media, remove it from the media with a soft brush and place on a clean tissue. Remove any food stuck to the larva with a wet soft brush and dry it on the tissue.
    4. Move the cleaned fly to a tissue under the objective lens of a wide field microscope.
    5. Adjust the focus of the microscope to find a clear view of the fly. Identify the right developmental stage of the larva by its structural characteristics with the microscope.
    6. Position the fly using the soft brush. Ensure the body is straight with the dorsal side facing upward to prepare for mounting on the glass slide by the dorsal side. Perform this step under the microscope.
    7. Ensure the larva is completely dry before mounting on the tape. Otherwise, the larva will not adhere to the tape.
    8. Stick the dorsal side of the positioned fly to the double side tape on the glass slide with moderate pressure. Note that too much pressure may kill the fly and too little force will lead to fly movement during imaging.
  2. Optical Imaging of Drosophila at Larval Stages (L2 and L3) with OCM
    NOTE: A broad lumen of the heart tube can be found located in the segments between A5 to A8 at the larval stages (Figure 1). The transverse OCM M-mode images (2D + time) were acquired at the A7 segment of the heart tube for each larva to facilitate the systolic and diastolic analysis.
    1. Place the mounted larva on the adjustable sample stage of the OCM system along the y-transverse direction with the dorsal side facing upward below the objective lens. A small hole in the sample stage is necessary for placing the larva to avoid its contact with the stage plane.
    2. Adjust the sample stage to move the heart tube of the fruit fly to the focal plane of the imaging beam. To easily find the A7 segment, find the posterior region of the heart tube with the real time cross sectional OCM images in the image acquisition software. Then move the stage forward until the A7 segment is visible.
    3. Set parameters of the image acquisition software to 100 A-scans per B-scan (frame), 100 B-scans, and the scanner voltage to cover ~ 0.28 mm in the x-transverse direction, and 0 V in the y-transverse direction. Click the "start" button in the software to acquire the background noise data for background subtraction by blocking the sample beam path with a dark cloth.
      NOTE: 3 of the 100 frames can be used for the background subtraction.
    4. Set parameters of the data acquisition software to 128 A-scans per B-scan, 4096 B-scans, and the scanner voltage to cover ~ 0.28 mm in the x-transverse direction, and 0 V in the y-transverse direction. Click the "start" button in the software to acquire the transverse M-mode images across the A7 segment of the fly heart tube over a region covering 0.28 x 0.57 mm2 for about 30 sec.
    5. Block the imaging beam using a dark cloth during the data saving process to avoid lengthy exposure of the fly heart to the imaging light.
    6. Repeat the measurement for 5 times to get reliable measurement of the heart function.
    7. Set parameters of the image acquisition software to 400 A-scans per B-scan, 800 B-scans, and the scanner voltage to cover ~ 1.7 mm in the x-transverse direction, and ~ 4 mm in the y-transverse direction. Move the stage in both directions to ensure the entire fruit fly can be imaged. Click the "start" button in the software to acquire one dataset to obtain images of the fruit fly in 3 dimensions. Note: The 3D fly structure can be rendered using Amira 3D software
    8. Use a wet soft brush to moisten the measured fly and gently remove it from the glass slide. Move it into a separate tube for continuous development. Label the tube for longitudinal study through the next developmental stages.
  3. Image Drosophila at Pupal Stages
    NOTE: All the fruit flies were taken out for imaging from PD1 to PD5. As shown in the larva schematic in Figure 1b, a broad lumen remains in A5 to A8 segments of the heart tube until PD1. From PD2, a conical chamber starts to develop between A1 to A4 segments. To acquire consistent images and facilitate heart analysis, transverse M-mode images were obtained from the A7 segment at PD1, and from A1 segment after PD2, as marked in Figure 1b.
    1. Image Drosophila at PD1
      NOTE: Drosophila will have a white puparium for a short time window (0 - 1 hr) during PD1. This time window is ideal for performing optical imaging of early pupa because the high transparency leads to higher light penetration for the OCM imaging.
      1. As the fruit flies are found on the tube wall when they become pupa, remove the pupa from individual tubes for imaging at PD1 with a wet soft brush, and clean the pupa with the brush if there is food stuck to the body.
      2. Mount the fruit fly on a small glass slide directly with the wet brush and keep the dorsal side facing upward (Figure 1a). Make sure the glass slide is small enough to fit back into the tube once imaging at this stage is complete.
      3. Remove excessive water from the side of the fly body.
      4. Put the glass slide on the sample stage of the OCM system, keeping the fruit fly on top. Find clear real-time image of the A7 segment of the fly heart utilizing the same strategy described in the larva measurement.
      5. Set the same parameters of the data acquisition software as in section 3.2, and image the heartbeats at the A7 segment to acquire transverse M-mode and 3D images.
      6. After imaging, use a tweezer to place the glass slide with pupa back into the tube for continuous culture.
    2. Image Drosophila at PD2 to PD5 Stages
      NOTE: Since the specimen becomes more and more opaque during the pupal stages, the penetration depth of the imaging system will be reduced.
      1. Use a tweezer to carefully remove the glass slide mounted with the fly at PD2 from the tube for imaging. At PD2, the specimen shell becomes yellowish and the body becomes less transparent compared to PD1 (Figure 1).
      2. Put the slide on the sample stage of the OCM system.
      3. Adjust the sample stage to move the fly into the focal plane of imaging beam of the OCM system. Find the anterior end of the heart tube with real-time cross-sectional OCM image. Move ~ 50 µm back in the posterior direction to find the A1 segment of the heart tube.
        NOTE: At this point of heart development (PD2), the conical chamber will be very small and may not be beating.
      4. Collect transverse M-mode datasets from the A1 segment as well as 3D data using the same method as previous developmental stages.
      5. Put the slide back to the tube carefully for continuous culture.
        NOTE: At PD3, the color of the specimen in the shell is darker than that at PD2 stage. At PD4 stage, black stripes can be observed inside the shell of the specimens. Some flies will develop into adult from this stage in the following day, while others will evolve into PD5. At PD5 stage, black stripes are even more obviously seen in the fruit flies. These flies will become adults in the following day.
  4. Image Drosophila at the Adult Stage
    NOTE: At the adult stage, female and male flies can be distinguished by the size of the body and the color of the lower abdomen. Female adults have larger size, while males are smaller and dark-colored in the lower abdomen.
    1. Take the tube out from the incubator when the fruit fly develops into an adult, and transfer the adult fly to a ~ 45 ml empty vial.
    2. Dip the absorbent end (~ 1 cm length, ~ 3 mm diameter) of a wand into the anesthesia, put the wand into the vial, and plug the tube with a cluster of cotton to keep the anesthetic end just below the plugged cotton and to anesthetize the fly for 3 min. The duration of anesthesia depends on the size of the fly, and may vary between 2.5 to 3.5 min (for example: male for 2.5 min, female for 3 or 3.5 min).
    3. Prepare a glass slide with a piece of double sided tape.
    4. Move the anesthetized fly onto the glass slide with dorsal side facing upward using the soft brush.
    5. Separate the wings using a tweezer and stick the wings on the tape under a microscope to fix the fly and expose the heart region for imaging.
    6. Image the fly from the A1 segment of the fly heart (Figure 1). At the end of the experiment, the fly may be sacrificed.

4. Imaging Analysis16

  1. Develop Matlab programs to convert the 2D and 3D binary files collected with the image acquisition software to image files.
  2. Use ImageJ to identify the heart tube region in the transverse M-mode images and a magic wand algorithm to create a mask of the heart region for each transverse M-mode image. Segment the masked region and use a peak-finding algorithm to identify the systolic and diastolic locations. Calculate the time dependent heart diameter changes from the transverse M-mode images.
  3. Based on the acquired time dependent heart diameters, calculate the cardiac parameters such as HR, cardiac activity period (CAP),end diastole diameter (EDD), end systole diameter (ESD), end diastole area (EDA), and end systole area (ESA). Calculate the fractional shortening (FS) with Fractional shortening formula equation; FS=(EDD-ESD)/EDD×100; cardiac measurement.
  4. Use ImageJ to analyze the 3D OCM images to visualize the structural development of the fly heart.

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结果

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The longitudinal cardiac imaging was conducted using the fruit flies with the 24B-GAL4/+ strain at room temperature with OCM. Measurements were performed at L2, L3, and at 8 hr intervals from PD1 to PD4, and adult day 1 (AD1) to track the metamorphosis process (Table 1). Larva, early pupa, late pupa and adult flies were mounted on the glass slides as seen in Figure 1A. The segment features of the heart for larval and adult flies were shown in the schematic representations in Figu...

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讨论

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The rapid heartbeat of Drosophila, with a maximum HR around 400 bpm at larval and adult stages, requires high imaging speed to resolve the heart diastoles and systoles (no less than 80 frames/sec based on experiences). Due to the small heart chamber size and micron scale heart wall thickness (5 - 10 µm), a high spatial resolution (better than 2 µm) is required for resolving the heart tube structures. In this study, a high resolution and ultrahigh speed OCM system was developed, where a spectrometer wit...

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披露

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作者声明与当前研究没有相关的利益冲突。

致谢

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这项工作得到了利哈伊大学创业基金、NIH（R00EB010071 到 C.Z.、R15EB019704 到 C.Z. 和 A.L.、R03AR063271 到 A.L.、R01AG014713 和 R01MH060009 到 R.E.T.）、NSF（1455613 到 C.Z. 和 A.L.）、治愈阿尔茨海默病基金（到 R.E.T.）和马萨诸塞州总医院（A.L. 研究奖执行委员会）的支持。MC 和 Y.M. 得到了国家重点基础研究计划的支持中国（973 计划），资助号 2014CB340404。

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材料

本文使用的材料清单
姓名公司目录编号评论
定制 OCM 成像系统实验室
my Temp Mini 数字培养箱BenchmarkH2200-HC
盖玻片AmScope200PCS
棉球RITE AID
即时果蝇配方CAROLINA配方 4-24
酵母ActiveDry
显微镜SONYWILD M420
刷Loew-Cornell245B用于移动标本
Labview 软件National Instruments
ImageJ美国国立卫生
MatlabMathworks
镊子WihaAA SA固定水果飞翼
FlyNapCarolina Biological Supply Company4,224,898
苏格兰永久性双面胶带，3 M苏格兰
移液器FisherbrandMU18837
有机额外椰子油Spring Valley13183
显微镜载玻片CapitolBrandM3504-E
果蝇 样品瓶SEOH8401SS
全反式视网膜Sigma-Aldrich Co.R2500
在我们的开发子 研究院

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