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Super-resolution Imaging of Live BY2 Cells Using 3D-structured Illumination Microscopy
采用三维结构照明显微镜对活的BY2细胞进行超分辨率成像   

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本实验方案简略版
Plant Physiology
Aug 2015

Abstract

Light microscopy is the standard tool for studying sub-cellular structures however, owing to the diffractive properties of light, resolution is limited to 200 nm. Super-resolution microscopy methods circumvent this limit, offering greater resolution, particularly when studying fluorescently labeled sub-cellular structures. Super-resolution methods such as 3D-SIM (Structured Illumination Microscopy) fill a useful niche between confocal and electron microscopy. We have previously had success using fixed plant tissue samples with 3D-SIM (Bell and Oparka, 2014). However, sensitive structures can be altered by fixation and embedding procedures, so we developed a method for imaging live cells. In this protocol we used 3D-SIM to image the ER and Hechtian Strands in live, plasmolysed BY2 cells.

Materials and Reagents

  1. Microscope slides (Thermo Fisher Scientific)
  2. #1.5 coverslips (0.17 mm thick) (Thermo Fisher Scientific)
  3. BY2 cells expressing fluorescent marker
  4. 250 ml Erlenmeyer flasks (Thermo Fisher Scientific)
  5. Murashige and Skoog (MS) basal salt media (Sigma-Aldrich, catalog number: M5519 )
  6. Sucrose (Thermo Fisher Scientific)
  7. (2, 4-Dichlorophenoxy) acetic acid sodium salt monohydrate (Sigma-Aldrich, catalog number: D6679 )
  8. Calcofluor white/Fluorescent Brightener 28 (Sigma-Aldrich, catalog number: F3543 )
  9. 1 M D-Mannitol pure (Scientific Laboratory Supplies, catalog number: CHE1796 )
  10. Nail varnish
  11. BY2 growth media (see Recipes)
  12. Calcofluor White Stock solution (see Recipes)

Equipment

  1. Controlled temperature (28 °C) incubator or room
  2. Orbital shaker
  3. PersonalDV Deltavision Epi-fluorescence Inverted Microscope (GE Healthcare, Dharmacon)
  4. 3D-SIM microscope [DeltaVision OMX Blaze (GE Healthcare, Dharmacon) fitted with an Olympus PlanApo N 100x 1.42 NA oil objective]
  5. Edge sCMOS camera (PCO AG)

Software

  1. SoftWoRx 6.0 (GE)

Procedure

  1. Sample preparation
    1. Culture 40 ml of BY2 cell lines stably expressing a fluorescent reporter in 250 ml Erlenmeyer flasks with sterile Murashige and Skoog Basal Salts media supplemented with 3% sucrose and 2 μg ml-1 2,4-Dichlorophenoxyacetic acid. Grow cultures at 28 °C, in the dark, on an orbital shaker at 140 rpm. Aseptically sub-culture cells to fresh media weekly using a dilution of 1 ml of cells to 40 ml of media. Healthy cultures are pale yellow to light beige in colour and there should be sufficient growth that the cell suspension is more viscous than the media (Figure 1).
    2. At 4 days post-subculture, pipette 1 ml of cells into an Eppendorf tube and then stain cell walls (if required) with a final concentration of 3.5 μg ml-1 Calcofluor white for 5 min at room temperature. Excess stain is removed by allowing the cells to settle to the bottom of the tube and exchanging the media for fresh, twice.
    3. Plasmolyse cells by removing 450 μl of media and replacing it with 450 μl of 1 M Mannitol.
      Note: Plasmolysis is not required, but enabled us to observe plasmodesmata more accurately.
    4. Incubate at room temperature for 10 min, gently inverting the tube every 2 min to keep cells in suspension and then pipette 40 μl of cells onto a microscope slide. Lay a rectangular coverslip gently on top. Remove excess media by gentle pressure using a folded flat piece of absorbent tissue paper.
      Note: It is important not to damage the cells, so the pressure must be light.
    5. Seal all edges of the coverslip with nail varnish and begin imaging as soon as the varnish has dried.
    6. Locate candidate cells for imaging using a PersonalDV DeltaVision live-cell imaging system, which has stage coordinates mapped to the OMX. Use low level bright field illumination and mark the position on the slide using point visiting tool.
    7. Expose all marked cells to the appropriate wavelength of fluorescent light to check the calcofluor stain (abs350 nm/em455 nm) has worked well and that the cells are expressing a good level of fluorescent reporter (Figure 2). It is vital to keep this exposure as brief as possible in order to minimize bleaching prior to 3D-SIM imaging. The point visiting function allows the marked cells to be found swiftly on the OMX, thus avoiding unnecessary exposure to light.

  2. 3D-SIM Imaging
    1. We acquire 3D-SIM images using a DeltaVision OMX Blaze fitted with an Olympus PlanApo N 100x 1.42 NA oil objective. The 3D-SIM imaging protocol is based on that described in Schermelleh et al. (2010). Place the slide on the OMX and apply immersion oil. To minimise spherical aberration and optimize illumination modulation contrast, adjust the type of immersion oil to match the Refractive Index (RI) of the sample and imaging depth. Ideally, BY2 cells would be mounted in glycerol or similar to maintain the RI of the oil immersion objective but to keep the ER in good condition the cells must be mounted in media. Cells also need to be as close to the coverslip as possible.
    2. Find the selected cells using the Point List and then the Spiral Mosaic function to center the cell in the image. Determine top and bottom limits of z-stack quickly and efficiently, continuing to minimize the cell’s exposure to light.
    3. In the OMX 3D-SIM system, light from solid state lasers (405, 488 and 564 nm), shuttered by high speed tilt mirrors and coupled into a broadband single mode optical fiber is split into three beams. 3D-interference patterns in the sample plane are generated by focusing the beams onto the back focal plane of the objective lens. Striped illumination patterns are shifted by five phase steps and rotated by 3 angles (-60°, 0° and +60°), providing a set of 15 images per unprocessed z-section. Interference patterns are phase shifted by directing the outer two beams through a separate pair of windows with individual tilt control. Phase of the interference pattern at the sample plane is shifted due to the change in the path length for the respective outer beam, while lateral refractive beam translation is canceled by tilting a given window pair in complementary directions. Angles of pattern orientation are shifted by a tilt mirror, directing the three beams pattern to one of three mirror clusters; the beam pattern from each of the three rotation paths is redirected back to a common exit path by reflecting a second time from the tilt mirror. For descriptive diagrams see http://microscopy.lifesci.dundee.ac.uk/omx/omx.html.
    4. Select the lowest possible laser power and exposure times for each channel to minimize photo bleaching. Adjust exposure times similarly, typically between 100 and 200 ms, and also adjust the power of each laser to achieve optimal intensities of between 1,000 and 3,000 counts in a raw image acquired by a 15-bit dynamic range Edge sCMOS camera. Acquire image stack.
    5. Unprocessed image stacks are composed of 15 images per z-section (five phase-shifted images per each of three interference pattern angles). The microscope must be routinely calibrated by measuring channel specific optical transfer functions (OTFs) to optimize both lateral and axial image resolution.
    6. Adjust images from the different color channels, recorded on separate cameras, with the SoftWorx 6.0 alignment tool, based on alignment parameters obtained from calibration measurements with 100 nm-diameter TetraSpeck beads. Reconstruct super-resolution 3D image stacks with SoftWoRx 6.0 using channel specific OTFs and Wiener filter setting of 0.002.

Representative data


Figure 1. Healthy 4-day old BY2 cell culture


Figure 2. Representative image showing the resolution obtainable with 3D-SIM using plasmolysed live BY2 cells. A. Standard confocal image of BY2 cells expressing RTN6:GFP (green) and the cell wall stained with Calcofluor White (blue). B. Pseudo-coloured 3D-SIM image of the boxed region in A showing high resolution of the Hechtian strands at high magnification.

Notes

A 3D-SIM microscope takes considerable expertise to operate, with different manufacturers using bespoke software specifically designed for their hardware. Therefore, the 3D-SIM imaging part of this protocol is provided as a guide only. Each experiment will take considerable optimization of all 3D-SIM parameters as outlined in the protocol.

Recipes

  1. BY2 growth media
    0.43% MS Basal Salts media
    3% sucrose
    2 μg ml-1 2, 4-Dichlorophenoxyacetic acid
    ddH2O
    Sterilize the media by autoclaving
  2. Calcofluor White Stock solution
    170 μg ml-1 Calcofluor white
    Dissolve in ethanol
    Stored in the dark at -20 °C

Acknowledgments

Use of the Deltavision OMX Blaze microscope at Dundee University was supported by an MRC Next Generation Optical Microscopy Award (Ref: MR/K015869/1). Development of this protocol was part of a project funded by grant BB/J004987/1 from the British Biotechnology and Biological Sciences Research Council (BBSRC) to Karl Oparka. We are grateful for the expert assistance of Dr. Markus Posch. Elements of this protocol have been adapted from those previously described in Bell and Oparka (2015) and Knox et al. (2015).

References

  1. Bell, K. and Oparka, K. (2015). Preparative methods for imaging plasmodesmata at super-resolution. Methods Mol Biol 1217: 67-79.
  2. Knox, K., Wang, P., Kriechbaumer, V., Tilsner, J., Frigerio, L., Sparkes, I., Hawes, C. and Oparka, K. (2015). Putting the squeeze on Plasmodesmata: A role for reticulons in primary plasmodesmata formation. Plant Physiol 168(4): 1563-1572.
  3. Schermelleh, L., Heintzmann, R. and Leonhardt, H. (2010). A guide to super-resolution fluorescence microscopy. J Cell Biol 190(2): 165-175.

简介

光学显微镜是用于研究亚细胞结构的标准工具,然而,由于光的衍射性质,分辨率限于200nm。 超分辨率显微镜方法规避这一限制,提供更大的分辨率,特别是当研究荧光标记的亚细胞结构。 超分辨率方法,如3D-SIM(结构照明显微镜)填充共聚焦和电子显微镜之间有用的利基。 我们以前已经成功使用固定植物组织样本与3D-SIM(贝尔和Oparka,2014)。 然而,敏感结构可以通过固定和嵌入程序改变,所以我们开发了一种成像活细胞的方法。 在这个协议中,我们使用3D-SIM成像ER和Hechtian链在活的,plasmolysed BY2细胞。

材料和试剂

  1. 显微镜载玻片(Thermo Fisher Scientific)
  2. #1.5盖玻片(0.17mm厚)(Thermo Fisher Scientific)
  3. 表达荧光标记的BY2细胞
  4. 250ml锥形瓶(Thermo Fisher Scientific)
  5. Murashige和Skoog(MS)基础盐培养基(Sigma-Aldrich,目录号:M5519)
  6. 蔗糖(Thermo Fisher Scientific)
  7. (2,4-二氯苯氧基)乙酸钠盐一水合物(Sigma-Aldrich,目录号:D6679)
  8. Calcofluor白/荧光增白剂28(Sigma-Aldrich,目录号:F3543)
  9. 1 M D-甘露醇纯(Scientific Laboratory Supplies,目录号:CHE1796)
  10. 指甲油
  11. BY2生长培养基(参见食谱)
  12. Calcofluor白色库存解决方案(参见配方)

设备

  1. 控制温度(28°C)孵化器或房间
  2. 轨道振动器
  3. PersonalDV Deltavision荧光反转显微镜(GE Healthcare,Dharmacon)
  4. 3D-SIM显微镜[DeltaVision OMX Blaze(GE Healthcare,Dharmacon),配有Olympus PlanApo N 100x 1.42 NA油目标物]
  5. Edge sCMOS摄像机(PCO AG)

软件

  1. SoftWoRx 6.0(GE)

程序

  1. 样品准备
    1. 培养40ml稳定表达荧光报道分子的BY2细胞系 ?在具有无菌Murashige和Skoog Basal的250ml Erlenmeyer烧瓶中 补充有3%蔗糖和2μg/ml的2,4-二氯苯氧基乙酸的盐培养基。在28℃,黑暗中培养培养物 140rpm的轨道振荡器。无菌培养细胞至新鲜 每周使用1ml细胞稀释至40ml培养基。健康 ?文化是淡黄色到浅米色的颜色,应该有 足够的生长使细胞悬浮液比粘性更粘稠 媒体(图1)。
    2. 在4天后亚培养,吸管1毫升 细胞进入Eppendorf管,然后染色细胞壁(如果需要) 最终浓度为3.5μg/ml的Calcofluor白色5分钟 室内温度。通过允许细胞除去过量的染色 沉降到管的底部并且将培养基更换为新鲜, 两次。
    3. 通过去除450μl的培养基并用450μl的1M甘露醇代替它来分离质粒细胞 注意:不需要溶血,但是使我们能够更准确地观察plasmodesmata。
    4. 在室温孵育10分钟,轻轻倒转管 每2分钟保持细胞悬浮,然后吸取40微升的细胞 到显微镜载玻片上。在上面轻轻地放置一个矩形盖玻片。 使用折叠的平片轻轻地压下多余的介质 吸收薄纸。
      注意:重要的是不要损坏细胞,所以压力必须很轻。
    5. 用指甲油密封盖玻片的所有边缘,并在清漆干燥后立即开始成像
    6. 使用PersonalDV DeltaVision定位用于成像的候选细胞 活细胞成像系统,其具有映射到OMX的载物台坐标。 ?使用低级明亮场照明并标记位置 使用点访问工具滑动
    7. 将所有标记的单元格公开 适当波长的荧光灯来检查calcofluor 染色( abs 350nm/ 455nm)已经工作良好,并且细胞 表达良好水平的荧光报告物(图2)。这是至关重要的 以使这种暴露尽可能简短以便最小化 在3D-SIM成像之前漂白。点访问功能允许 在OMX上迅速找到标记的单元格,从而避免 不必要的曝光。

  2. 3D-SIM成像
    1. 我们使用DeltaVision OMX Blaze获得3D-SIM图像 Olympus PlanApo N 100x 1.42 NA油目标。 3D-SIM成像 协议基于Schermelleh等人(2010)中描述的方法。地点 ?在OMX上滑动并涂抹浸油。最小化球面 像差和优化照明调制对比度,调整 类型的浸油以匹配样品的折射率(RI) 和成像深度。理想地,BY2细胞将安装在甘油或 类似于保持油浸物镜的RI但保持 ER处于良好状态时,细胞必须安装在培养基中。细胞也 需要尽可能接近盖玻片。
    2. 找出 使用点列表和螺旋马赛克功能选择单元格 以使图像中的单元格居中。确定的上限和下限 z-stack快速有效,继续最小化单元格 曝光。
    3. 在OMX 3D-SIM系统中,光从固体 (405,488和564nm),由高速倾斜反射镜关闭 ?并耦合成宽带单模光纤被分离 三光束。生成样本平面中的3D干涉图案 通过将光束聚焦到物镜的后焦平面上。 条纹照明图案偏移五个相位步长和 旋转3个角度(-60°,0°和+ 60°),提供一组15个图像 每个未处理的z截面。干扰模式相移 引导外部两个光束通过单独的一对窗口 个人倾斜控制。样品处的干涉图样的相位 ?平面由于路径长度的变化而偏移 相应的外光束,而横向折射光束平移 通过在给定的窗口对在互补方向上倾斜来取消。 图案取向的角度由倾斜反射镜偏移,引导 所述三个光束图案化为三个反射镜组中的一个;梁 来自三个旋转路径中的每一个的图案被重定向回到a 公共出口路径通过从所述倾斜反射镜反射第二时间。对于 说明图请参见 http://microscopy.lifesci.dundee.ac.uk/omx/omx.html。
    4. 选择 最低可能的激光功率和每个通道的曝光时间 最小化照片漂白。通常,调整曝光时间类似 在100和200 ms之间,并且还调整每个激光器的功率 在原料中实现1,000和3,000之间的最佳强度 图像由15位动态范围边缘sCMOS摄像机采集。获得 图像堆栈。
    5. 未处理的图像堆栈由15个图像组成 (每三个干涉的五个相移图像) 图案角度)。显微镜必须定期校准 测量信道特定光学传递函数(OTF)以进行优化 ?包括横向和轴向图像分辨率。
    6. 调整图像 ?不同的颜色通道,记录在单独的相机上,与 SoftWorx 6.0对齐工具,基于从中获得的对齐参数 ?用100nm直径的TetraSpeck珠进行校准测量。 使用SoftWoRx 6.0重建超分辨率3D图像堆栈 信道特定OTF和维纳滤波器设置为0.002。

代表数据


图1.健康的4天老的BY2细胞培养


A.表达RTN6的GFP细胞的BY2细胞的标准共聚焦图像:GFP(绿色)和用Calcofluor白色染色的细胞壁(图2)。图2.代表性图像显示使用质谱分离的活BY2细胞的3D-蓝色)。 B.在A中的盒装区域的伪彩色3D-SIM图像,其以高放大倍数显示高分辨率的Hechtian股线。

笔记

3D-SIM显微镜需要相当多的专业知识来操作,不同的制造商使用专门为其硬件设计的定制软件。因此,该协议的3D-SIM成像部分仅作为指导。每个实验将采取协议中概述的所有3D-SIM参数的相当大的优化。

食谱

  1. BY2生长培养基
    0.43%MS基础盐介质
    3%蔗糖 2μgml -1,2-二氯苯氧基乙酸 ddH sub 2 O
    通过高压灭菌
    灭菌培养基
  2. Calcofluor白色股票解决方案
    170μg/ml -1 Calcofluor白色
    溶于乙醇
    储存在-20°C的黑暗中

致谢

在Dundee大学使用Deltavision OMX Blaze显微镜由MRC下一代光学显微镜奖(参考:MR/K015869/1)支持。该协议的开发是由英国生物技术和生物科学研究委员会(BBSRC)向Karl Oparka授权BB/J004987/1资助的项目的一部分。我们感谢Markus Posch博士的专家协助。该协议的元素已经改编自先前在Bell和Oparka(2015)和Knox等人(2015)中描述的那些。

参考文献

  1. Bell,K。和Oparka,K。(2015)。 在超分辨率下成像plasmodesmata的制备方法。 /em> 1217:67-79。
  2. Knox,K.,Wang,P.,Kriechbaumer,V.,Tilsner,J.,Frigerio,L??.,Sparkes,I.,Hawes,C.and Oparka,K。 对Plasmodesmata的挤压:网状结构在原代等离子体形成中的作用 Plant Physiol 168(4):1563-1572。
  3. Schermelleh,L.,Heintzmann,R。和Leonhardt,H。(2010)。 超分辨荧光显微镜的指南 J Cell Biol em> 190(2):165-175。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Bell, K., Oparka, K. and Knox, K. (2016). Super-resolution Imaging of Live BY2 Cells Using 3D-structured Illumination Microscopy. Bio-protocol 6(1): e1697. DOI: 10.21769/BioProtoc.1697.
  2. Knox, K., Wang, P., Kriechbaumer, V., Tilsner, J., Frigerio, L., Sparkes, I., Hawes, C. and Oparka, K. (2015). Putting the squeeze on Plasmodesmata: A role for reticulons in primary plasmodesmata formation. Plant Physiol 168(4): 1563-1572.
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