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Force Measurement on Mycoplasma mobile Gliding Using Optical Tweezers
使用光镊测量运动支原体的滑行运动力   

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Abstract

Dozens of Mycoplasma species, belonging to class Mollicutes form a protrusion at a pole as an organelle. They bind to solid surfaces through the organelle and glide in the direction by a unique mechanism including repeated cycles of bind, pull, and release with sialylated oligosaccharides on host animal cells. The mechanical characters are critical information to understand this unique mechanism involved in their infectious process. In this protocol, we describe a method to measure the force generated by Mycoplasma mobile, the fastest gliding species in Mycoplasma. This protocol should be useful for the studies of many kinds of gliding microorganisms.

Keywords: Mycoplasma(支原体), Optical tweezers(光镊), Force(力), Avidin-biotin(抗生物素蛋白 - 生物素), Bead(珠子)

Background

Surface motility systems are spread over many bacterial species and they are not well elucidated compared to bacterial flagella and eukaryotic motor proteins (Jarrell and McBride, 2008), although potentially they can give us critical information to understand the survival strategy of bacteria. To elucidate a motility mechanism, we need information about the structure of machinery, the flow of energy, and the mechanical characters including speed and force. Optical tweezers are a special method used for micromanipulations or force measurements in the piconewton range under microscopy, by which an object with a diffractive index different from the medium is trapped at the center of focused laser beam (Ashkin et al., 1986). This method has greatly contributed to clarifying the features of motility systems including myosin, dynein, and kinesin, and now an established method in the field of biophysics. Here, we provide a protocol on how to measure force generated by surface moving microorganisms, based on our studies (Miyata et al., 2002; Tanaka et al., 2016) for gliding mechanism of M. mobile the fastest gliding species in class Mollicutes. This is the first protocol for force measurement made using optical tweezers in bio-protocol.

Materials and Reagents

  1. 18 x 18 mm Square microscope cover slip (Matsunami Glass, catalog number: C218181 )
  2. 22 x 40 mm Square microscope cover slip (Matsunami Glass, catalog number: C022401 )
  3. Double-sided tape (NICHIBAN, NICETACKTM, catalog number: NW-5 )
  4. Mending tape (3M, Scotch®, catalog number: 810-3-15 )
  5. Filter paper ϕ70 (ADVANTEC, catalog number: 00021070 )
  6. 1.5 ml microtube
  7. M. mobile 163K strain (ATCC, catalog number: 43663 )
  8. Polystyrene beads 1.0 µm in diameter (Polybead® Carboxylate Microspheres 1.00 µm) (Polysciences, catalog number: 08226-15 )
  9. PolyLink Protein Coupling Kit for COOH Microspheres (Polysciences, catalog number: 24350-1 )
    1. PolyLink coupling buffer
    2. PolyLink EDAC
  10. Avidin from egg white (Sigma-Aldrich, catalog number: A9275 )
  11. PBS with 20 mM glucose (PBS/G)
  12. PBS with 40 mM glucose
  13. Sulfo-NHS-LC-LC-biotin (Ez-LinkTM Sulfo-NHS-LC-LC-biotin) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 21338 )
  14. Glucose
  15. Nail polish
  16. Heart infusion broth (BD, catalog number: 238400 )
  17. Yeast extract (BD, catalog number: 212750 )
  18. 10 N NaOH
  19. Horse serum (Thermo Fisher Scientific, GibcoTM, catalog number: 16050122 )
  20. Amphotericin B (Sigma-Aldrich, catalog number: A2942 )
  21. Ampicillin Na (Nacalai Tesque, catalog number: 02739-32 )
  22. Sodium phosphate (pH 7.3)
  23. NaCl
  24. Aluotto medium (see Recipes)
  25. Phosphate-buffered saline (PBS) (see Recipes)

Equipment

  1. Centrifuge (Sigma Laborzentrifugen, model: Sigma 1-14 )
  2. 25 cm2 tissue culture flask (AS ONE, catalog number: 2-8589-01 )
  3. 25 °C incubator (Tokyo Rikakikai, model: LTI-400E )
  4. Sonicator (Emerson Industrial Automation, BRANSON, model: 2510J-MT )
  5. Optical microscope (Olympus, model: IX71 )
  6. Optical tweezers system (Note 1)
  7. High speed charge-coupled device (DigiMo, model: LRH2500XE-1 )
  8. Power meter (Laser Power/Energy Meter) (Coherent, model: FieldMaxII-TOP, catalog number: 1098580 )
  9. Autoclave

Software

  1. ImageJ (http://rsbweb.nih.gov/ij/index.html)
  2. IGOR Pro 6.33J (WaveMetrics, Portland, OR)

Procedure

  1. Coating polystyrene beads with avidin
    1. Warm polystyrene beads, coupling buffer and PBS to room temperature (RT).
    2. Centrifuge 60 µl polystyrene bead suspension at 2,000 x g for 6 min at RT.
    3. Discard supernatant, resuspend the pellet in 400 µl of coupling buffer and centrifuge it at 2,000 x g for 6 min at RT.
    4. Discard supernatant, resuspend the pellet in 160 µl of coupling buffer.
    5. Just before use, dissolve 11.7 mg of EDAC in 50 µl of coupling buffer, add 20 µl of this solution to the suspension and incubate for 5 min at RT.
    6. Dissolve 0.4 mg of avidin in 10 µl of coupling buffer to be 0.13 mM as the final concentration, add the avidin solution to the suspension and incubate it with gently end-over-end mixing for 3 h at RT.
    7. Centrifuge the suspension at 2,000 x g for 6 min at RT, discard the supernatant and resuspend the pellet in 450 µl of PBS. Repeat this step five times.
    8. Transfer the suspension to new microtube and storage it at 4 °C until use (it can be stored for 6 months).

  2. Biotinylation of M. mobile cell surface
    1. Inoculate 1 ml of frozen stock of M. mobile into 10 ml of Aluotto medium in a 25 cm2 tissue culture flask and statically cultivate it at 25 °C to reach an optical density at 600 nm of 0.06-0.08.
    2. Collect 9 ml of cultured cells by centrifugation at 12,000 x g for 4 min at RT, discard supernatant, resuspend the cell pellet in 900 µl of PBS/G and centrifuge it at 12,000 x g for 4 min at RT (Note 2).
    3. Just before use, prepare a 0.5 mM of Sulfo-NHS-LC-LC-biotin solution by dissolving 0.3 mg of Sulfo-NHS-LC-LC-biotin in 1 ml of PBS/G.
    4. Discard supernatant of step B2, resuspend the cell pellet (Note 2) in 300 µl of Sulfo-NHS-LC-LC-biotin solution and incubate the cell suspension for 15 min at RT (Hiratsuka et al., 2006).
    5. Centrifuge the suspension at 12,000 x g for 4 min at RT, discard supernatant, and resuspend the cell pellet (Note 2) in 300 µl of PBS/G. Repeat this step three times.

  3. Force measurement by using optical tweezers
    1. Prepare a tunnel chamber (5-mm interior width, 18-mm length, 60-μm wall thickness) composed of two cover glasses from box, and double-sided tapes (Figure 1).


      Figure 1. Tunnel chamber (step C1)

    2. To coat the glass surface with glycoproteins, inject 20 µl of 10% horse serum in PBS/G into the tunnel chamber and incubate it for 15 min at RT (Note 3).
    3. Dilute the suspension of biotinylated cell with a 10 to 20-fold volume of PBS/G.
    4. Wash the tunnel chamber by flowing 40 µl of PBS/G, inject 20 µl of the diluted cell suspension into the tunnel chamber and incubate it for 15 min at RT.
    5. To remove floating cells, insert 20 µl of PBS/G into the tunnel chamber.
    6. Add the avidin-coated beads in PBS to the same volume of PBS containing 40 mM glucose, for adjusting glucose concentration to 20 mM.
    7. Dilute the bead suspension with PBS/G to get an appropriate bead density (10-20 beads per a 125.4 x 70.6 µm field of view) for the force measurements.
    8. Transfer 40 µl of bead suspension to a new microtube and sonicate it for 20 sec.
    9. Inject sonicated bead suspension into the tunnel chamber and seal both ends with nail polish.
    10. Set and fix the tunnel chamber by mending tapes onto the stage of inverted microscope equipped with optical tweezers (Figure 2).


      Figure 2. Setting tunnel chamber on microscope stage (step C10)

    11. Trap a floating bead near the bottom of glass surface by focused laser beam, start to record the bead movements at 200 frames per second and immediately attached the bead to the back end of the gliding cell through the avidin-biotin interaction. The cell pulls the bead from trap center of optical tweezers and finally stalls (Video 1). The recording should be continued at least 30 sec after the start of stall (Note 4). After recording, turn off the laser beam.
    12. Remove the tunnel chamber and objective lens, turn on the laser beam to the same power as the step C11 and measure the applied laser power of the parallel light, which was achieved by the removal of objective lens, with using a power meter.
    13. Trace the bead movement from the trap center of optical tweezers and calculate the force from the distance between centers of the bead and the laser beam, by using ImageJ and IGOR Pro, as previously described (Tanaka et al., 2016).

      Video 1. Trapping of bead and attachment to gliding cell (step C11). Focused point of laser beam is indicated by a red circle, marked ‘Trap center’. At 5 sec, a flowing bead came from the right side and trapped at the beam. At 18 sec, a gliding cell was moved into the field by stage handling. At 27 sec, the bead was attached to the gliding cell. The cell pulled the trapped bead and stalled from 33 sec to the end.

Data analysis

The calculation method for bead positioning is described in Thompson et al. (2002).

Notes

  1. Construction of optical tweezers was described in Tanaka et al. (2016). Similar systems are also commercially available (Laser Optical tweezers – Mini type Basic model/without shutter, SIGMAKOKI, catalog number: LMS-M1064-2000. OTM200 Optical Tweezer Add-On, THORLABS, catalog number: OTM200).
  2. Suspension of cell pellet
    More than 100 ups and downs with the pipette is recommended to separate cells completely.
  3. Heat treatment of horse serum
    For M. mobile growth and gliding, the horse serum should be inactivated by heat treatment at 56 °C for 30 min.
  4. Available data size for ImageJ v1.43u is limited around 1 GB. The data size of movie depends on the recording time, the field size, and the frame rate. One example is 180 sec, 4 x 4 µm square, and 200 frames per second.

Recipes

  1. Aluotto medium
    2.1% heart infusion broth
    0.56% yeast extract
    0.035% 10 N NaOH
    Note: Dissolve the above three reagents as a mixture, autoclave, cool to lower than 37 °C, and add below three reagents in clean bench.
    10% horse serum
    0.025% amphotericin B
    0.005% ampicillin Na
  2. Phosphate-buffered saline (PBS)
    75 mM sodium phosphate (pH 7.3)
    68 mM NaCl
    Sterilize by using filter or autoclave

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research on Innovative Area, ‘Harmonized Supramolecular Motility Machinery and Its Diversity’ (grant 24117002 to M. Miyata) and by a grant-in-aid for scientific research (B) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (grant 24390107 to M. Miyata).

References

  1. Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E. and Chu, S. (1986). Observation of a single-beam gradient force optical trap for dielectric particles. Opt Lett 11(5): 288.
  2. Hiratsuka, Y., Miyata, M., Tada, T. and Uyeda, T. Q. (2006). A microrotary motor powered by bacteria. Proc Natl Acad Sci U S A 103(37): 13618-13623.
  3. Jarrell, K. F. and McBride, M. J. (2008). The surprisingly diverse ways that prokaryotes move. Nat Rev Microbiol 6(6): 466-476.
  4. Miyata, M., Ryu, W. S. and Berg, H. C. (2002). Force and velocity of mycoplasma mobile gliding. J Bacteriol 184(7): 1827-1831.
  5. Tanaka, A., Nakane, D., Mizutani, M., Nishizaka, T. and Miyata, M. (2016). Directed binding of gliding bacterium, Mycoplasma mobile, shown by detachment force and bond lifetime. MBio 7(3).
  6. Thompson, R. E., Larson, D. R. and Webb, W. W. (2002). Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82(5): 2775-2783.

简介

属于类 Mollicutes 的数十种支原体物种作为细胞器在极点形成突起。它们通过细胞器与固体表面结合,并通过独特的机制沿着方向滑动,包括在宿主动物细胞上重复的结合,拉伸和释放与唾液酸化寡糖的循环。机械特征是了解传染病过程中涉及到的独特机制的关键信息。在本协议中,我们描述了一种测量由支原体(Mycoplasma)中最快的滑翔物种支原体移动产生的力的方法。该方案对于许多种滑动微生物的研究应该是有用的。

背景 表面运动系统分布在许多细菌物种上,与细菌鞭毛和真核细胞运动蛋白相比并不能很好地阐明(Jarrell和McBride,2008),尽管它们可能给我们提供了解细菌生存策略的关键信息。为了阐明动力机制,我们需要关于机械结构,能量流动以及包括速度和力在内的机械特性的信息。光学镊子是用于在显微镜下在微米范围内的微观操作或力测量的特殊方法,通过该技术,具有与介质不同的衍射指数的物体被捕获在聚焦激光束的中心(Ashkin等人,1986)。这种方法大大有助于阐明肌球蛋白,动力蛋白和驱动蛋白的运动系统的特征,现在已经成为生物物理学领域的一个方法。在这里,我们根据我们的研究(Miyata等人,2002; Tanaka等人,2016),提供了如何测量表面移动微生物产生的力的方案,对于M的滑翔机制。移动类 Mollicutes 中最快的滑翔物种。这是使用生物协议中的光学镊子进行力测量的第一个协议。

关键字:支原体, 光镊, 力, 抗生物素蛋白 - 生物素, 珠子

材料和试剂

  1. 18×18毫米方形显微镜盖(松本玻璃,目录号:C218181)
  2. 22 x 40毫米方形显微镜盖滑(松本玻璃,目录号:C022401)
  3. 双面胶带(NICHIBAN,NICETACK TM ,目录号:NW-5)
  4. 修补胶带(3M,Scotch ®,目录号:810-3-15)
  5. 滤纸 70(ADVANTEC,目录号:00021070)
  6. 1.5 ml微管
  7. M。移动 163K株(ATCC,目录号:43663)
  8. 直径1.0μm的聚苯乙烯珠(Polybead<>羧酸盐微球1.00μm)(Polysciences,目录号:08226-15)
  9. PolyLink Protein Coupling Kit for COOH Microspheres(Polysciences,catalog number:24350-1)
    1. PolyLink耦合缓冲区
    2. PolyLink EDAC
  10. 来自蛋清的抗生物素蛋白(Sigma-Aldrich,目录号:A9275)
  11. 具有20mM葡萄糖的PBS(PBS/G)
  12. 具有40mM葡萄糖的PBS//
  13. Sulfo-NHS-LC-LC-生物素(Ez-Link Sulfo-NHS-LC-LC-生物素)(Thermo Fisher Scientific,Thermo Scientific TM,目录号: 21338)
  14. 葡萄糖
  15. 指甲油
  16. 心输液汤(BD,目录号:238400)
  17. 酵母提取物(BD,目录号:212750)
  18. 10 N NaOH
  19. 马血清(Thermo Fisher Scientific,Gibco TM ,目录号:16050122)
  20. 两性霉素B(Sigma-Aldrich,目录号:A2942)
  21. 氨苄青霉素钠(Nacalai Tesque,目录号:02739-32)
  22. 磷酸钠(pH 7.3)
  23. NaCl
  24. Aluotto中等(见食谱)
  25. 磷酸盐缓冲盐水(PBS)(见食谱)

设备

  1. 离心机(Sigma Laborzentrifugen,型号:Sigma 1-14)
  2. 25厘米组织培养瓶(AS ONE,目录号:2-8589-01)
  3. 25℃培养箱(东京Rikakikai,型号:LTI-400E)
  4. 超声波发生器(艾默生工业自动化,BRANSON,型号:2510J-MT)
  5. 光学显微镜(奥林巴斯,型号:IX71)
  6. 光学镊子系统(注1)
  7. 高速电荷耦合器件(DigiMo,型号:LRH2500XE-1)
  8. 功率计(激光功率/能量计)(相干型号:FieldMaxII-TOP,目录号:1098580)
  9. 高压灭菌器

软件

  1. ImageJ( http://rsbweb.nih.gov/ij/index。 html
  2. IGOR Pro 6.33J(WaveMetrics,Portland,OR)

程序

  1. 涂布聚苯乙烯珠与抗生物素蛋白
    1. 温热聚苯乙烯珠,偶联缓冲液和PBS至室温(RT)
    2. 在室温下将60μl聚苯乙烯珠悬浮液离心60分钟6分钟
    3. 弃去上清液,将沉淀重悬于400μl偶联缓冲液中,并在室温下以2,000×g离心6分钟。
    4. 弃去上清液,将沉淀重悬于160μl偶联缓冲液中
    5. 使用前,将11.7mg EDAC溶于50μl偶联缓冲液中,加入20μl该溶液至悬浮液中,并在室温下孵育5 min。
    6. 将0.4mg的抗生物素蛋白溶解在10μl偶联缓冲液中,为最终浓度为0.13mM,将抗生物素蛋白溶液加入到悬浮液中,并在室温下轻轻的端到端混合孵育3小时。
    7. 在室温下以2,000×g离心悬浮液6分钟,弃去上清液并将沉淀重悬于450μlPBS中。重复此步骤五次。
    8. 将悬浮液转移到新的微管,并将其储存在4°C直到使用(可以储存6个月)。

  2. M的生物素化。移动细胞表面
    1. 接种1ml冷冻原料的M。在25cm 2组织培养瓶中移动到10ml的Aluotto培养基中,并在25℃静态培养以达到0.06-0.08的600nm的光密度。 >
    2. 通过在室温下以12,000xg离心收集9ml培养细胞4分钟,弃去上清液,将细胞沉淀重悬在900μlPBS/G中,并以12,000xg离心> 4分钟(注2)。
    3. 在使用之前,通过将0.3mg Sulfo-NHS-LC-LC-生物素溶解在1ml PBS/G中制备0.5mM的Sulfo-NHS-LC-LC-生物素溶液。
    4. 丢弃步骤B2的上清液,将细胞沉淀(注2)重悬于300μl的Sulfo-NHS-LC-LC-生物素溶液中,并在室温下孵育细胞悬浮液15分钟(平冢等)。 ,2006)
    5. 在室温下以12,000×g离心悬浮液4分钟,弃去上清液,并将细胞沉淀(注2)重悬于300μlPBS/G中。重复此步骤三次。

  3. 使用光学镊子强制测量
    1. 准备一个隧道室(5毫米内部宽度,18毫米长,60毫米壁厚)由两个盖玻璃从盒子和双面胶带组成(图1)。


      图1.隧道室 (步骤C1)

    2. 用糖蛋白涂覆玻璃表面,将20μl10%马血清的PBS/G注入隧道室,并在室温下孵育15分钟(注3)。
    3. 用10至20倍体积的PBS/G稀释生物素化细胞的悬浮液。
    4. 通过流动40μlPBS/G清洗隧道室,将20μl稀释的细胞悬浮液注入隧道室,并在室温下孵育15分钟。
    5. 要移除浮游细胞,将20μlPBS/G插入隧道室
    6. 将PBS中的抗生物素蛋白包被的珠子加入到含有40mM葡萄糖的相同体积的PBS中,以将葡萄糖浓度调节至20mM。
    7. 用PBS/G稀释珠悬浮液,以获得适当的珠密度(10-20珠/125.4×70.6μm视场)用于力测量。
    8. 将40μl珠悬浮液转移到新的微管,并超声处理20秒。
    9. 将超声波珠悬浮液注入隧道室,并用指甲油密封两端
    10. 通过将胶带修补到装有光学镊子的倒置显微镜台上来设置和固定隧道室(图2)。


      图2.在显微镜平台上设置隧道室 (步骤C10)

    11. 通过聚焦激光束将玻璃表面底部附近的浮珠捕获,开始以200帧/秒的速度记录珠子的运动,并通过抗生物素蛋白 - 生物素相互作用立即将珠粒附着在滑翔细胞的后端。细胞从光镊的陷阱中心拉出珠子,最后停止(视频1)。记录应在开始停止后至少30秒继续(注4)。录制后,关闭激光束。
    12. 拆下隧道室和物镜,将激光束打开与步骤C11相同的功率,并测量通过使用功率计去除物镜实现的平行光的应用激光功率。 />
    13. 跟踪来自光学镊子的陷阱中心的珠子移动,并使用ImageJ和IGOR Pro,如前所述(Tanaka等人),从珠粒和激光束的中心距离算出力。 ,2016)
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      视频1.将小珠和附件捕获到滑翔单元格 (步骤C11)。激光束的聚焦点由红色圆圈表示,标记为"陷阱中心"。 5秒钟,流动的珠子从右侧流出并被束缚在梁上。在18秒时,通过舞台处理将滑翔单元移动到场中。在27秒时,将小珠附着在滑翔细胞上。电池拉动被困珠并从33秒停止到最后。
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数据分析

珠粒定位的计算方法在Thompson等人描述。 (2002)。

笔记

  1. 在Tanaka等人中描述了光学镊子的构造。 (2016)。类似的系统也可商购(激光光学镊子 - 迷你型基本型/无快门,SIGMAKOKI,目录号:LMS-M1064-2000。OTM200光学镊子附件,THORLABS,目录号:OTM200)。
  2. 细胞沉淀悬浮液
    推荐使用移液器超过100次起伏分离细胞。
  3. 马血清热处理
    对于 M。移动生长和滑行,马血清应在56℃热处理30分钟灭活。
  4. ImageJ v1.43u的可用数据大小限制在1 GB左右。电影的数据大小取决于录制时间,字段大小和帧速率。一个例子是180秒,4 x 4平方米,每秒200帧。

食谱

  1. Aluotto中等
    2.1%心脏输液肉汤
    0.56%酵母提取物
    0.035%10N NaOH
    注意:将上述三种试剂作为混合物溶解,高压灭菌,冷却至37℃以下,并在洁净台上加入三种试剂。
    10%马血清
    0.025%两性霉素B
    0.005%氨苄青霉素钠
  2. 磷酸盐缓冲盐水(PBS)
    75 mM磷酸钠(pH 7.3)
    68 mM NaCl
    使用过滤器或高压灭菌器灭菌

致谢

这项工作得到了创新区"协调超分子运动机械及其多样性"科学研究资助(Mi Miata的授予24117002)和科学研究援助(B)的支持。日本教育部,文化部,体育部,科学技术部(授予日本宫田市授予24390107)。

参考文献

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  2. 平冢,Y.,Miyata,M.,Tada,T.和Uyeda,TQ(2006)。由细菌驱动的微电动机。 Proc Natl Acad Sci USA 103(37):13618-13623。
  3. Jarrell,KF和McBride,MJ(2008)。  令人惊奇的多种方式原核细胞移动。 Nat Rev Microbiol 6(6):466-476。
  4. Miyata,M.,Ryu,WS and Berg,HC(2002)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/11889087"target = "_blank">支原体移动滑行的力和速度。 J Bacteriol 184(7):1827-1831。
  5. Tanaka,A.,Nakane,D.,Mizutani,M.,Nishizaka,T。和Miyata,M。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/27353751"target ="_ blank">滑移细菌的定向结合,支原体移动,由分离力和结合寿命所示。 MBio 7(3)。
  6. Thompson,RE,Larson,DR和Webb,WW(2002)。 
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引用:Mizutani, M. and Miyata, M. (2017). Force Measurement on Mycoplasma mobile Gliding Using Optical Tweezers. Bio-protocol 7(3): e2127. DOI: 10.21769/BioProtoc.2127.
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