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Quantitative 3D Time Lapse Imaging of Muscle Progenitors in Skeletal Muscle of Live Mice
活小鼠骨骼肌中肌肉祖细胞的定量三维延时成像   

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Abstract

For non-optically clear mammalian tissues, it is now possible to use multi-photon microscopy to penetrate deep into the tissue and obtain detailed single cell images in a live animal, i.e., intravital imaging. This technique is in principle applicable to any fluorescently marked cell, and we have employed it to observe stem cells during the regenerative process. Stem cell-mediated skeletal muscle regeneration in the mouse model has been classically studied at specific time points by sacrificing the animal and harvesting the muscle tissue for downstream analyses. A method for direct visualization of muscle stem cells to gain real-time information over a long period in a live mammal has been lacking. Here we describe a step-by-step protocol adapted from Webster et al. (2016) to quantitatively measure the behaviors of fluorescently labeled (GFP, EYFP) muscle stem and progenitor cells during homeostasis as well as following muscle injury.

Keywords: Muscle stem cell(肌肉干细胞), Muscle progenitor(肌肉祖细胞), Muscle regeneration(肌肉再生), Ghost fiber(幽灵纤维), Live imaging(实时成像), Multi-photon microscopy(多光子显微镜), Second harmonic generation(二次谐波产生)

Background

Long-term in vivo imaging of stem and progenitor cells was first used for hair follicles during continuous physiological regeneration without surgical procedure (Rompolas et al., 2012). By contrast, stem cells for skeletal muscles are largely quiescent and inactive during the normal homeostatic state. An injury to the muscle is necessary to activate muscle stem cells to mount a regenerative process. In vitro live imaging of muscle stem/progenitor cells has been widely used to study them in artificial settings. To understand muscle stem cell behavior during regeneration in their native environment, we developed a method to image them during skeletal muscle regeneration. Our method allows up to 8 h of continuous imaging per session daily following injury. This is the first time that skeletal muscle stem cells have been observed in vivo in an injured/regenerative environment (Webster et al., 2016).

Materials and Reagents

  1. Lab tape (VWR, catalog number: 89097 )
  2. Razor blades for shaving hair (VWR, catalog number: 55411-060 )
  3. Kimwipes (ULINE)
  4. Transfer pipette (Globe Scientific, catalog number: 137038 )
  5. Cyanoacrylate based adhesive that bonds skin to glass (i.e., Loctite glass glue; Amazon.com; Scognamiglio et al., 2016)
  6. Mouse with GFP or YFP expression in Pax7-expressing muscle stem cells (or cell types of interest); GFP and YFP Cre-reporter mice and Pax7-Cre-ERT2 mouse strains are available at Jackson Laboratory (THE JACKSON LABORATORY, catalog numbers: 021847 , 006148, and 012476, respectively)
  7. Isoflurane (Patterson Veterinary Supply, IsoFlo®, catalog number: 07-806-3204 ; To be applied directly into the vapor chamber of equipment list 4; indicated by arrow in Figure 2)
  8. EtOH, 70% (Decon Labs, catalog number: V1401 )
  9. Distilled water (in house)

Equipment

  1. An inverted Leica SP5 (or equivalent) equipped with a Leica 25x/0.95 water objective, a 35 mm culture dish holder attached to the stage, and nondescanned detectors with a dichroic mirror separating the detectable spectrum (430-550 nm) at 495 nm (Figure 1)


    Figure 1. Inverted Multi-photon setup. The imaging setup includes an inverted confocal microscope with 25x water objective fitted with a culture dish holder, multi-photon laser and computer.

  2. Chameleon Vision laser (Coherent)
  3. Heating pad with enough area to cover the animal on the microscope stage (Sunbeam 756-500 Heating Pad from Amazon.com)
  4. Animal anesthesia system equipped with induction chamber as well as tubing with nose-cone (VetEquip, catalog number: 901806 ). Please consult with the operation manual prior to use
  5. 35 mm fluorodish (World Precision Instruments, catalog number: FD35-100 )
  6. Metal spatula (VWR, catalog number: 82027-530 )
  7. Heat protective glove (VWR)
  8. Bunsen burner (VWR, catalog number: 89038-528 )
  9. Laminar flow fume hood
  10. Fine tip forceps (Dumont #5 forceps) (Fine Science Tools, catalog number: 11252-30 )
  11. Fine scissors for cutting skin (straight, 11.5 cm) (Fine Science Tools, catalog number: 14058-11 )
  12. Spring scissors for cutting fascia (8 mm blades) (Fine Science Tools, catalog number: 15009-08 )

Software

  1. Leica SP5 software
  2. Fiji (Fiji is an updated version of ImageJ, an open source image processing software; Schindelin et al., 2012)
  3. Imaris (Bitplane, version 7.6.4 for Windows X64)

Procedure

Ethical statement: All procedures discussed here are in accordance with and were approved by the Carnegie Institution for Science Institutional Animal Care and Use Committee.

  1. Intravital imaging setup
    1. Turn on inverted confocal microscope and multi-photon (MP) laser. Set MP to 920 nm for GFP detection, or 940 nm for EYFP detection.
    2. Start Leica imaging software.
    3. Turn on heating pad and warm to 35 °C.
    4. Begin oxygen flow to anesthesia machine and then begin filling induction chamber with 1.7% isoflurane (by adjusting the turn dial on top of the container to 1.7) and the flow-meter indicator ball to 1 (for 1 L/min flow rate) (See Figure 2).
    5. Place mouse in induction chamber to begin anesthetization (Figure 2) and continue onto making modified fluorodish. The anesthetized mouse can stay in the induction chamber for this duration (~20 min). If the fluorodish preparation takes longer, the mouse can be left in the induction chamber without concern, as they will continue to be placed under similar anesthesia condition during imaging. 
       

      Figure 2. Anesthetization setup. This consists of anesthesia machine with regulator, induction chamber and nose-cone (pictured in Figure 4).

  2. Prepare modified fluorodish for mounting on mouse’s leg
    1. Wearing a heat protective glove, heat metal spatula over Bunsen burner in a fume hood.
    2. Use heated spatula to melt and remove a 2-3 cm section of the fluorodish wall. Take care not to melt and deform the bottom of the fluorodish, which can be problematic when fitting the fluorodish into the microscope stage holder (see step 4e). It is important to leave part of the fluorodish to secure and stabilize it in the stage holder.
    3. Repeat steps 2a and 2b and remove a second section from the opposite side of the fluorodish.
    4. This can be done ahead of time; multiple fluorodishes can be prepared and stored for future use (Figure 3).


      Figure 3. Modified fluorodish

  3. Mount fluorodish on mouse
    1. Move mouse from induction chamber to procedure surface where anesthesia tubing with attached nose-cone has been fastened with tape (Figure 4).
    2. Secure mouse’s head by placing a small piece of tape over the head across to the nose-cone.
    3. Check level of anesthetization by observing the mouse. The mouse should maintain a steady rate of respiration but not react to slight pinches with forceps on the skin of the leg. If the mouse’s respiration is very slow, decrease the Isoflurane to 1.3% to 1.5%. If the mouse flinches when leg is pinched, then wait longer for mouse to enter deeper state of anesthetization before proceeding.
    4. Use a razor blade to shave hair from skin covering the tibialis anterior (TA) muscle. Begin shaving at the ankle and work up to the knee. Remove any lose hair from the lower leg. This can be done by washing the skin with 70% EtOH followed by Kimwipes to remove loose hairs.
    5. Ready adhesive and set aside.
    6. Cut a thin strip of skin from the region over the TA by pulling a small piece of skin up with forceps and cutting with scissors, beginning near the ankle and cutting up towards the knee. Do not remove too much skin, only enough to expose a 1-2 cm2 area over the TA.
    7. Remove the fascia covering the TA by gently pulling up on the membrane at the side of the TA and then cutting with scissors. One may avoid damaging the muscle by using forceps to pull the membrane at the edge of the TA without making direct contact with tissue that is to be imaged.
    8. Place the modified fluorodish on the mouse’s leg so that the surface of the dish is flush with the exposed muscle and the mouse’s upper leg/body and lower leg/ankle fit in the spaces in the dish wall that have been removed. Secure the dish in place by holding the mouse’s leg in contact with the glass bottom of the dish. The remaining sides of the modified fluorodish should run parallel with the length of the mouse’s limb to be imaged.
    9. While holding the dish in place, apply adhesive where the skin of the leg is in contact with the glass. Apply adhesive around the entire perimeter and let the adhesive set for 90 sec or more while holding the dish in place.


      Figure 4. Fluorodish mounted on mouse. Pseudo-colored white ring indicates position to apply adhesive, performed using nose-cone anesthesia.

    10. Begin filling the induction chamber with 1.7% isoflurane at 1 L/min. Maintain flow to the nose-cone.
    11. Once adhesive has set, return mouse to induction chamber and stop flow to the nose-cone.
  4. Position mouse for intravital imaging
    1. Tape the anesthesia tube with nose-cone to the microscope stage.
    2. If using a water immersion objective, apply water to the surface of the objective.
    3. Switch delivery of 1.7% isoflurane at 1 L/min to the nose-cone outlet.
    4. Remove mouse from induction chamber and place on microscope stage, placing the head of the mouse into the nose-cone. Secure the head to the nose-cone with tape. The mouse should be positioned on its abdomen. When placed on its back, lungs often collapse prematurely terminating imaging (~1-2 h).
    5. Place the fluorodish in the culture dish holder affixed to the stage (Figure 5I).
    6. Raise the objective lens into position and identify the imaging field using the epifluorescence function of the Leica SP5. A piece of tape may be placed across the lower portion of the mouse’s body (attached on either side to the stage) to reduce movement from respiration.
    7. Place heating pad over mouse (Figure 5II).
    8. Reduce anesthesia to 1.3% isoflurane at 1 L/min.


      Figure 5. Mouse positioned for intravital imaging. I. Stage setup. A) Exposed TA muscle for imaging mounted to modified fluorodish. B) Nose-cone anesthesia apparatus adhered to stage. C) Culture dish holder. D) 25x water objective with H2O (pictured below field of view). II. Microscope setup with positioned heating pad.

  5. Image capture parameters and analyses
    1. Capture serial optical sections at 3 μm steps to a total depth of 150-200 μm every 3 min for up to 8 h. Scan fields of 0.31 to 0.62 mm2 at 400 Hz. Following imaging, the mouse is sacrificed. Due to the adhesive used, it is not possible to remove fluorodish without causing additional tissue damage for proper suture, and the fluorodish is too bulky to be left on the mouse without affecting mobility.
    2. Image sequence files (.lif) can be opened in Fiji and converted to .tiff stacks or image sequences for analysis.

Data analysis

Data storage, transfer, and processing:

  1. A Leica generated .lif file can range from 3 to 8 GB.
  2. A single .tiff image sequence (converted from .lif using Fiji) is about 3 GB (3D time lapse).
  3. A single .tiff stack (3D time lapse) is ≤ 1 GB.
  4. A single .tiff stack (2D time lapse) is ≤ 100 MB.
  5. An .avi movie is ≤ 20 MB.
  6. Image sequences can be aligned using the Fiji plugin, ImageJ 2.0.0 ‘Correct 3D drift’.
  7. Drift-corrected image sequences can be analyzed further with Fiji (e.g., long axes and division angle measurements of cells, as well as measurements of cell migration direction). Imaris 7.6.4 is used for 4D rendering, cell tracking, migration statistics, and 4D movies. 
  8. The processed data (images and videos) and statistical analyses are published in Webster et al. (2016), which can be found at: http://www.cell.com/cell-stem-cell/fulltext/S1934-5909(15)00503-2

Notes

  1. Obstructions to light transmission include hair, red blood cells, the epimysium, and adhesive. These can be avoided by removing all lose hair from the leg during step 3d, minimizing skin removal and damage to vasculature during steps 3e and 3f, removing all membrane covering the exposed TA in step 3f, and ensuring that adhesive does not cover the exposed tissue in step 3i.
  2. Mounting the fluorodish on the leg can be quite challenging and may require some practice. Fitting a P1000 pipette tip on the adhesive tube can allow for easier access and delivery of adhesive to the interface of skin and glass at the perimeter of the exposed TA.
  3. A shroud placed over the stage/microscope may be necessary to block ambient light from causing high background signal, especially when using very sensitive non-descanned detectors (Figure 6).
  4. For long duration imaging sessions, repeated applications of water are needed. To overcome the hassle of pausing the experiment and applying water to the objective lens, a water delivery objective lens ring cover and pump apparatus can be used (New Era Pump Systems, Inc.).
  5. This protocol can be adapted to other fluorescent proteins (i.e., tdT, CFP) by adjusting MP wavelength and detection filters accordingly.
  6. All reagents used in this protocol were purchased and used directly without any dilution or mixing.


    Figure 6. Imaging with shroud

Acknowledgments

The protocol reported here was supported by the NIAMS of the NIH under award numbers F32AR065366 (to MTW) and RO1AR060042 (to C-MF), as well as by intramural funds of the Carnegie Institution (to TH). This protocol is a modified version derived from a previous publication in Cell Stem Cell (cited in the reference, Webster et al., 2016).

References

  1. Rompolas, P., Deschene, E. R., Zito, G., Gonzalez, D. G., Saotome, I., Haberman, A. M. and Greco, V. (2012). Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration. Nature 487: 496-499.
  2. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  3. Scognamiglio, F., Travan, A., Rustighi, I., Tarchi, P., Palmisano, S., Marsich, E., Borgogna, M., Donati, I., de Manzini, N. and Paoletti, S. (2016). Adhesive and sealant interfaces for general surgery applications. J Biomed Mater Res B Appl Biomater 104(3): 626-639.
  4. Webster, M. T., Manor, U., Lippincott-Schwartz, J. and Fan, C. M. (2016). Intravital imaging reveals ghost fibers as architectural units guiding myogenic progenitors during regeneration. Cell Stem Cell 18(2): 243-252.

简介

对于非光学清晰的哺乳动物组织,现在可以使用多光子显微镜深入渗透到组织中,并且在活体动物(即,即活体成像)中获得详细的单细胞图像。这种技术原则上适用于任何荧光标记的细胞,我们已经使用它来观察再生过程中的干细胞。小鼠模型中的干细胞介导的骨骼肌再生已经在特定时间点通过牺牲动物和收获肌肉组织进行下游分析进行了经典研究。一直缺乏直接观察肌肉干细胞以在活体哺乳动物中长时间获得实时信息的方法。在这里,我们将介绍一种从Webster等人修改的分步骤协议。 (2016),以定量测量稳态期间荧光标记(GFP,EYFP)肌肉干细胞和祖细胞的行为以及肌肉损伤后的行为。

背景干细胞和祖细胞的长期体内成像首先在无外科手术的连续生理再生中用于毛囊(Rompolas等人,2012)。相比之下,骨骼肌的干细胞在正常的内稳态期间主要是静止的和无活性的。需要肌肉的损伤来激活肌肉干细胞来安装再生过程。肌肉干细胞/祖细胞的体外成像已被广泛用于人造环境中的研究。为了了解在自然环境中再生过程中的肌肉干细胞行为,我们开发了一种在骨骼肌再生过程中对其进行成像的方法。我们的方法允许高达8小时的连续成像每次每次伤害后。这是在受伤/再生环境(Webster等人,2016)中首次观察到体内骨骼肌干细胞。

关键字:肌肉干细胞, 肌肉祖细胞, 肌肉再生, 幽灵纤维, 实时成像, 多光子显微镜, 二次谐波产生

材料和试剂

  1. 实验室胶带(VWR,目录号:89097)
  2. 剃须刀剃须刀(VWR,目录号:55411-060)
  3. Kimwipes(ULINE)
  4. 转移移液器(Globe Scientific,目录号:137038)
  5. 将皮肤粘合到玻璃上的基于氰基丙烯酸酯的粘合剂(即,乐泰玻璃胶; Amazon.com; Scognamiglio等人,2016)
  6. 在表达Pax7的肌肉干细胞(或感兴趣的细胞类型)中具有GFP或YFP表达的小鼠; GFP和YFP Cre报告小鼠和Pax7-Cre-ERT2小鼠品系可以在Jackson Laboratory(THE JACKSON LABORATORY,目录号:021847,006148和012476)分别获得
  7. 异氟烷(Patterson Veterinary Supply,IsoFlo ®,目录号:07-806-3204;直接应用于设备清单4的蒸气室;由图2中的箭头表示)
  8. EtOH,70%(Decon Labs,目录号:V1401)
  9. 蒸馏水(室内)

设备

  1. 装有Leica 25x/0.95水体物镜的反向Leica SP5(或等效物),附着在舞台上的35mm培养皿保持架以及在495nm处分离可检测光谱(430-550nm)的二向色镜的非扫描检测器(图1)


    图1.反向多光子设置成像设置包括一个倒置的共聚焦显微镜,配有25x水样,配有培养皿支架,多光子激光和计算机。

  2. Chamelion Vision激光(相干)
  3. 具有足够面积的加热垫覆盖显微镜平台上的动物(Sunbeam 756-500来自Amazon.com的加热垫)
  4. 装有感应室的动物麻醉系统以及鼻锥管(VetEquip,目录号:901806)。使用前请咨询操作说明书
  5. 35 mm fluorodish(世界精密仪器,目录号:FD35-100)
  6. 金属铲(VWR,目录号:82027-530)
  7. 防护手套(VWR)
  8. 本生灯(VWR,目录号:89038-528)
  9. 层流通风柜
  10. 精细镊子(Dumont#5镊子)(精细科学工具,目录号:11252-30)
  11. 用于切割皮肤的精细剪刀(直,11.5厘米)(精细科学工具,目录号:14058-11)
  12. 用于切割筋膜的弹簧剪刀(8毫米刀片)(精细科学工具,目录号:15009-08)

软件

  1. 徕卡SP5软件
  2. 斐济(斐济是ImageJ的更新版本,开源图像处理软件; Schindelin等人,2012)
  3. Imaris(Bitplane,版本7.6.4,适用于Windows X64)

程序

道德声明:本文讨论的所有程序均符合卡内基科学机构动物保护和使用委员会的批准。

  1. 活检成像设置
    1. 打开反相共聚焦显微镜和多光子(MP)激光。将GFP设置为920 nm进行GFP检测,或将940 nm用于EYFP检测。
    2. 启动Leica成像软件。
    3. 打开加热垫并加热至35°C。
    4. 开始氧气流到麻醉机,然后开始用1.7%异氟烷填充诱导室(通过将容器顶部的转盘调整到1.7),将流量计指示器球调整到1(1L/min流速)(见图2)
    5. 将鼠标置于诱导室中以开始麻醉(图2),并继续进行修饰的氟化物。麻醉的小鼠可以停留在感应室中持续这段时间(约20分钟)。如果氟牙膏制剂需要更长时间,则可以将鼠标留在感应室中,而不用担心,因为它们将在成像期间继续放置在类似的麻醉条件下。 
       

      图2.麻醉设置这包括具有调节器,感应室和鼻锥的麻醉机(如图4所示)。

  2. 准备修改的氟染色以安装在鼠标的腿上
    1. 穿着防护手套,在通风橱里的本生灯上加热金属铲。
    2. 使用加热刮刀熔化并去除2-3厘米的氟壁。注意不要使氟染色体的底部熔化和变形,当将荧光剂装入显微镜台架时(参见步骤4e),这可能是有问题的。重要的是留下一部分氟牙膏以固定和稳定在舞台上。
    3. 重复步骤2a和2b,并从氟染色的相对侧移除第二部分。
    4. 这可以提前完成;可以制备和储存多种荧光素以备将来使用(图3)

      图3.修改的氟化物

  3. 在小鼠上安装荧光
    1. 将鼠标从诱导室移动到手术表面,其中带有附着鼻锥的麻醉管已用胶带固定(图4)。







    2. 通过观察鼠标检查麻醉水平。鼠标应保持稳定的呼吸速率,但不会对腿部皮肤上的镊子产生轻微的夹持作用。如果老鼠的呼吸非常缓慢,将异氟烷降至1.3%至1.5%。如果当腿部被挤压时鼠标退缩,则等待更长时间才能进入更深的麻醉状态,然后继续。
    3. 使用剃须刀刮去头皮以覆盖胫前肌(TA)肌肉。开始在脚踝处剃须,然后上膝盖。从小腿去除任何失去的头发。这可以通过用70%EtOH洗涤皮肤,然后用Kimwipes洗涤去除松散的毛发。
    4. 准备粘合剂并放在一边。
    5. 用镊子将一小块皮肤从剪刀上切开,然后从脚踝附近开始向膝盖切开,从TA上方的区域切出一条薄薄的皮肤。不要去除太多的皮肤,只能在TA上露出1-2厘米的 2 区域。
    6. 通过轻轻拉起TA侧面的膜,然后用剪刀切割,去除覆盖TA的筋膜。人们可以通过使用镊子在TA的边缘拉动膜而不与要成像的组织直接接触来避免损伤肌肉。
    7. 将修改后的氟化物放置在鼠标的腿部上,使得表面与暴露的肌肉齐平,鼠标的上腿/身体和小腿/脚踝适合已移除的碗壁上的空间。通过握住鼠标的腿与盘的玻璃底部接触来将盘固定在适当位置。修饰的荧光的剩余侧面应与待成像的小鼠肢体的长度平行。
    8. 在将盘保持在适当位置时,涂抹粘合剂,使皮肤与玻璃接触。在整个周边涂抹粘合剂,并将粘合剂固定在90秒以上,同时将盘固定在适当位置。


      图4.安装在鼠标上的氟化物伪彩色白色环表示使用鼻锥麻醉进行粘合剂的位置。

    9. 开始以1升/分钟用1.7%异氟烷填充感应室。保持流向鼻锥。
    10. 粘合剂固定后,将鼠标移至感应室,停止流向鼻锥。
  4. 定位鼠标进行活体成像
    1. 将具有鼻锥的麻醉管贴到显微镜载物台上
    2. 如果使用水浸物镜,将水施加到物镜表面。
    3. 将1.7%异氟烷以1升/分钟的速度交付给鼻锥出口。
    4. 从感应室取出鼠标,放在显微镜平台上,将鼠标头放入鼻锥。用胶带把头固定在鼻锥上。鼠标应位于其腹部。当放置在背部时,肺常常会过早终止成像(〜1-2小时)。
    5. 将氟化物置于固定在舞台上的培养皿中(图5I)
    6. 使用Leica SP5的落射荧光功能将物镜提升到位置并识别成像场。一条胶带可以放置在鼠标的身体的下部(连接到舞台的两侧),以减少呼吸的运动。
    7. 将加热垫放在鼠标上(图5II)
    8. 以1升/分钟将麻醉减至1.3%异氟烷。


      图5.用于活体成像的鼠标。 I.舞台设置。 A)暴露的TA肌肉用于成像安装到修饰的荧光。 B)鼻锥麻醉器附着于舞台。 C)培养皿架。 D)具有H 2 O 5的25倍水体(如下图所示)。二,显微镜设置与定位加热垫。

  5. 图像捕捉参数和分析
    1. 以3μm的步长捕获串联光学部分,总共深度为150-200μm,每3分钟长达8 h。在400Hz下扫描场为0.31至0.62mm 2。成像后,处死小鼠。由于使用的粘合剂,不可能除去氟染,而不会对适当的缝合造成额外的组织损伤,并且氟化物太笨重,不会留在小鼠上而不影响移动性。
    2. 图像序列文件(.lif)可以在斐济打开并转换成.tiff堆栈或图像序列进行分析。

数据分析

数据存储,传输和处理:

  1. 徕卡生成的.lif文件的范围可以是3到8 GB。
  2. 单个.tiff图像序列(使用斐济从.lif转换)约为3 GB(3D时间流逝)。
  3. 单个.tiff堆栈(3D时间流逝)≤1 GB。
  4. 单个.tiff堆栈(2D时间流逝)≤100MB。
  5. .avi电影不超过20 MB。
  6. 图像序列可以使用Fiji插件对齐,ImageJ 2.0.0'正确的3D漂移'。
  7. 可以使用斐济(例如,细胞的长轴和分割角测量以及细胞迁移方向的测量)进一步分析漂移校正的图像序列。 Imaris 7.6.4用于4D渲染,单元格跟踪,迁移统计和4D电影。
  8. 已处理的数据(图像和视频)和统计分析发布在Webster等人。 (2016),可以在以下网址找到: http://www.cell.com/cell-stem-cell/fulltext/S1934-5909(15)00503-2

笔记

  1. 光透射的障碍包括头发,红细胞,epimysium和粘合剂。这可以通过在步骤3d中从脚上脱掉所有失去的毛发来避免,在步骤3e和3f中最小化皮肤移除和损伤脉管系统,在步骤3f中去除覆盖暴露的TA的所有膜,并确保粘合剂不覆盖暴露的组织在步骤3i。
  2. 将氟染色固定在腿上可能是相当具有挑战性的,可能需要一些练习。在粘合剂管上安装P1000吸头可以使粘接剂更容易地接触到外露TA的周边的皮肤和玻璃界面。
  3. 放置在舞台/显微镜上的护罩可能需要阻挡环境光引起高背景信号,特别是在使用非常敏感的非下行探测器时(图6)。
  4. 对于长时间成像会话,需要重复使用水。为了克服暂停实验和向物镜施加水的麻烦,可以使用输水物镜环盖和泵装置(New Era Pump Systems,Inc.)。
  5. 通过相应地调整MP波长和检测滤波器,可以将该协议适用于其他荧光蛋白(即,例如,tdT,CFP)。
  6. 本方案中使用的所有试剂均为直接购买和直接使用,无任何稀释或混合。


    图6.使用护罩成像

致谢

这里报告的方案得到NIH的NIAMS的支持,获奖号为F32AR065366(MTW)和RO1AR060042(至C-MF)以及卡内基学会(至TH)的校内基金。该协议是衍生自Cell Stem Cell之前的出版物的修改版本(在引用中引用,Webster等人,2016)。

参考文献

  1. Rompolas,P.,Deschene,ER,Zito,G.,Gonzalez,DG,Saotome,I.,Haberman,AM and Greco,V。(2012)。< a class ="ke-insertfile"href ="http ://www.ncbi.nlm.nih.gov/pubmed/22763436"target ="_ blank">干细胞的活体成像和生理性毛囊再生中的后代行为自然 487:496-499。
  2. Schindelin,J.,Arganda-Carreras,I.,Frize,E.,Kaynig,V.,Longair,M.,Pietzsch,T.,Preibisch,S.,Rueden,C.,Saalfeld,S.,Schmid,B 。,Tinevez,JY,White,DJ,Hartenstein,V.,Eliceiri,K.,Tomancak,P。和Cardona,A.(2012)。  斐济:用于生物图像分析的开源平台。 Nat方法 9(7 ):676-682。
  3. Scognamiglio,F.,Travan,A.,Rustighi,I.,Tarchi,P.,Palmisano,S.,Marsich,E.,Borgogna,M.,Donati,I.,de Manzini,N。和Paoletti, (2016)。普通外科应用的粘合剂和密封剂界面。 J Biomed Mater Res B Appl Biomater 104(3):626-639。
  4. Webster,MT,Manor,U.,Lippincott-Schwartz,J.and Fan,CM(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26686466"target ="_ blank">活体成像显示鬼纤维作为在再生期间引导肌原性祖细胞的构建单位。细胞干细胞 18(2):243-252。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Webster, M. T., Harvey, T. and Fan, C. (2016). Quantitative 3D Time Lapse Imaging of Muscle Progenitors in Skeletal Muscle of Live Mice. Bio-protocol 6(24): e2066. DOI: 10.21769/BioProtoc.2066.
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