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Visualization of Cell Complexity in the Filamentous Cyanobacterium Mastigocladus laminosus by Transmission Electron Microscopy (TEM)
透射电镜(TEM)法观察分析丝状蓝细菌层理鞭枝藻细胞复杂度   

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Abstract

The cyanobacterium Mastigocladus laminosus (M. laminosus) is one of the most morphologically complex prokaryotes. It forms long chains of cells that are connected via septal junction complexes; such complexes allow diffusion of metabolites and regulators between neighboring cells. Cellular division occurs in multiple planes, resulting in the formation of true branches, and cell differentiation leads to the formation of specialized cell types for nitrogen fixation (heterocysts) and culture dispersal (hormogonia and necridia). Here, we describe a detailed protocol for the preparation of M. laminosus for TEM in order to visualize the ultrastructural properties of the organism. The presented preparation method is based on adding potassium permanganate as fixative which has been shown to increases the contrast of membranes (Luft, 1956), making it suitable for studies in cyanobacteria where the visualization of the photosynthetic membranes is important.

Keywords: Ultrastructure(超微结构), Fischerella(侧生藻属), Heterocyst(异形胞), Hormogonia(段殖体), Branching cyanobacteria(分枝蓝藻)

Materials and Reagents

  1. Liquid culture of Mastigocladus laminosus
  2. Potassium phosphate monobasic (KH2PO4) (Thermo Fisher Scientific, catalog number: BP362 )
  3. Sodium phosphate dibasic (Na2HPO4) (Thermo Fisher Scientific, Acros Organics, catalog number: 204855000 )
  4. Glutaraldehyde (25%, EM grade) (Agar Scientific, catalog number: R1020 )
  5. Low gelling temperature agarose (Sigma-Aldrich, catalog number: A9414 )
  6. Potassium permanganate (KMnO4) (VWR International, BDH, catalog number: 296444N )
  7. 100% ethanol
  8. Propylene oxide (Agar Scientific, catalog number: R1080 )
  9. Araldite CY212 (Agar Scientific, catalog number: R1042 )
  10. Methyl nadic anhydride (MNA) (Agar Scientific, catalog number: R1083 )
  11. Dodecenylsuccinic anhydride (DDSA) (Agar Scientific, catalog number: R1052 )
  12. Benzyldimethylamine (BDMA) (Agar Scientific, catalog number: R1061 )
  13. Sodium tetraborate (borax) (Sigma-Aldrich, catalog number: 221732 )
  14. Toluidine blue (Sigma-Aldrich, catalog number: 89640 )
  15. Uranyl acetate [UO2(CH3COO)2]*2 H2O] (SPI Supplies, catalog number: 02624-AB )
  16. Lead (II) nitrate [Pb(NO3)2] (Sigma-Aldrich, catalog number: 228621 )
  17. Tri-sodium citrate (TAAB, catalog number: S011 )
  18. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 )
  19. 0.125 M Sørensen's phosphate buffer (PB) (see Recipes)
  20. 4% (v/v) glutaraldehyde in PB (see Recipes)
  21. 2% (w/v) low gelling temperature agarose (see Recipes)
  22. 2% (w/v) potassium permanganate (see Recipes)
  23. Araldite (see Recipes)
  24. 1% (w/v) toluidine blue (see Recipes)
  25. Uranyl acetate (see Recipes)
  26. Reynold's lead citrate stain (see Recipes)

Equipment

  1. Centrifuge
  2. Copper grids (300 mesh) (Agar Scientific, catalog number: G2740C )
  3. Dental wax
  4. Disposable plastic Pasteur pipettes
  5. Disposable polyethylene beaker
  6. Eppendorf tubes (1.5 ml)
  7. Glass or diamond knife
  8. Hotplate
  9. Light microscope
  10. Oven (60 °C)
  11. Razor blades
  12. Rotator
  13. Rubber embedding mould
  14. Sealable glass vials (7 ml)
  15. Shaker
  16. Spatula
  17. Sterilization filters (pore size: 0.22 µm)
  18. Syringe
  19. TEM (JOEL, model: JEM-1230 )
  20. Tweezers
  21. Ultramicrotome (Reichert Ultracut E)
  22. Vortexer
  23. SPI Slide-A-GridTM storage box (SPI Slide-A-GridTM, catalog number: 02450-AB )

Procedure

Note: Some chemicals used in this protocol are highly toxic and should be used only under the fume hood with adequate precautions. Please check the MSDS before using them.
Preparation of samples for ultra-thin section transmission electron microscopy requires multiple days. The timeline for the different phases of preparation is shown in Table 1.

Table 1. Timeline for preparation of samples for transmission electron microscopy
Days 1-7      Growth of M. laminosus (A)
Day 8           Fixation and pre-embedding (B); Storage for up to 3 months possible
Days 9-11    Dehydration and embedding in Araldite (C); Indefinite storage possible
Day 12        Sectioning and post-staining (D); Storage for up to 1 year possible
Day 13        Visualization at the transmission electron microscope

  1. Growth of M. laminosus
    M. laminosus was grown in gas washing bottles filled with Castenholz D or ND medium (lacking combined nitrogen) (Castenholz, 1988) by bubbling with sterile air. Cultures were kept at 40 °C under constant white light at approximately 20 µE/m2/s until a dense cell mat was visible at the surface (Figure 1).


    Figure 1. Dense cell mat of M. laminosus grown in Castenholz D medium

  2. Fixation and pre-embedding
    1. Transfer a piece of the cell mat (corresponding to a volume of approximately 100 µl) into an Eppendorf tube using a 1 ml pipette.
    2. Add 1 ml of PB, centrifuge for 2 min at 3,000 x g and remove supernatant.
    3. Resuspend the cells in 500 µl of 4% (v/v) glutaraldehyde (toxic!) and leave the tube standing for 2 h at room temperature.
    4. Centrifuge cells for 2 min at 3,000 x g and remove supernatant. Wash cells 3x in 1 ml of PB with 10 min of incubation time between each step to remove the fixative. Resuspend cells by inverting the tube.
    5. Remove supernatant after the final centrifugation step.
    6. Resuspend cells in an approximately equal volume of 2% (w/v) liquid agarose (place vial at 60 °C ca. 30 min prior usage to melt the agarose and keep it fluid until usage). Leave sample at room temperature until it is fully polymerized. It is possible to stop the protocol at this step. Samples can be stored in the fridge for up to 3 months.
    7. Cut the cell pellet with a razor blade into small cubes of about 1-2 mm length.
    8. Transfer cubes to a glass vial and add 2% (w/v) potassium permanganate solution to fully cover the cubes.
    9. Incubate for 12 h under gentle shaking at 4 °C.
    10. Remove potassium permanganate solution and wash the sample several times for 10 min with distilled water until the supernatant remains clear.

  3. Dehydration and embedding in Araldite
    Note: All incubation steps were performed using a rotator with a rotation speed of 3 rpm.
    1. Dehydrate cells by adding 30%, 50%, 70% and 90% (v/v) ethanol for 15 min and 3x 100% (v/v) ethanol for 20 min. Remove ethanol after each incubation step.
    2. Incubate the sample for 5 min in propylene oxide (toxic!), remove supernatant and repeat washing step with propylene oxide for 5 min to remove remaining ethanol. Remove supernatant.
    3. Add freshly prepared Araldite until the cubes are fully covered and incubate for 1 h.
    4. Remove Araldite and replace by a fresh solution of Araldite. Incubate over night.
    5. Transfer samples to an embedding mould which is filled with fresh Araldite solution and remove all air bubbles with tweezers.
    6. Polymerize samples by placing the Araldite filled embedding moulds for 48 h at 60 °C. After polymerization samples can be stored indefinitely.

  4. Sectioning and post-staining
    Note: Sectioning requires a lot of experience. Contacting an expert is recommended for beginners.
    1. Trim blocks with a razor blade and cut sections of 1 µm thickness with an ultramicrotome.
    2. Transfer a section to a drop of distilled water on a glass slide by using an eyelash attached to a matchstick or a metal loop. Dry section on a hotplate (circa 60 °C).
    3. Stain sections with 1% (w/v) toluidine blue for 30 sec on hotplate.
    4. Drain excess stain and rinse section briefly with distilled water.
    5. Dry on a hotplate and localize cells in a light microscope.
    6. Reduce size of block further to get the smallest possible block area including the sample of interest and prepare ultrathin sections of approximately 70 nm thickness.
    7. Transfer the sections to a copper grid.
    8. Place droplets of saturated aqueous uranyl acetate (toxic!) onto a piece of dental wax in a Petri dish and float the grids (sections downwards) on the droplets for 4 min (Video 1).

      Video 1.

    9. Wash the grids by dipping approximately 10 times into distilled water. Repeat three times. (Video 1)
    10. Blot off excess water with the edge of a piece of filter paper, and transfer the grids to droplets of Reynold's lead citrate placed on dental wax. Stain for 4 min (Video 1).
    11. Wash with distilled water as described in step 9.
    12. Blot off excess water and leave the sections to dry before viewing at the TEM. Grids can be used for TEM at any time. For long-term storage (up to 1 year) grids are kept in grid storage boxes (e.g. SPI Slide-A-GridTM storage box). A sample micrograph is shown in Figure 2.


      Figure 2. Electron micrograph of an ultra-thin section through a trichome of M. laminosus grown in Castenholz ND medium. Under nitrogen deprivation M. laminosus differentiates photosynthetically active vegetative cells (veg) into nitrogen-fixing heterocysts (het) which possess an additional multilayered envelope (env) as gas diffusion barrier to reduce the entrance of O2 into heterocysts. When filaments break by the formation of necridia ("releasing cells") a small fraction of the former cell remains attached to the trichome (cb). Different membranes are highlighted by black arrows: thylakoid membrane (tm), cytoplasmic membrane (cm) and outer membrane (om). Scale bar, 2 µm

Recipes

  1. 0.125 M Sørensen's phosphate buffer (PB) (pH 7.2-7.4)
    450 ml 0.2 M sodium phosphate dibasic (28.4 g in 1,000 ml distilled water)
    150 ml 0.2 M potassium phosphate monobasic (27.2 g in 1,000 ml distilled water)
    381 ml distilled water
    Stored at 4 °C
  2. 4% (v/v) glutaraldehyde in PB
    4 ml 25% glutaraldehyde
    21 ml PB
    Stored at 4 °C
    Use within 1 month
  3. 2% (w/v) low gelling temperature agarose
    Add 0.2 g agarose to 10 ml PB
    Place in the oven at 80 °C and stir occasionally until the agarose is fully dissolved
    Stored at 4 °C
  4. 2% (w/v) potassium permanganate
    Add 2 g potassium permanganate to 10 ml distilled water
    Dissolve by vortexing and filter
    Prepare freshly before usage
  5. Araldite
    40 ml Araldite CY212
    60 ml dodecenylsuccinic anhydride
    2 ml methyl nadic anhydride
    1 ml benzyl dimethylamine
    Stir with a spatula for at least 5 min in a disposable polyethylene beaker; introduce as little air as possible
    Leave for 1 h at room temperature
    Prepare freshly before usage
  6. 1% (w/v) toluidine blue
    Add 1 g toluidine blue to 100 ml 1% (w/v) sodium tetraborate in distilled water
    Dissolve by boiling and stir occasionally
    Filter and stored at room temperature
  7. Uranyl acetate
    Add 1 g uranyl acetate to 100 ml distilled water
    Shake vigorously and leave for 48 h to dissolve at room temperature
    If all the uranyl acetate has dissolved, add a further 1 g and shake vigorously
    Leave for a further 48 h at room temperature
    Repeat until no more uranyl acetate dissolves and some undissolved reagent remains
  8. Reynold's lead citrate stain
    Dissolve 2.66 g lead nitrate in 30 ml distilled water
    Dissolve 3.52 g tri-sodium citrate in 30 ml distilled water
    Combine solutions and shake vigorously for 30 sec every 5 min over a period of 30 min
    Add 16 ml 1 M sodium hydroxide solution
    Mix until solution is clear
    Add distilled water to a final volume of 100 ml
    Stored at 4 °C

Acknowledgments

This work was supported by a college studentship of Queen Mary University of London.

References

  1. Castenholz, R. W. (1988). [3] Culturing methods for cyanobacteria. Methods in Enzymology 167: 68-93.
  2. Luft, J. H. (1956). Permanganate; a new fixative for electron microscopy. J Biophys Biochem Cytol 2(6): 799-802.
  3. Nurnberg, D. J., Mariscal, V., Parker, J., Mastroianni, G., Flores, E. and Mullineaux, C. W. (2014). Branching and intercellular communication in the Section V cyanobacterium Mastigocladus laminosus, a complex multicellular prokaryote. Mol Microbiol 91(5): 935-949.

简介

蓝藻(Mastigocladus laminosus)( M。laminosus )是最具形态复杂的原核生物之一。 它形成通过间隔连接复合物连接的细胞的长链; 这种复合物允许代谢物和调节剂在相邻细胞之间扩散。 细胞分裂发生在多个平面,导致真正的分支的形成,细胞分化导致固氮(heterocysts)和文化扩散(hormogonia和necridia)的专门细胞类型的形成。 在这里,我们描述了用于制备 M的详细方案。 层状结构用于TEM,以便可视化生物体的超微结构特性。 所提出的制备方法基于添加高锰酸钾作为固定剂,已显示增加膜的对比度(Luft,1956),使其适合于在其中光合膜的可视化是重要的蓝细菌的研究。

关键字:超微结构, 侧生藻属, 异形胞, 段殖体, 分枝蓝藻

材料和试剂

  1. 液体培养的胸膜肺炎
  2. 磷酸二氢钾(KH 2 PO 4)(Thermo Fisher Scientific,目录号:BP362)
  3. 磷酸氢二钠(Na 2 HPO 4)(Thermo Fisher Scientific,Acros Organics,目录号:204855000)
  4. 戊二醛(25%,EM级)(Agar Scientific,目录号:R1020)
  5. 低胶凝温度琼脂糖(Sigma-Aldrich,目录号:A9414)
  6. 高锰酸钾(KMnO 4)(VWR International,BDH,目录号:296444N)
  7. 100%乙醇
  8. 环氧丙烷(Agar Scientific,目录号:R1080)
  9. Araldite CY212(Agar Scientific,目录号:R1042)
  10. 甲基纳迪克酸酐(MNA)(Agar Scientific,目录号:R1083)
  11. 十二碳烯基琥珀酸酐(DDSA)(Agar Scientific,目录号:R1052)
  12. 苄基二甲胺(BDMA)(Agar Scientific,目录号:R1061)
  13. 四硼酸钠(硼砂)(Sigma-Aldrich,目录号:221732)
  14. 甲苯胺蓝(Sigma-Aldrich,目录号:89640)
  15. 乙酸铀[UO 2(CH 3 COO)2] 2 H 2 O](SPI Supplies, 目录号:02624-AB)
  16. 硝酸铅(II)[Pb(NO 3)2](Sigma-Aldrich,目录号:228621)
  17. 柠檬酸三钠(TAAB,目录号:S011)
  18. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:221465)
  19. 0.125 MSørensen的磷酸盐缓冲液(PB)(参见配方)
  20. 4%(v/v)戊二醛在PB中(参见配方)
  21. 2%(w/v)低胶凝温度琼脂糖(参见Recipes)
  22. 2%(w/v)高锰酸钾(参见配方)
  23. Araldite(参见食谱)
  24. 1%(w/v)甲苯胺蓝(参见配方)
  25. 醋酸铀(见配方)
  26. 雷诺铅柠檬酸盐染色(参见配方)

设备

  1. 离心机
  2. 铜网(300目)(Agar Scientific,目录号:G2740C)
  3. 牙科蜡
  4. 一次性塑料巴斯德移液器
  5. 一次性聚乙烯烧杯
  6. Eppendorf管(1.5ml)
  7. 玻璃或金刚石刀
  8. Hotplate
  9. 光学显微镜
  10. 烤箱(60°C)
  11. 剃刀刀片
  12. 旋转器
  13. 橡胶嵌入模具
  14. 可密封的玻璃小瓶(7ml)
  15. 振动器
  16. 小铲
  17. 灭菌过滤器(孔径:0.22μm)
  18. 注射器
  19. TEM(JOEL,型号:JEM-1230)
  20. 镊子
  21. 超微切片机(Reichert Ultracut E)
  22. Vortexer
  23. SPI Slide-A-Grid TM 存储盒(SPI Slide-A-Grid TM ,目录号:02450-AB)

程序

注意:本协议中使用的一些化学物质毒性很大,只能在通风橱下使用足够的预防措施。请在使用前检查MSDS。
超薄切片透射电子显微镜的样品制备需要多天。不同制备阶段的时间表如表1所示
表1.透射电子显微镜样品制备时间表
第1-7天      M.的生长。 laminosus (A)
第8天          固定和预嵌入(B);最多可存储3个月
第9-11天    脱水和包埋在Araldite(C);无限期存储
第12天       切片和后染色(D);最多可存储1年
第13天       在透射电子显微镜下可视化

  1. M.的生长。 laminosus
    M。 通过用无菌空气鼓泡,在充满Castenholz D或ND培养基(缺乏组合氮)的气体洗涤瓶(Castenholz,1988)中生长。 将培养物在约20μE/m 2/s的恒定白光下保持在40℃,直到在表面可见致密的细胞垫(图1)。


    图1. M的密集单元格。 在Castenholz D培养基中生长的

  2. 固定和预嵌入
    1. 转移一块细胞垫(相当于一卷的 大约100μl)使用1ml移液管置于Eppendorf管中
    2. 加入1ml PB,在3,000×g离心2分钟,除去上清液
    3. 将细胞重悬在500μl的4%(v/v)戊二醛(有毒!)中,并将管在室温下静置2小时。
    4. 在3,000×g离心细胞2分钟并除去上清液。 洗   细胞在1ml PB中3x,每个步骤之间具有10分钟的温育时间   以去除固定剂。 通过翻转管重悬细胞。
    5. 在最终离心步骤后除去上清液。
    6. 重悬细胞在大约等体积的2%(w/v)液体 琼脂糖(在60℃下放置小瓶,使用前约30分钟以熔化琼脂糖 并保持流体直到使用)。 将样品在室温下保存 它完全聚合。 可以在此停止协议 步。 样品可以存储在冰箱中最多3个月。
    7. 用剃刀刀片将细胞沉淀切成约1-2毫米长的小立方体
    8. 将立方体转移到玻璃小瓶中,加入2%(w/v)高锰酸钾溶液以完全覆盖立方体
    9. 在4℃下轻轻振荡孵育12小时
    10. 取出高锰酸钾溶液,并洗几次样品 用蒸馏水洗涤10分钟,直到上清液保留 明确。

  3. 脱水和嵌入Araldite
    注意:所有孵育步骤都使用旋转速度为3rpm的旋转器进行。
    1. 通过加入30%,50%,70%和90%(v/v)乙醇脱水细胞15分钟   和3x 100%(v/v)乙醇洗涤20分钟。 每次后除去乙醇 孵育步骤
    2. 在环氧丙烷中孵育样品5分钟   (中毒!),去除上清液,并用丙烯重复洗涤步骤 氧化物5分钟以除去剩余的乙醇。 除去上清液。
    3. 加入新鲜制备的Araldite,直到立方体完全覆盖,孵育1小时
    4. 删除Araldite,更换为新鲜的Araldite溶液。 孵育过夜。
    5. 将样品转移到填充有新鲜的包埋模具中 Araldite溶液,并用镊子清除所有气泡
    6. 通过放置Araldite填充嵌入模具聚合样品 在60℃下48小时。 聚合后样品可无限期储存。

  4. 切片和后染色
    注意:切片需要很多经验。 建议初学者与专家联系。
    1. 修剪刀片用剃刀刀片切割,切片厚度为1μm,用超薄切片机
    2. 将部分转移到玻璃载玻片上的一滴蒸馏水中 使用附在火柴或金属环上的睫毛。 干部 (约60℃)。
    3. 在加热板上用1%(w/v)甲苯胺蓝染色30秒
    4. 用蒸馏水短暂排出多余的污渍和冲洗部分
    5. 在电热板上干燥,并在光学显微镜中定位细胞
    6. 进一步减小块的大小以获得最小可能的块面积   包括感兴趣的样品和准备超薄切片 约70nm厚度
    7. 将部分转移到铜网格。
    8. 将饱和的乙酸铀酰水滴(有毒!)滴在a 一块牙科蜡在培养皿和浮动网格(部分 向下)在液滴上4分钟(视频1)。

      视频1
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      获取Adobe Flash Player

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      获取Adobe Flash Player

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    9. 通过将约10次浸入蒸馏水中来洗涤网格。 重复三次。 (视频1)
    10. 用一块滤纸的边缘刮掉多余的水,和   将网格转移到放置在雷诺兹柠檬酸铅上的液滴 牙科蜡。 着色4分钟(视频1)。
    11. 如步骤9所述,用蒸馏水清洗。
    12. 在观察之前,喷掉多余的水,并让部分干燥 在TEM。网格可以随时用于TEM。长期储存  (最多1年)网格保存在网格存储盒(例如 SPI Slide-A-Grid TM 存储盒)。样品显微照片如图2所示。


      图2.通过毛细管的超薄切片的电子显微照片。在Castenholz ND培养基中生长的 。在氮剥夺下。 laminosus 区分 光合活性营养细胞(veg)转化为固氮 杂合体(het),其具有额外的多层包膜 (env)作为气体扩散阻挡层以减少O 2入口进入 杂合子。当细丝通过坏死的形成破裂时 ("释放细胞"),前一细胞的一小部分保持附着  到毛状体(cb)。不同的膜用黑色突出显示 箭头:类囊体膜(tm),细胞质膜(cm)和外部 膜(om)。比例尺,2μm

食谱

  1. 0.125MSørensen的磷酸盐缓冲液(PB)(pH 7.2-7.4) 450ml 0.2M磷酸氢二钠(28.4g,在1,000ml蒸馏水中) 150ml 0.2M磷酸二氢钾(27.2g,在1,000ml蒸馏水中) 381ml蒸馏水
    储存在4°C
  2. 4%(v/v)戊二醛的PB
    4ml 25%戊二醛
    21 ml PB
    储存在4°C
    1个月内使用
  3. 2%(w/v)低凝胶化温度琼脂糖 加入0.2 g琼脂糖至10 ml PB
    放在80°C的烤箱中,偶尔搅拌直到琼脂糖完全溶解
    储存在4°C
  4. 2%(w/v)高锰酸钾 将2g高锰酸钾加入10ml蒸馏水中
    涡旋和过滤溶解
    在使用前新准备
  5. Araldite
    40ml Araldite CY212
    60ml十二烯基琥珀酸酐 2ml甲基纳迪克酸酐
    1ml苄基二甲胺
    在一次性聚乙烯烧杯中用刮刀搅拌至少5分钟; 引入尽可能少的空气
    在室温下放置1小时
    在使用前新准备
  6. 1%(w/v)甲苯胺蓝
    将1克甲苯胺蓝加入100ml蒸馏水中的1%(w/v)四硼酸钠中 通过煮沸和偶尔搅拌溶解
    过滤并在室温下贮存
  7. 乙酸乙烯酯
    将1 g醋酸双氧铀添加到100 ml蒸馏水中
    剧烈振荡,在室温下放置48小时使其溶解 如果所有的乙酸双氧铀已经溶解,再加入1g并剧烈摇动
    在室温下再放置48小时
    重复,直到没有更多的醋酸铀溶解,一些未溶解的试剂保持
  8. 雷诺铅柠檬酸盐染色
    将2.66克硝酸铅溶于30毫升蒸馏水中 将3.52g柠檬酸三钠溶于30ml蒸馏水中
    合并溶液,并在30分钟内每5分钟强力振荡30秒 加入16ml 1M氢氧化钠溶液
    混合直到溶液澄清
    加入蒸馏水至终体积为100ml 储存在4°C

致谢

这项工作是由伦敦女王玛丽大学的大学学生支持。

参考文献

  1. Castenholz,R.W。(1988)。 [3]蓝藻培养方法 方法 167:68-93。
  2. Luft,J.H。(1956)。 高锰酸盐; 一种用于电子显微镜的新固定剂。 2(6):799-802。
  3. Nurnberg,D.J.,Mariscal,V.,Parker,J.,Mastroianni,G.,Flores,E.and Mullineaux,C.W。(2014)。 V部蓝藻菌复合多细胞原核生物中的枝状细胞和细胞间通讯。 Mol Microbiol 91(5):935-949
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引用:Nürnberg, D. J., Mastroianni, G., Mullineaux, C. W. and McPhail, G. D. (2014). Visualization of Cell Complexity in the Filamentous Cyanobacterium Mastigocladus laminosus by Transmission Electron Microscopy (TEM). Bio-protocol 4(23): e1305. DOI: 10.21769/BioProtoc.1305.
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