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PEA-CLARITY: Three Dimensional (3D) Molecular Imaging of Whole Plant Organs
全植株的PEA-CLARITY三维分子成像   

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参见作者原研究论文

本实验方案简略版
Scientific Reports
Sep 2015

Abstract

Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration. Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in tobacco and Arabidopsis leaves. Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material. Therefore, this protocol facilitates protein localisation of intact tissue in 3D whilst retaining cellular structure.

Background

Fixation and embedding of plant tissue for molecular interrogation using techniques such as histological staining, immunohistochemistry or in situ hybridisation has been the foundation of cell biology studies for decades. Applying these techniques for 3D tissue analysis is seriously limited by the need to section the tissue, image each section, and then reassemble the images into a 3D representation of the structures of interest. Here we present a fundamental shift from the two dimensional plane to that of three dimensions whilst retaining molecular structures of interest without the need to section the plant tissue. Recent advances in fixation and ‘clearing’ techniques such as SeeDB, ScaleA2, 3DISCO, CLARITY and its recent variant PACT enabled intact imaging of whole embryos, brains and other organs in mouse and rat models. The new CLARITY system fixes and binds tissues within an acrylamide mesh structure. Proteins and nucleic acids are covalently linked to the acrylamide mesh by formaldehyde, then optically interfering lipid structures of animal cell membranes are removed using detergent (SDS). This renders such tissue optically transparent and suitable for deep imaging of up to ~5 mm using confocal microscopy.

Materials and Reagents

  1. 50 ml conical tube
  2. 1.5 ml microfuge tubes
  3. Aluminum foil
  4. Parafilm (Sigma-Aldrich, catalog number: P-7793 )
  5. Lint free paper
  6. 1.5 ml Protein LoBind tubes (Eppendorf, catalog number: 0030108116 )
  7. Glass microscope slide
  8. Glass microscope coverslip
  9. Nicotiana tabacum (Hanson and Köhler, 2001)
  10. 16% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710 )
  11. Sodium azide (NaN3) (Sigma-Aldrich, catalog number: S-2002 )
  12. 0.005% NaN3 in PBS (N3PBS)
  13. Triton X-100 (Sigma-Aldrich, catalog number: T-9284 )
  14. Dulbecco's phosphate buffered saline (DPBS, autoclaved) (Thermo Fisher Scientific, GibcoTM, 21600-010 )
  15. 0.1% Triton X-100 in PBS (PBST)
  16. Vaseline (Unilever, VASELINE®)
  17. BluTack (Bostic)
  18. Rubisco antibody (rabbit) (Gift - Spencer Whitney, Whitney and Andrews, 2001)
  19. Cy5 secondary AB (anti-rab) (Abcam, catalog number: Ab6564 )
  20. Propidium iodide (Sigma-Aldrich, catalog number: P-4864 )
  21. Calcofluor white (Sigma-Aldrich, catalog number: F-3543 )
  22. 40% acrylamide (Bio-Rad Laboratories, catalog number: 161-0140 )
  23. 2% bis acrylamide (Bio-Rad Laboratories, catalog number: 161-0142 )
  24. VA-044 initiator (Wako Pure Chemical Industries, catalog number: 017-19362 )
  25. Deionized and distilled water (ddH2O)
  26. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L-3771 )
  27. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B-6768 )
  28. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S-8045 )
  29. α-amylase (Megazyme, catalog number: E-ANAAM )
  30. α-L-arabinofuranosidase (Megazyme, catalog number: E-ABFCJ )
  31. β-mannanase (Megazyme, catalog number: E-BMACJ )
  32. Cellulase (Megazyme, catalog number: E-CELBA )
  33. Pectate lyase (Megazyme, catalog number: E-PLYCJ )
  34. Xyloglucanase (Megazyme, catalog number: E-XEGP )
  35. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C-5670 )
  36. Hydrogel solution (200 ml) (see Recipes)
  37. SDS clearing solution (1 L) (see Recipes)
  38. Enzyme treatment solution (10 ml) (see Recipes)

Notes:

  1. Please refer to MSDS before conducting protocol as paraformaldehyde (PFA), acrylamide, sodium dodecyl sulfate (SDS) and sodium azide (NaN3) are known irritants, sensitizers, carcinogens and neurotoxins. The use of personal protective equipment (PPE) is imperative whilst undertaking this protocol.
  2. Any specific IgG primary antibody and respective secondary antibody can be used with this protocol.
  3. Protocol can be paused and samples stored at any stage from step D onwards in either SDS clearing solution or N3PBS.

Equipment

  1. Vacuum pump at -100 kPa
  2. Fume hood
  3. 4 °C fridge
  4. 37 °C water bath
  5. Weigh balance
  6. 37 °C incubator shaker
  7. Leica SP8 confocal microscope/lightsheet microscope or equivalent
  8. Long working distance objectives greater than 2 mm

Software

  1. Leica Applications Suite - Fluorescence (LAS-AF) software

Procedure

  1. Plant harvesting
    1. Harvest mature, fully expanded leaves at the end of the dark period to minimise starch accumulation. Replication and position within the leaf of excised disk will differ depending on experimental design.

  2. Fixation
    1. Excise ~20 N. tabacum 7 mm leaf disks of each line from fully expanded leaves before immediately placing each sample into a single 50 ml conical tube containing ice cold hydrogel solution. If samples are required to be individually separated, use 1.5 ml tubes for each individual sample.
    2. Place tissue under vacuum at -100 kPa (in fume hood) for 1-2 h on ice and in darkness if fluorophores are present to facilitate infiltration of hydrogel and removal of gas.
    3. Transfer to a 4 °C fridge overnight.
      Note: If fluorophores are present, keep samples in darkness throughout protocol, i.e., wrap tube(s) in aluminium foil.

  3. Hydrogel polymerization (Figure 1.1)
    1. Carefully remove individual leaf disks from the 50 ml conical tube and place a single disk into 1.5 ml microfuge tubes containing 1 ml fresh chilled hydrogel solution and keep on ice.
    2. Place samples under vacuum for 15 min to remove excess gas from the transferring of samples.
    3. Completely fill 1.5 ml tubes with hydrogel solution taking care to remove any air bubbles before sealing with Parafilm.
    4. Float sealed tubes in a 37 °C water bath overnight to polymerize.

  4. Tissue clearing (Figure 1.2)
    1. Remove polymerized leaf disks from 1.5 ml tubes and separate excess hydrogel carefully from the sample with lint free paper (see Note below).
    2. Wash samples in separate 50 ml conical tubes of SDS clearing solution and change 3 times daily for 2 days to remove excess unbound PFA and acrylamide at room temperature. Take care to dispose of this solution correctly.
    3. 50 ml of SDS clearing solution was replaced daily in each tube for a period of 4-6 weeks (or until clear) at 37 °C with very gentle agitation.
      Note: Peel away excess hydrogel by placing sample onto clean area of lint free paper and pull apart slowly. Repeat this process until all excess hydrogel is removed from the surface of the leaf disk. The sample is quite robust at this stage and even perpendicular trichome parturitions will remain after removal of excess hydrogel if completed with care.

  5. Enzyme treatment (Figure 1.3)
    1. Extensively wash samples in 50 ml N3PBS (see Materials and Reagents section) with 3 changes each day for 3 days at room temperature.
      Note: SDS is an inhibitor of enzymatic activity and improper washing will result in incomplete enzymatic degradation of the cell wall.
    2. Transfer individual sample to a 1.5 ml Protein LoBind tube containing 1 ml of enzyme mix ([EM], see Materials and Reagents section) and keep at 37 °C for 5-7 days with very gentle agitation.
      1. During enzyme treatment a vacuum of -100 kPa is applied in 3 x 5 min bursts, 3 x daily to help facilitate enzyme infiltration.
      2. Replace solution with fresh EM on the third day.
    3. Carefully remove samples after 5-7 days then wash in 50 ml PBST with three changes over a 24 h period with gentle agitation.

  6. Immunolocalization
    1. Dilute primary/secondary antibodies to desired concentration in PBST.
      1. Transfer sample into 1.5 ml Protein LoBind tube containing 1 ml desired primary antibody/s concentration at 37 °C for 5 days with very gentle agitation.
      2. During antibody treatment a vacuum of -100 kPa is applied in 3 x 5 min bursts, 3 x daily to help facilitate primary antibody infiltration.
    2. Wash 3 times with 50 ml PBST for 24 h period.
    3. Follow steps E2a and E2b above for secondary antibody/s.
    4. Wash 3 times with 50 ml N3PBS for a 24 h period.
      Note: For multiple rounds of immunohistochemistry using the same sample, repeat protocol steps D1 and D2 to strip previous antibody after imaging then re-probe using a different set of primary and secondary antibodies as outlined on protocol steps F1-F4.


      Figure 1. Clearing, mounting and imaging of N. tabacum leaf. Left panel: 1. Fresh leaf disc from a fully expanded N. tabacum leaf. 2. Fixed, hydrogel embedded, passively cleared leaf disk. 3. Cleared cell wall enzyme treated leaf disk for immunohistochemistry and imaging. 4. Example of mounting procedure for confocal imaging. Scale bar = 1 mm. Right panel: A. CLSM 3D projection of a passively cleared, cell wall enzyme treated (PEA-CLARITY) Sv-40 (nuclear localised GFP-green) N. tabacum leaf, immunostained with tobacco RuBisCO primary and Cy5 secondary antibodies (red). The 3D projection is shown in (B) and the x, y, z slices are shown in (A, C, D) respectively. E. CLSM 3D projection of a passively cleared (without cell wall enzyme digestion) N. tabacum leaf showing nuclei stained with propidium iodide (red), and cell walls stained with calcofluor white (green). The 3D projection was generated with Leica Applications Suite - Fluorescence (LAS-AF) software. See, Palmer et al. (2015) for further details.

  7. Imaging preparation (Figure 1.4)
    Note: During this protocol samples were mounted in PBS however, as described in (Palmer et al., 2015) other mounting mediums such as Focus Clear (Chung et al., 2013) and RIMS (Yang et al., 2014) can be used to optically match the hydrogel to enhance image clarity. If using other mounting mediums, then samples will need to be incubated prior to mounting.
    1. Using a small piece of BluTack create a well by rolling the putty into a long cylindrical shape and apply to a glass microscope slide so the sides of the well are just higher than the sample thickness (~2 mm).
    2. Seal the outer rim of the BluTack well and the microscope slide with a thin layer of Vaseline.
    3. Half fill well with PBS (or mounting medium).
    4. Place sample into well and cover with PBS (or mounting medium).
    5. Place a glass microscope coverslip over the well ensuring there are no air bubbles.
    6. Samples are now ready for imaging.

Data analysis

All images displayed in this article are raw images taken from the Leica SP8 (Figures 1A-1E). Unprocessed 3D reconstructions were performed in the Leica Applications Suite - Fluorescence (LAS-AF) software.

Recipes

  1. Hydrogel solution (200 ml)

    Note: Store at 4 °C.

  2. SDS clearing solution (1 L)


  3. Enzyme treatment solution [EM] (10 ml)

    Note: Make fresh, do not store. Only use enzymes that have passed a size separation gel quality control procedure to assess purity such as those listed from Megazyme.

Acknowledgments

We would like to thank Mark Talbot for helpful suggestions during CLSM measurements, David McCurdy for providing the Sv-40 N. tabacum line, Spencer Whitney for providing the RuBisCO antibody, Vivien Rolland for initial CLSM investigations, and Joe Enright for growing the plants.

References

  1. Chung, K., Wallace, J., Kim, S. Y., Kalyanasundaram, S., Andalman, A. S., Davidson, T. J., Mirzabekov, J. J., Zalocusky, K. A., Mattis, J., Denisin, A. K., Pak, S., Bernstein, H., Ramakrishnan, C., Grosenick, L., Gradinaru, V. and Deisseroth, K. (2013). Structural and molecular interrogation of intact biological systems. Nature 497(7449): 332-337.
  2. Hanson, M. R. and Kohler, R. H. (2001). GFP imaging: methodology and application to investigate cellular compartmentation in plants. J Exp Bot 52(356): 529-539.
  3. Palmer, W. M., Martin, A. P., Flynn, J. R., Reed, S. L., White, R. G., Furbank, R. T. and Grof, C. P. (2015). PEA-CLARITY: 3D molecular imaging of whole plant organs. Sci Rep 5: 13492.
  4. Whitney, S. M. and Andrews, T. J. (2001). The gene for the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit relocated to the plastid genome of tobacco directs the synthesis of small subunits that assemble into Rubisco. Plant Cell 13(1): 193-205.
  5. Yang, B., Treweek, J. B., Kulkarni, R. P., Deverman, B. E., Chen, C. K., Lubeck, E., Shah, S., Cai, L. and Gradinaru, V. (2014). Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158(4): 945-958.

简介

在这里我们报告CLARITY技术适应植物组织与添加的酶降解,以提高光学清除和促进抗体探针穿透。植物 - 酶辅助(PEA)-CLARITY,允许在烟草和拟南芥叶中的污渍,表达的荧光蛋白和IgG抗体的深度光学可视化。酶处理使得抗体能够穿透进入整个组织,而不需要材料的任何切片。因此,该方案促进3D中完整组织的蛋白定位,同时保留细胞结构。

[背景] 使用组织学染色等技术固定和包埋植物组织进行分子探查,免疫组织化学或原位杂交几十年来一直是细胞生物学研究的基础。将这些技术应用于3D组织分析严重受限于需要切片组织,对每个部分成像,然后将图像重新组装成感兴趣结构的3D表示。这里我们提出从二维平面到三维的基本转变,同时保留感兴趣的分子结构,而不需要切片植物组织。固定和"清除"技术,如SeeDB,ScaleA2,3DISCO,CLARITY和其最近的变种PACT的最新进展使小鼠和大鼠模型中完整成像的整个胚胎,大脑和其他器官。新的CLARITY系统固定并结合丙烯酰胺网状结构内的组织。蛋白质和核酸通过甲醛与丙烯酰胺网状物共价连接,然后使用洗涤剂(SDS)除去动物细胞膜的光学干扰脂质结构。这使得这样的组织是光学透明的,并且适合于使用共聚焦显微镜的高达〜5mm的深度成像

材料和试剂

  1. 50ml锥形管
  2. 1.5 ml微量离心管
  3. 铝箔
  4. 石蜡膜(Sigma-Aldrich,目录号:P-7793)
  5. 无绒纸
  6. 1.5ml蛋白LoBind管(Eppendorf,目录号:0030108116)
  7. 玻璃显微镜载玻片
  8. 玻璃显微镜盖玻片
  9. Nicotiana tabacum (Hanson和Köhler,2001)
  10. 16%多聚甲醛(Electron Microscopy Sciences,目录号:15710)
  11. 叠氮化钠(NaN 3)(Sigma-Aldrich,目录号:S-2002)
  12. 0.005%NaN 3在PBS(N 3 PBS)中的溶液
  13. Triton X-100(Sigma-Aldrich,目录号:T-9284)
  14. Dulbecco's磷酸盐缓冲盐水(DPBS,高压灭菌)(Thermo Fisher Scientific,Gibco TM,,21600-010)
  15. 0.1%Triton X-100的PBS(PBST)
  16. Vaseline(Unilever,VASELINE ®
  17. BluTack(Bostic)
  18. Rubisco抗体(兔)(礼品 - Spencer Whitney,Whitney和Andrews,2001)
  19. Cy5二级AB(抗-rab)(Abcam,目录号:Ab6564)
  20. 碘化丙啶(Sigma-Aldrich,目录号:P-4864)
  21. Calcofluor白(Sigma-Aldrich,目录号:F-3543)
  22. 40%丙烯酰胺(Bio-Rad Laboratories,目录号:161-0140)
  23. 2%双丙烯酰胺(Bio-Rad Laboratories,目录号:161-0142)
  24. VA-044引发剂(Wako Pure Chemical Industries,目录号:017-19362)
  25. 去离子和蒸馏水(ddH 2 O)
  26. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L-3771)
  27. 硼酸(H 3 BO 3)(Sigma-Aldrich,目录号:B-6768)
  28. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S-8045)
  29. α-淀粉酶(Megazyme,目录号:E-ANAAM)
  30. α-L-阿拉伯呋喃糖苷酶(Megazyme,目录号:E-ABFCJ)
  31. β-甘露聚糖酶(Megazyme,目录号:E-BMACJ)
  32. 纤维素酶(Megazyme,目录号:E-CELBA)
  33. 果胶裂解酶(Megazyme,目录号:E-PLYCJ)
  34. 木葡聚糖酶(Megazyme,目录号:E-XEGP)
  35. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C-5670)
  36. 水凝胶溶液(200ml)(参见配方)
  37. SDS清除溶液(1 L)(参见配方)
  38. 酶处理溶液(10ml)(见配方)

注意:

  1. 在进行多聚甲醛(PFA),丙烯酰胺,十二烷基硫酸钠(SDS)和叠氮化钠(NaN3)是已知的刺激剂,敏化剂,致癌物质和神经毒素之前,请参考MSDS。在实施此协议时,必须使用个人防护设备(PPE)。
  2. 任何特定的IgG一级抗体和各自的二级抗体可用于该方案。

  3. 可以暂停协议并且将样品从步骤D开始的任何阶段存储在SDS清除溶液或N <

设备

  1. 真空泵在-100 kPa
  2. 通风橱
  3. 4°C冰箱
  4. 37°C水浴
  5. 权衡余额
  6. 37℃恒温摇床
  7. Leica SP8共聚焦显微镜/lightheet显微镜或等同物
  8. 长工作距离目标大于2毫米

软件

  1. Leica应用套件 - 荧光(LAS-AF)软件

程序

  1. 植物收获
    1. 在黑暗期结束时收获成熟,完全扩展的叶子,以最小化淀粉积累。根据实验设计,在切除的椎间盘叶中的复制和位置将不同
  2. 固定
    1. Excise〜20 em。 tabacum从完全膨胀叶片的每条线的7mm叶片,然后立即将每个样品放入含有冰冷水凝胶溶液的单个50ml锥形管中。如果样品需要单独分离,则对每个样品使用1.5 ml试管
    2. 如果存在荧光团以便于水凝胶渗透和除去气体,将组织置于-100kPa(通风橱中)的真空下在冰上和黑暗中放置1-2小时。
    3. 转移至4°C冰箱过夜。
      注意:如果存在荧光团,则在整个方案中将样品保存在黑暗中,即用铝箔包裹试管。

  3. 水凝胶聚合(图1.1)
    1. 小心地从50毫升锥形管中删除单个叶盘,并将单个磁盘放入1.5毫升微量离心管,含有1毫升新鲜的冷冻水凝胶溶液,并保持在冰上。
    2. 将样品在真空下放置15分钟,以除去样品转移过量的气体。
    3. 用水凝胶溶液完全填充1.5 ml管,注意在用石蜡密封前除去任何气泡
    4. 将密封的管在37℃水浴中过夜漂浮聚合
  4. 组织清除(图1.2)
    1. 从1.5 ml管中取出聚合的叶盘,并用无绒纸小心地从样品中分离过量的水凝胶(见下面的注释)。
    2. 洗涤样品在单独的50毫升锥形管的SDS清除溶液,并改变3次,每天2天,以去除过量未绑定的PFA和丙烯酰胺在室温。注意正确处理此解决方案。
    3. 在37℃下,在非常温和的搅拌下,在每个管中每天更换50ml SDS清除溶液4-6周(或直到透明)。
      注意:通过将样品放置在无绒纸的干净区域上并且缓慢拉开,剥离多余的水凝胶。重复此过程,直到所有过量的水凝胶从叶盘的表面去除。在这个阶段,样品是相当坚固的,甚至垂直毛发分娩后,如果仔细完成,在去除多余的水凝胶后仍会保留。

  5. 酶处理(图1.3)
    1. 在50ml Nil 3 PBS(参见材料和试剂部分)中广泛洗涤样品,每天3次,在室温下3天。
      注意:SDS是酶活性的抑制剂,不当的洗涤将导致细胞壁的酶不完全降解。
    2. 将单个样品转移到含有1ml酶混合物([EM],参见材料和试剂部分)的1.5ml蛋白LoBind管中,并在非常温和的搅拌下在37℃保持5-7天。
      1. 在酶处理期间,-1​​00kPa的真空以3×5分钟的爆发施用,每天3次,以帮助促进酶渗透。
      2. 第三天用新鲜的EM替代溶液。
    3. 5-7天后小心取出样品,然后在24小时内用温和搅拌,在50ml PBST中用三次更换洗涤。

  6. 免疫定位
    1. 在PBST中稀释初级/次级抗体至所需浓度。
      1. 将样品转移到1.5 ml含有1 ml所需一级抗体浓度的蛋白LoBind管中,37°C,5天,轻轻搅动。
      2. 在抗体治疗期间,以3×5分钟的爆发施加-100kPa的真空,每天3次以帮助促进初级抗体浸润。
    2. 用50 ml PBST洗涤3次,每次24小时
    3. 按照上述步骤E2a和E2b进行二抗。
    4. 用50ml N 3 PBS洗涤3次,每次24小时 注意:对于使用相同样品的多轮免疫组织化学,重复方案步骤D1和D2以在成像后剥离前抗体,然后使用如方案步骤F1-F4所述的不同组的一抗和二抗重新探测。 br />

      图1.普通烟叶的清除,安装和成像。左图:1.完全膨胀的新鲜叶片。 tabacum 叶。 2.固定,水凝胶嵌入,被动清除叶盘。 3.清除细胞壁酶处理的叶盘用于免疫组织化学和成像。 4.共焦成像的安装程序的示例。比例尺= 1mm。右图:A.被动清除的细胞壁酶处理(PEA-CLARITY)Sv-40(核定位GFP-绿色)N的CLSM 3D投影。烟草RuBisCO原代和Cy5二次免疫染色(红色)。 3D投影在(B)中示出,x,y,z切片分别在(A,C,D)中示出。 E.CLSM被动清除(无细胞壁酶消化)N的3D投影。 (红色)染色的细胞核和用calcofluor白(绿色)染色的细胞壁。使用Leica应用套件 - 荧光(LAS-AF)软件生成3D投影。参见,Palmer等人。 (2015)了解更多详情
  7. 成像准备(图1.4)
    注意:在该方案期间,样品被固定在PBS中,然而,如(Palmer等人,2015)所述的其他固定介质如Focus Clear(Chung等人,2013)和RIMS(Yang等人,2014 )可用于光学匹配水凝胶以增强图像清晰度。如果使用其他固定介质,则样品需要在安装前孵育。
    1. 使用一小块BluTack通过将腻子滚动成长圆柱形状并应用于玻璃显微镜载片,使得孔的侧面刚好高于样品厚度(〜2mm),从而产生孔。
    2. 密封BluTack井的外边缘和显微镜载玻片与一薄层凡士林。
    3. 用PBS(或固定介质)填满一半。
    4. 将样品放入孔中,并用PBS(或封固剂)盖上
    5. 将玻璃显微镜盖玻片盖在井上,确保没有气泡。
    6. 样品现在可以成像。

数据分析

本文中显示的所有图像都是从Leica SP8(图1A-1E)拍摄的原始图像。未处理的3D重建在徕卡应用套件 - 荧光(LAS-AF)软件中进行。

食谱

  1. 水凝胶溶液(200ml)
    注意:存储在4°C。

  2. SDS清洗溶液(1L)


  3. 酶处理溶液[EM](10ml)

    注意:使新鲜,不要存储。只使用已通过大小分离凝胶质量控制程序的酶来评估纯度,例如Megazyme列出的那些。

致谢

我们要感谢Mark Talbot在CLSM测量期间提供有用的建议,David McCurdy提供Sv-40 N。 tabacum,用于提供RuBisCO抗体的Spencer Whitney,用于初始CLSM研究的Vivien Rolland和用于生长植物的Joe Enright。

参考文献

  1. Chung,K.,Wallace,J.,Kim,SY,Kalyanasundaram,S.,Andalman,AS,Davidson,TJ,Mirzabekov,JJ,Zalocusky,KA,Mattis,J.,Denisin,AK,Pak, ,H.,Ramakrishnan,C.,Grosenick,L.,Gradinaru,V.and Deisseroth,K.(2013)。  完整生物系统的结构和分子探测

    497(7449):332-337。
  2. Hanson,MR和Kohler,RH(2001)。  GFP成像:用于研究植物中细胞分区的方法和应用。 52(356):529-539。
  3. Palmer,W​​M,Martin,AP,Flynn,JR,Reed,SL,White,RG,Furbank,RT and Grof,CP(2015)。  PEA-CLARITY:3D植物器官的3D分子成像。 Sci Rep 5:13492.
  4. Whitney,SM and Andrews,TJ(2001)。  基因的核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)小亚基重定位到烟草的质体基因组指导组装成Rubisco的小亚基的合成。植物细胞 (1):193-205
  5. (a,b,c,d,c,d,d,d,d,d,d,d) "ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25088144"target ="_ blank">透明完整组织通过全身清除的单细胞表型。 158(4):945-958。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Palmer, W. M., Martin, A. P., Flynn, J. R., Reed, S., White, R., Furbank, R. T. and Grof, C. P. (2016). PEA-CLARITY: Three Dimensional (3D) Molecular Imaging of Whole Plant Organs. Bio-protocol 6(21): e2000. DOI: 10.21769/BioProtoc.2000.
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