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Cryo-focused Ion Beam Sample Preparation for Imaging Vitreous Cells by Cryo-electron Tomography
低温聚焦离子束方法制备样本用于低温电子断层扫描术观察玻璃化细胞   

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Abstract

Cryo-electron tomography (CET) is a well-established technique for imaging cellular and molecular structures at sub-nanometer resolution. As the method is limited to samples that are thinner than 500 nm, suitable sample preparation is required to attain CET data from larger cell volumes. Recently, cryo-focused ion beam (cryo-FIB) milling of plunge-frozen biological material has been shown to reproducibly yield large, homogeneously thin, distortion-free vitreous cross-sections for state-of-the-art CET. All eukaryotic and prokaryotic cells that can be plunge-frozen can be thinned with the cryo-FIB technique. Together with advances in low-dose microscopy, this has shifted the frontiers of in situ structural biology. In this protocol we describe the typical steps of the cryo-FIB technique, starting with fully grown cell cultures. Three recently investigated biological samples are given as examples.

Keywords: Focused ion beam(聚焦离子束), Cryo-electron microscopy(低温电子显微镜), Tomography(断层), Chlamydomonas(衣藻), Yeast(酵母)

Materials and Reagents

  1. Biological material
    Note: The FIB-milling procedure is compatible with a wide range of cell types, including single-celled eukaryotes, prokaryotes, and mammalian cells such as HeLa and neuronal cultures. The following examples are used in this protocol:
    1. A Chlamydomonas reinhardtii (C. reinhardtii) wild-type strain CC-124 (137c) (Chlamydomonas Resource Center, University of Minnesota, Minneapolis, MN)
    2. B Chlamydomonas reinhardtii strain mat3-4 (Umen and Goodenough, 2001)
    3. C Yeast strain Saccharomyces cerevisiae (S. cerevisiae)
  2. Tris-acetate-phosphate (TAP) medium (Harris et al., 2009; Culture media recipe is available at http://www.chlamy.org/media.html)
  3. Lugol’s iodine solution (Sigma-Aldrich, catalog number: 62650-100ML-F )
  4. Liquid nitrogen
  5. Ethane/propane high purity gas mixture (37% ethane) (Linde)
  6. Filter paper
  7. YPD liquid medium (see Recipes)

Equipment

  1. R 2/1 Holey Carbon-coated 200-mesh copper grids (Quantifoil Micro Tools GmbH, catalog number: Q2100CR1 )
  2. Tweezers
  3. Screwdrivers
  4. Light microscope
  5. Teflon sheets (custom made, cut from larger sheets in the exact shape and size as the filter paper used for plunge-freezing)
  6. Glass petri dish (9-12 cm)
  7. Glass microscopy slides (Thermo Fisher Scientific, catalog number: 10144633B )
  8. Cryo grid boxes (custom made, or FEI, Eindhoven, The Netherlands)
  9. Cryo AutoGrid boxes (custom made, or FEI, Eindhoven, The Netherlands)
  10. AutoGrids (custom made and modified for FIB work, see Figure 1a)
  11. Clip rings (FEI, Eindhoven, The Netherlands, see Figure 1b)
  12. Liquid nitrogen Dewars
  13. Personal protection equipment for work with liquid nitrogen (safety glasses, face shield, and cold-resistant gloves)
  14. Hemocytometer (Neubauer)
  15. Pipets and tips
  16. Clipping tools (custom made, or FEI, Eindhoven, The Netherlands)
  17. Personal protective equipment for work with liquid nitrogen and liquid ethane/propane
  18. Plasma cleaner (Harrick, model: PDC-3XG )
  19. Vitrobot Mark 4 (FEI)
  20. Focused ion beam microscope (FEI, model: DB Quanta 3D FEG ), Cryo-system (Quorum Technologies, model: PP3000T ), Cryo-stage (custom made, Max Planck Institute)
  21. Transmission electron microscope I. (FEI, model: Tecnai G2 Polara , FEG 300kV), Post-column energy-filter (Gatan, model: HR-GIF 2002 ), Direct detection camera (Gatan, model: K2 Summit ), SerialEM software (Mastronarde, 2005)
  22. Transmission electron microscope II. (FEI, model: Titan Krios, FEG 300kV), post-column energy-filter (Gatan, model: 968 Quantum K2 ) Direct detection camera (Gatan, model: K2 Summit), SerialEM software (Mastronarde, 2005)


Figure 1. AutoGrids modified for cryo-FIB.
a) Top and b) bottom side of an AutoGrid holding a clipped TEM grid. The flat top side shows the cutout required for cryo-FIB milling. c) The FIB shuttle loaded with two AutoGrids. Detailed view showing a properly oriented AutoGrid loaded into the shuttle. Note that the AutoGrid cutout faces up.

Software

  1. FIB software

Procedure

Note: Working with liquid nitrogen and liquid ethane/propane mixture is potentially dangerous. Personal protective equipment should be used.

  1. Vitrification by plunge-freezing
    Note: Standard protocols can be used for plunge-freezing cells (e.g. Iancu et al., 2006). For proper vitrification (freezing in non-crystalline ice) and cell density, parameters must be optimized for each specific cell type.
    1. Cell concentration is determined by counting using a hemocytometer. (C. reinhardtii cells are fixed with Lugol’s iodine solution for the counting process in order to stop cell motility. A Lugol’s solution to cell culture of ratio 1:10 is sufficient.)
    2. The concentration of the cells is diluted to 200-750 cells/μl in fresh TAP for the C. reinhardtii wild-type strain, 500-4,000 cells/μl in fresh TAP for the C. reinhardtii mat3-4 strain, and 10,000-15,000 cells/μl for S. cerevisiae cells in YPD medium.
    3. The carbon-coated 200-mesh copper TEM grids placed carbon side up on a glass slide are glow discharged by 30 sec plasma cleaning. The slide is then stored in a glass petri dish until plunging, which should proceed immediately.
    4. The cells are plunge-frozen using the Vitrobot:
      1. The Vitrobot is set to 90% humidity, blot force 10, 7 to 10 sec blot time, room temperature (C. reinhardtii cells) or 27 °C (S. cerevisiae cells).
      2. 3.5 μl of the diluted cell culture is pipetted onto TEM grids inside the Vitrobot.
      3. The grids are blotted from the reverse side using Teflon sheets on both sides and filter paper on the backside. Then they are immediately plunged into the liquid ethane/propane mixture at liquid nitrogen temperature.
      4. The plunge-frozen grids are then stored in sealed boxes under liquid nitrogen until used.

  2. Sample thinning using cryo-FIB milling
    1. Cooling the FIB system from room temperature to cryo conditions.
      1. Before starting the cooling, the vacuum in the FIB chamber and in the prep-chamber must be lower than 4 x 10-6 mbar to avoid contamination from water vapor.
      2. The N2 gas pipeline between the liquid nitrogen Dewar and the FIB chamber (vacuum isolated line) is pumped to 2 x 10-2 mbar.
      3. The nitrogen gas flow for the stage and anti-contaminator are adjusted to 3.8 L/min and 4.2 L/min, respectively. These values ensure that temperatures of 182 °C and 192 °C are reached after cooling, respectively.
      4. The cooling is started by slowly inserting the cooling rod into the liquid nitrogen Dewar.
    2. Sample clipping and loading the AutoGrids into the FIB shuttle.
      Note: Clipping and loading is started when the cryo-stage and anti-contaminator reach temperatures below -160 °C. Using a surgical mask can minimize water vapor contamination of the work area.
      1. The shuttle and the clipping support base are placed into the loading box (Figure 2).
      2. The loading box is filled with liquid nitrogen.
      3. After boiling has finished, the double wall tank is filled with liquid nitrogen by slightly tilting the loading box (Figure 2).
      4.  The sample boxes from the storage Dewar are transferred into the loading box and opened there. Transfer time should be minimized to avoid contamination of the sample.
      5. All tools that will contact the TEM grids are pre-cooled by liquid nitrogen in the loading box immediately before use.


      Figure 2. Sample clipping and loading. a-c) The sample loading box with double-walled reservoir (9). The second reservoir is filled with liquid nitrogen through the filling gaps (2, 8). The AutoGrid notch (6) aids the fast and easy handling of Autogrids under liquid nitrogen. The clipping tool (1) and tweezers can be cooled in the round bore (7). The shuttle (3) (in loading position), sample storage box (4) and clipping metal support (5) are immersed under liquid nitrogen.

      1. The AutoGrid is placed into the clipping metal support with the flat side facing down. It must be centered properly. The TEM grid is removed from the sample box and placed into the AutoGrid with cells facing down. The grid is then clipped into the AutoGrid with a clip ring using the clipping tool (Figure 1b).
      2. As the shuttle has space for two AutoGrids, step B2f) is repeated for the second AutoGrid.
      3. The AutoGrids are loaded into the shuttle and oriented such that the cutouts are facing up (Figure 1c). The shuttle is then flipped back into the transfer position.

    3. Shuttle transfer onto the FIB cryo-stage.
      1. The temperatures of the cryo-stage and anti-contaminator should be between -194 °C and -165 °C. The temperature of the anti-contaminator should be lower than the temperature of the cryo-stage.
      2. The stage is moved to the transfer position.
      3. The loading box (Figure 3c) is placed onto the pumping station (Figure 3a) and covered.
      4. The transfer unit (Figure 3b) is placed onto the cover of the loading box, and the transfer unit’s insertion rod is attached to the shuttle.
      5. The pumping of the transfer unit and loading box is started. After one minute, the shuttle is lifted out of the loading box into the transfer unit. The transfer unit valve is closed, the pumping is stopped and the loading station is vented (Figure 3d).


      Figure 3. Sample transfer system. a) The Quorum cryo-system with the transfer unit (1). The pumping station (2) with the loading box inside. b) The sample transfer unit with the small transfer chamber (3) and the shuttle (4) attached to the transfer rod. c) The loading box (5) inside the pumping station (7). Here, the shuttle is in the transfer position (6). d) The transfer unit attached to the loading box for shuttle pick-up/drop-off. The small transfer chamber (3) is closed/opened using the transfer unit valve (8).

      1. The transfer unit is attached to the airlock of the FIB (“prep chamber” connected to the turbo pump of the Quorum system).
      2. The airlock is pumped to the vacuum level required to safely open the airlock valves and the transfer unit valve.
      3. The shuttle is transferred onto the cryo-stage with the insertion rod.
      4. The insertion rod is detached from the shuttle and retracted, then the airlock valves and the transfer unit valve are closed. The airlock is vented and the transfer unit is removed.

    4. Lamella milling.
      Note: See Video 1 for an overview of the milling procedure. For help with basic FIB operation, press the F1 key in the FIB software.
      1. Before starting the milling, the vacuum in the FIB chamber at cryo condition must be lower than 2 x 10-6 mbar to avoid contamination from water vapor.
      2. The FIB user interface of the FIB microscope is active.
      3. The column valves are opened and ion beam emission is started (“Beam on” or “Wake up” buttons are pressed).
      4. Electron and ion beams are in the operational state.
        Note: The following parameters enable rapid milling of the bulk cellular material, followed by fine milling and polishing of the lamella.
      5. The scanning parameters for the electron beam are set to: 5.0 kV beam energy, 12 pA beam current, 1,024 x, 884 or 1,536 x 1,024 scan resolution, 1 µs dwell time. Scanning is started.
      6. Scan rotation for both beams is set to 180 degrees.
      7. Low magnification (70x) is set and the stage is moved to one of the two AutoGrids.
      8. The grid bar is scanned at increased zoom to adjust focus, astigmatism and other required alignments for the electron beam.
      9. The stage is linked to a focused working distance (“Link Z to FWD” button).
      10. The stage is moved to the eucentric height (coincidence point) of the microscope (ion and electron beams display the same location).
      11. The stage rotation is adjusted to align the AutoGrid cutout with the incident direction of the ion beam. This is achieved by facing the cutout towards the bottom of the electron image (Figure 4a).
      12.  Organometallic platinum deposition is performed on both AutoGrids using the in situ gas injection system to protect the lamella surface and reduce curtaining effects (protocol adapted from Hayles et al., 2007):
        1. Stage parameters are set to 0° tilt and 3 mm below eucentric height.
        2. The stage is adjusted such that the AutoGrid faces the gas injection system (GIS). For this, a relative stage rotation of 180° is performed using the compucentric rotation. The AutoGrid is then centered at the GIS position in the electron image (Figure 4b).
        3. The GIS temperature is set to 26 °C.
        4. The GIS needle is inserted and opened for 4 to 8 sec.
        5. The GIS needle is retracted.
        6. The stage is moved back to the initial position where it was before the procedure.


      Figure 4. Lamella milling. a). Scanning electron microscope (SEM) image of an AutoGrid aligned with the incident direction of the ion beam. b). The AutoGrid is centered at the GIS position in the SEM image and the GIS needle is inserted. c). SEM image of a TEM grid with Chlamydomonas cells. d). SEM image of two target Chlamydomonas cells before FIB milling. e). FIB-induced secondary electron (FIB SE) image of the two target cells with two rectangle standard milling patterns as a starting point for the milling procedure. f). FIB SE image showing the milling step at 50pA where patterns on both sides are made slimmer (but pattern width remains constant) and shifted closer to the lamella edge. g, i). FIB SE and h) SEM images of the finished lamella after cryo-FIB milling. See Video 1 for an overview of cryo-FIB milling.

      Video 1. Cryo-FIB milling of a vitreous Chlamydomonas cell

      1. The stage is tilted to the milling position with the ion beam almost parallel (typically 6 to 12°) to the grid surface. For a shuttle that is 7° pre-tilted, and a desired milling angle of 10° with respect to TEM grid surface, the stage is tilted to 17°.
      2. The ion beam parameters are set to: 30.0 kV beam energy, 10 pA beam current, 1,024 x 884 or 1,536 x 1,024 scan resolution, 1 µs dwell time.
      3. The focus and astigmatism of the ion beam are adjusted while scanning the grid bar at increased zoom.
      4. A target cell is localized in both electron and ion beam images (Figure 4c-e).
        Note: The three meshes closest to the edge of the TEM grid are often not accessible in the TEM microscope. Generally, central meshes are preferred.
      5. The eucentric height is adjusted using stage movement in the z-direction so that the ion beam and the electron beam show the same sample surface.
        Note: The eucentric height must be separately adjusted for each milling position.
      6. Two parallel rectangular standard milling patterns are drawn (Figure 4e). With pre-tilt correction disabled, the size of the pattern is approximately 10 x 6 µm and the distance between the patterns is more than 5 µm. The milling direction is set to top-to-bottom for one pattern and to bottom-to-top for the other, so that the milling direction of the pattern is always towards the lamella edge. The milling parameters are set to: Ice material (or Si), 1 µs dwell time, 60% overlap.
      7. The ion beam current is changed to 0.3 nA, and a fast single scan is taken (50 ns dwell time) to verify the proper milling position.
      8. The milling process is started. The milling is stopped as soon as the material is completely removed, as observed by live imaging of the scanned area.
      9. Iteratively, the beam current is reduced, pattern heights on both sides are made slimmer (while keeping pattern widths constant) and patterns are shifted closer to the lamella edge (Figure 4f and Video 1). Typical steps for the beam current are 0.1 nA, 50 pA and then 30 pA.
      10. At a sample thickness of about 500 nm, the final milling is performed using 30 pA and optionally the cleaning-cross-section pattern. The target lamella thickness is between 300 and 100 nm (Figure 4g). During this step, thickness is estimated using electron images (Figure 4h-i).
      11. It is possible to prepare multiple lamellas (~10) during one FIB session. For each new lamella, first set the ion beam current back to 10 pA and then repeat steps B4p-v).
        Note: To minimize time-dependent ice contamination, it is recommended to perform steps B4p-U at all sample locations first, then afterwards perform step B4v for each lamella.

    5. Shuttle transfer out of the FIB
      1. The column valves are closed (“Beam on\off” buttons, or “Sleep” button to end the FIB session).
      2. The stage is moved to the transfer position.
      3. The loading box is filled with liquid nitrogen and then placed onto the pumping station.
      4. The transfer unit is docked to the airlock of the FIB.
      5. The airlock is pumped to the vacuum level required to safely open the airlock valves. The airlock valves and the transfer unit valve are then opened.
      6. The shuttle is retrieved from the cryo-stage with the insertion rod and retracted out of the FIB chamber into the transfer unit.
      7. The airlock valves and the transfer unit valve are closed.
      8. The airlock is vented.
      9. The transfer unit is attached to the loading box.
      10. The loading station is pumped for 50 sec. Then the transfer unit valve is opened, and the shuttle is inserted into the loading box with the liquid nitrogen. The pumping is stopped and the loading station is vented.
      11. The insertion rod is detached from the shuttle and the transfer unit is removed.
      12. AutoGrids are removed from the shuttle and stored under liquid nitrogen in storage boxes suitable for AutoGrids.

    6. Warming the FIB system
      1. The cooling rod is taken out of the liquid nitrogen Dewar.
      2. When the cryo-stage and anti-contaminator have reached a temperature of about 20 °C, the gas flow is decreased to 0.5 L/min.

  3. Cryo-ET
    1. The AutoGrid sample is loaded into the cryo-TEM microscope under cryo conditions using the standard procedure (see FEI user manual). When loading the AutoGrid, the AutoGrid cutout (and thus the milling direction of the lamellas) must be aligned perpendicular to the tilt axis of the stage.
    2. The lamella positions are localized using low magnification imaging.
    3. Tilt-series acquisition is performed using SerialEM software under low-dose conditions (<100 e/Å2 cumulative dose). K2 images are recorded at 2° tilt increments, with −4 μm to −8 μm defocus, typically with pixel sizes of 3 to 5 Å.
    1. 1. YPD liquid medium
      1% yeast extract
      2% peptone
      2% D-glucose
      Sterilize by autoclaving

Representative data

Figure 5 shows examples of final cryo-FIB-prepared lamellas of Chlamydomonas and yeast cells. Samples of this quality are reproducibly produced by the described technique (Rigort et al., 2012) and have been used for data collection published elsewhere (Engel et al., 2014; Villa et al., 2013).


Figure 5. TEM images of final lamellas. a) Cross-section of a Chlamydomonas cell, with a lamella thickness of 100 nm. b) Cross-section through several yeast cells, with a lamella thickness of 250 nm.

Recipes

  1. YPD liquid medium
    1% yeast extract
    2% peptone
    2% D-glucose
    Sterilize by autoclaving

Acknowledgments

We thank Alexander Rigort and Felix Bäuerlein for their contributions to developing the cryo-FIB technique (Rigort et al., 2012), and Elizabeth Villa for fruitful scientific discussions. This work was supported by the European Commission's 7th Framework Programme grant agreements ERC-2012-SyG_318987-ToPAG and HEALTH-F4-2008-201648/PROSPECTS, the Deutsche Forschungsgemeinschaft Excellence Clusters CIPSM and SFB 1035, the Federal Ministry of Education and Research (BMBF), an inter-institutional research initiative of the Max Planck Society, a postdoctoral research fellowship from the Alexander von Humboldt Foundation (to BDE), and by EMBO and HFSP postdoctoral research fellowships.

References

  1. Engel, B. D., Schaffer, M., Kuhn Cuellar, L., Villa, E., Plitzko, J. M. and Baumeister, W. (2015). Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography. Elife 4: e04889.
  2. Harris, E. H., Stern, D. B. and Witman, G. B. (2009). The chlamydomonas sourcebook. Academic Press, Elsevier, 2000.
  3. Hayles, M. F., Stokes, D. J., Phifer, D. and Findlay, K. C. (2007). A technique for improved focused ion beam milling of cryo-prepared life science specimens. J Microsc 226(Pt 3): 263-269.
  4. Iancu, C. V., Tivol, W. F., Schooler, J. B., Dias, D. P., Henderson, G. P., Murphy, G. E., Wright, E. R., Li, Z., Yu, Z., Briegel, A., Gan, L., He, Y. and Jensen, G. J. (2006). Electron cryotomography sample preparation using the Vitrobot. Nat Protoc 1(6): 2813-2819.
  5. Mastronarde, D. N. (2005). Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152(1): 36-51.
  6. Rigort, A., Villa, E., Bauerlein, F. J., Engel, B. D. and Plitzko, J. M. (2012). Integrative approaches for cellular cryo-electron tomography: correlative imaging and focused ion beam micromachining. Methods Cell Biol 111: 259-281.
  7. Umen, J. G. and Goodenough, U. W. (2001). Control of cell division by a retinoblastoma protein homolog in Chlamydomonas. Genes Dev 15(13): 1652-1661.
  8. Villa, E., Schaffer, M., Plitzko J. M. and Baumeister, W. (2013). Opening windows into the cell: Focused-ion-beam milling for cryo-electron tomography. Curr Opin Struc Biol 23(5), 771–777.

简介

低电子层析成像(CET)是用于在亚纳米分辨率下成像细胞和分子结构的成熟技术。 由于该方法仅限于比500nm薄的样品,因此需要合适的样品制备以从较大的细胞体积获得CET数据。 最近,冷冻生物材料的低温聚焦离子束(cryo-FIB)研磨已被证明可再现地产生大的,均匀的,无失真的玻璃体横截面用于最先进的CET。 可以使用cryo-FIB技术稀释所有可以浸入冷冻的真核细胞和原核细胞。 连同低剂量显微镜的进展,这已经转移了原位结构生物学的前沿。 在本协议中,我们描述了冷冻FIB技术的典型步骤,从完全生长的细胞培养开始。 最近研究的三个生物样品作为例子。

关键字:聚焦离子束, 低温电子显微镜, 断层, 衣藻, 酵母

材料和试剂

  1. 生物材料
    注意:FIB研磨程序与广泛的细胞类型相容,包括单细胞真核生物,原核生物和哺乳动物细胞例如HeLa和神经元培养物。 本协议使用以下示例:
    1. 衣原体( C。reinhardtii )野生型菌株CC-124                      (137c)(Chlamydomonas Resource Center,University of Minnesota,                      Minneapolis,MN)
    2. B mat3-4 (Umen和Goodenough,2001)
    3. C酵母菌株酿酒酵母(酿酒酵母)
  2. Tris-乙酸 - 磷酸(TAP)培养基(Harris等人,2009; Culture media recipe at available at http://www.chlamy.org/media.html
  3. Lugol碘溶液(Sigma-Aldrich,目录号:62650-100ML-F)
  4. 液氮
  5. 乙烷/丙烷高纯度气体混合物(37%乙烷)(Linde)
  6. 过滤纸
  7. YPD液体培养基(见配方)

设备

  1. R 2/1多孔碳涂覆的200目铜网格(Quantifoil Micro Tools GmbH,目录号:Q2100CR1)
  2. 镊子
  3. 螺丝刀
  4. 光学显微镜
  5. 特氟龙片材(定制,从较大的片材切割成与用于切入冻结的滤纸精确的形状和尺寸)
  6. 玻璃培养皿(9-12厘米)
  7. 玻璃显微镜载玻片(Thermo Fisher Scientific,目录号:10144633B)
  8. Cryo网格框(定制的,或FEI,Eindhoven,荷兰)
  9. Cryo AutoGrid框(定制,或FEI,Eindhoven,荷兰)
  10. AutoGrids(为FIB工作定制和修改,见图1a)
  11. 夹环(FEI,Eindhoven,荷兰,见图1b)
  12. 液氮杜瓦瓶
  13. 使用液氮工作的个人防护设备(安全眼镜,面罩和防寒手套)
  14. 血细胞计数器(Neubauer)
  15. 移液器和提示
  16. 剪切工具(定制,或FEI,Eindhoven,荷兰)
  17. 使用液氮和液体乙烷/丙烷的个人防护设备
  18. 等离子体清洁器(Harrick,型号:PDC-3XG)
  19. Vitrobot Mark 4(FEI)
  20. 聚焦离子束显微镜(FEI,型号:DB Quanta 3D FEG),Cryo系统(Quorum Technologies,型号:PP3000T),Cryo-stage(定制,Max Planck Institute)
  21. 透射电子显微镜I.(FEI,型号:Tecnai G2 Polara,FEG 300kV),柱后能量过滤器(Gatan,型号:HR-GIF 2002),直接检测相机(Gatan,型号:K2 Summit),SerialEM软件 Mastronarde,2005)
  22. 透射电子显微镜II。 (FEI,型号:Titan Krios,FEG 300kV),柱后能量过滤器(Gatan,型号:968 Quantum K2)直接检测相机(Gatan,型号:K2 Summit),SerialEM软件(Mastronarde,


图1.为cryo-FIB修改的AutoGrids。 a)顶部和b)AutoGrid的底部,持有剪辑的TEM网格。 平顶侧显示了cryo-FIB铣削所需的切口。 c)装有两个AutoGrids的FIB梭子。 详细视图显示正确定向的AutoGrid加载到班车。 请注意,AutoGrid开口朝上。

软件

  1. FIB软件

程序

注意:使用液氮和液体乙烷/丙烷混合物是潜在的危险。 应使用个人防护装备。

  1. 插入冻结玻璃化
    注意:标准方案可用于冻结细胞(例如Iancu等人,2006)。为了适当的玻璃化(在非结晶冰中冻结)和细胞密度,必须针对每种特定细胞类型优化参数。
    1. 通过使用血细胞计数器计数来测定细胞浓度。                     ( C。reinhardtii 细胞用Lugol的碘溶液固定                     计数过程,以停止细胞运动。卢戈的解决方案                     细胞培养物比例1:10就足够了。)
    2. 浓度                     将细胞在新鲜TAP中稀释至200-750个细胞/μl。                         莱茵衣藻野生型菌株,500-4,000个细胞/μl的新鲜TAP                         英格兰氏菌 菌株,以及10,000-15,000个细胞/μl。酿酒酵母细胞在YPD培养基中
    3. 碳涂层200目铜TEM栅格                      将放置的碳面朝上放在载玻片上30秒钟发光放电                      等离子清洗。 然后将玻片保存在玻璃培养皿中,直到                      暴跌,应立即进行。
    4. 使用Vitrobot将细胞冷冻冻存:
      1. 将Vitrobot设置为90%湿度,印迹力10,7至10秒印迹                              时间,室温(细胞)或27℃(酿酒酵母细胞)。
      2. 将3.5μl稀释的细胞培养物吸移到Vitrobot内部的TEM网格上。
      3. 网格使用特氟隆片从背面吸干                              两侧和滤纸在背面。 然后他们立即 在液氮下浸入液体乙烷/丙烷混合物中                              温度。
      4. 然后将插入冷冻网格在液氮下储存在密封箱中直到使用。

  2. 使用cryo-FIB铣削的样品稀化
    1. 将FIB系统从室温冷却至低温条件。
      1. 在开始冷却之前,在FIB室中的真空和 预备室必须低于4×10 -6 -6毫巴,以避免污染                              从水蒸气。
      2. 液氮杜瓦瓶和FIB室(真空隔离管线)之间的N 2气体管道被泵送到2×10 -2 -2mbar。
      3. 阶段和抗污染物的氮气流为                              分别调节至3.8L/min和4.2L/min。 这些值确保                              冷却后达到182℃和192℃的温度,                              分别
      4. 通过将冷却杆缓慢地插入液氮杜瓦瓶中开始冷却。
    2. 示例剪辑并将AutoGrids加载到FIB班次 注意:当cryo-stage和                          抗污染物达到温度低于-160°C。 使用手术                          面罩可以最小化工作区域的水蒸汽污染。
      1. 梭子和夹子支撑基座放置在装载箱(图2)中。
      2. 装载箱充满液氮。
      3. 煮沸完成后,双壁罐装满                              通过稍微倾斜装载箱(图2)使液氮
      4.  来自存储装置Dewar的样本框被传输到加载                              盒子和打开那里。 传输时间应尽量避免                              样品污染。
      5. 所有将接触TEM网格的工具在使用前立即在装载箱中用液氮预冷却


      图2.样品剪切和加载 a-c)样品加载框                     双壁储层(9)。第二储存器填充有液体                     氮通过填充间隙(2,8)。 AutoGrid凹槽(6)有助于                     在液氮下快速和容易地处理Autogrids。的                     (1)和镊子可以在圆孔(7)中冷却。的                     穿梭(3)(装载位置),样品存储盒(4)和夹具                     金属载体(5)浸入液氮中。

      1. 的                             AutoGrid放置在具有平坦侧面的夹持金属支撑件中                             面向下。它必须正确居中。从中移除TEM网格 样品盒并放入AutoGrid,细胞面朝下。 的                              网格然后被剪辑到带有夹环的AutoGrid中                              剪切工具(图1b)
      2. 由于梭车有两个AutoGrid的空间,因此对第二个AutoGrid重复步骤B2f)
      3. AutoGrids装载到班车和定向,使                              切口朝上(图1c)。 然后往返翻转                              进入转移位置。

    3. 穿梭运输到FIB低温阶段。
      1. 低温阶段和抗污染物的温度应该是                              在-194℃和-165℃之间。 抗污染物的温度                              应低于低温阶段的温度
      2. 舞台移动到转换位置。
      3. 装载箱(图3c)被放置在泵站(图3a)上并被覆盖
      4. 传送单元(图3b)放置在盖子上                              装载箱,并且传送单元的插入杆附接到装载箱                              穿梭。
      5. 传送单元和装载箱的泵送                              开始。 一分钟后,将梭子从装载箱中提出                              进入转移单元。 传输单元阀关闭,泵送 停止并且装载站被排气(图3d)。


      图3.示例传输系统。a)Quorum cryo系统                     (1)。泵站(2)内有装料箱。                     b)具有小传送室(3)和样品传送单元的样品传送单元                     穿梭(4)连接到传输杆。 c)装载箱(5)内                     泵站(7)。这里,梭子处于转移位置                     (6)。 d)转移单元连接到装载箱用于穿梭                     拾取/放下。小传送室(3)使用关闭/打开                     转移单元阀(8)
      1. 传送单元连接到FIB的气锁("准备室",连接到Quorum系统的涡轮泵)。
      2. 气闸被泵送至所需的真空度,以安全地打开气锁阀和转换单元阀
      3. 穿梭器用插入杆转移到低温阶段。
      4. 然后,插入杆从梭上分离并缩回                              气闸阀和传送单元阀关闭。 气闸                              并且移除传送单元。

    4. 薄片铣削 注意:有关铣削步骤的概述,请参阅视频1。 求助                          使用基本FIB操作,在FIB软件中按F1键。
      1. 在开始研磨之前,在冷冻室中的FIB室中的真空                              条件必须低于2×10 -6 mbar,以避免污染                              水蒸气
      2. FIB显微镜的FIB用户界面已激活。
      3. 打开柱阀,开始离子束发射(按下"Beam on"或"Wake up"按钮)。
      4. 电子和离子束处于操作状态。
        注意:以下参数允许快速铣削块体                                  细胞材料,随后精细研磨和抛光                                  薄片。
      5. 设置电子束的扫描参数                              到:5.0kV束能量,12pA束电流,1,024x,884或1,536x 1,024扫描分辨率,1μs停留时间。 开始扫描。
      6. 两个光束的扫描旋转设置为180度
      7. 设置低倍率(70x),舞台移动到两个AutoGrids之一。
      8. 以增加的变焦扫描网格条以调整电子束的聚焦,散光和其它所需的对准
      9. 舞台与重点工作距离相关("Link Z to FWD"按钮)。
      10. 载物台移动到的重心高度(重合点)                              显微镜(离子和电子束显示相同的位置)
      11. 调整平台旋转以使AutoGrid开口与                             离子束的入射方向。这是通过面对                             朝向电子图像的底部(图4a)
      12.  有机金属铂沉积使用两个AutoGrids进行                             原位气体注入系统保护薄片表面和                             减少视觉效果(由Hayles等人于2007年修订的协议):
        1. 舞台参数设置为0°倾斜,低于偏心高度3mm。
        2. 调整载物台以使AutoGrid面向气体                                     注入系统(GIS)。为此,180°的相对级旋转                                     使用同心旋转进行。 AutoGrid然后居中                                     在电子图像中的GIS位置(图4b)
        3. GIS温度设置为26°C
        4. GIS针插入并打开4至8秒。
        5. GIS针缩回。
        6. 载物台移回到其在程序之前的初始位置。


      图4.薄板铣削。 a)。 扫描电子显微镜(SEM)图像                      的自动格栅与离子束的入射方向对准。 b)。                      AutoGrid以SEM图像中的GIS位置为中心 GIS针插入。 C)。具有衣藻细胞的TEM网格的SEM图像。 d)。 FIB之前的两个靶细胞衣藻细胞的SEM图像                     铣削。 e)。 FIB诱导的二次电子(FIB SE)图像                     靶细胞以两个矩形标准铣削图案为起点                     点用于铣削过程。 F)。 FIB SE图像显示铣削                     步骤在50pA,其中两侧的图案变得更苗条(但图案                     宽度保持恒定)并且移动得更靠近薄片边缘。 g,i)。                     FIB SE和h)在cryo-FIB研磨后成品薄片的SEM图像。                     有关cryo-FIB铣削的概述,请参见视频1
      视频1. Cryo-FIB研磨玻璃体衣藻细胞


      1. 用离子束将载物台倾斜到铣削位置                             平行(通常为6至12°)。对于穿梭                             是预倾斜7°,并且相对于TEM具有10°的期望铣削角 网格表面,舞台倾斜到17°
      2. 离子束                              参数设置为:30.0kV束能量,10pA束电流,1,024x                              884或1,536 x 1,024扫描分辨率,1μs停留时间
      3. 在以增大的变焦扫描网格条时,调整离子束的焦点和像散。
      4. 靶细胞定位在电子和离子束图像中(图4c-e)。
        注意:最靠近TEM网格边缘的三个网格经常                                  在TEM显微镜中不可接近。 通常,中心网格是                                  首选。
      5. 使用平台移动来调整偏心高度                              在z方向上,使得离子束和电子束显示 相同的样品表面。
        注意:必须为每个铣削位置单独调整中心高度。
      6. 绘制两个平行的矩形标准铣削图案(图                             4e)。禁用预倾斜校正时,图案的大小为                             大约10×6μm,并且图案之间的距离更大                             大于5μm。对于一种模式,铣削方向设置为从上到下                             并从底部到顶部为另一个,使铣削方向                             图案总是朝向薄片边缘。铣削参数                             设置为:冰材料(或Si),1μs停留时间,60%重叠
      7. 离子束电流改变为0.3nA,并且快速单次扫描                             采取(50 ns停留时间)以验证正确的铣削位置
      8. 开始铣削过程。一旦停止,铣削就停止                             材料被完全去除,如通过实时成像所观察到的 扫描区域
      9. 迭代地,束电流减小,图案                             两侧的高度变得更细(同时保持图案宽度                             恒定),并且图案更靠近薄片边缘移动(图4f                             和视频1)。束流的典型步骤为0.1 nA,50 pA和                             然后30 pA。
      10. 在约500nm的样品厚度下,                             使用30pA并任选地进行研磨                             清洁横截面图案。目标薄片厚度在之间                             300和100nm(图4g)。在该步骤期间,估计厚度                             使用电子图像(图4h-i)
      11. 可以准备                             在一个FIB期间多个薄片(〜10)。对于每个新的薄片, 扫描区域
      12. 迭代地,束电流减小,图案                             两侧的高度变得更细(同时保持图案宽度                             恒定),并且图案更靠近薄片边缘移动(图4f                             和视频1)。束流的典型步骤为0.1 nA,50 pA和                             然后30 pA。
      13. 在约500nm的样品厚度下,                             使用30pA并任选地进行研磨                             清洁横截面图案。目标薄片厚度在之间                             300和100nm(图4g)。在该步骤期间,估计厚度                             使用电子图像(图4h-i)
      14. 可以准备                             在一个FIB期间多个薄片(〜10)。对于每个新的薄片,... The loading box is filled with liquid nitrogen and then placed onto the pumping station.
      15. The transfer unit is docked to the airlock of the FIB.
      16. The airlock is pumped to the vacuum level required to safely open the airlock valves. The airlock valves and the transfer unit valve are then opened.
      17. The shuttle is retrieved from the cryo-stage with the insertion rod and retracted out of the FIB chamber into the transfer unit.
      18. The airlock valves and the transfer unit valve are closed.
      19. 气闸排气。
      20. 传送单元连接到装载箱。
      21. 装载站被泵送50秒。 然后转移单元                              阀打开,梭子插入装载箱中                              液氮。 泵送停止,装载站为                              通风。
      22. 插入杆与梭子分离,并且移除传送单元。
      23. 将AutoGrids从穿梭器中移出并在液氮下储存在适合于AutoGrids的储存箱中。

    5. 加热FIB系统
      1. 冷却杆从液氮杜瓦瓶中取出。
      2. 当低温阶段和抗污染物已经达到约20℃的温度时,气体流量降低至0.5L/min。

  3. Cryo-ET
    1. 将AutoGrid样品加载到cryo下的cryo-TEM显微镜中                      条件使用标准程序(见FEI用户手册)。 什么时候                      加载AutoGrid,AutoGrid剪切(从而铣削                      方向)必须垂直于倾斜定向                      轴的位置。
    2. 使用低放大率成像定位薄片位置
    3. 倾斜系列采集使用SerialEM软件进行                      低剂量条件(<100e /次 2累积剂量)。 K2图像是                      以2°倾斜增量记录,通常具有-4μm至-8μm散焦                      像素尺寸为3至5。
    1. 1. YPD液体介质
      1%酵母提取物
      2%蛋白胨
      2%D-葡萄糖 通过高压灭菌消毒

代表数据

图5显示了最终cryo-FIB制备的衣藻和酵母细胞的片层的实例。 这种质量的样品通过所述技术(Rigort等人,2012)可再现地产生,并且已经用于其他地方公开的数据收集(Engel等人,2014; Villa 等人,2013)。


图5.最终薄片的TEM图像。 a)衣藻厚度为100nm的衣藻细胞的横截面。 b)通过几个酵母细胞的横截面,片层厚度为250nm。

食谱

  1. YPD液体介质
    1%酵母提取物
    2%蛋白胨
    2%D-葡萄糖 通过高压灭菌消毒

致谢

我们感谢Alexander Rigort和FelixBäuerlein对开发cryo-FIB技术(Rigort等人,2012年)的贡献,以及Elizabeth Villa的富有成效的科学讨论。 这项工作得到了欧盟委员会第七框架计划赠款协议ERC-2012-SyG_318987-ToPAG和HEALTH-F4-2008-201648/PROSPECTS,德意志交易所卓越集群CIPSM和SFB 1035,联邦教育和研究部(BMBF) ),马克斯普朗克学会的机构间研究计划,亚历山大·冯·洪堡基金会(BDE)的博士后研究奖学金以及EMBO和HFSP博士后研究奖学金。

参考文献

  1. Engel,B.D.,Schaffer,M.,Kuhn Cuellar,L.,Villa,E.,Plitzko,J.M.and Baumeister,W。(2015)。 原位构建的衣藻叶绿体由原位/em> cryo-electron tomography。 4:e04889。
  2. Harris,E.H.,Stern,D.B.and Witman,G.B。(2009)。 衣原体。 Academic Press,Elsevier,2000。
  3. Hayles,M.F.,Stokes,D.J.,Phifer,D。和Findlay,K.C。(2007)。 一种用于改进冷冻制备的生命科学标本的聚焦离子束铣削的技术。 em> J Microsc 226(Pt 3):263-269。
  4. Iancu,CV,Tivol,WF,Schooler,JB,Dias,DP,Henderson,GP,Murphy,GE,Wright,ER,Li,Z.,Yu,Z.,Briegel, Y.和Jensen,GJ(2006)。 使用Vitrobot的电子cryotomography样品制备。 Nat Protoc 1(6):2813-2819。
  5. Mastronarde,D.N。(2005)。 使用样本运动的鲁棒预测的自动电子显微镜层析成像。结构生物 152(1):36-51。
  6. Rigort,A.,Villa,E.,Bauerlein,F.J.,Engel,B.D.and Plitzko,J.M。(2012)。 细胞低温电子层析成像的整合方法:相关成像和聚焦离子束微加工。 Methods Cell Biol 111:259-281。
  7. Umen,J.G。和Goodenough,U.W。(2001)。 通过白喉衣原体中的视网膜母细胞瘤蛋白同源物控制细胞分裂。< a> Genes Dev 15(13):1652-1661。
  8. Villa,E.,Schaffer,M.,Plitzko J.M.and Baumeister,W。(2013)。 打开窗口进入细胞:聚焦离子束铣削用于冷冻电子层析成像。 Curr Opin Struc Biol 23(5),771-777。
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Copyright Schaffer et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Schaffer, M., Engel, B. D., Laugks, T., Mahamid, J., Plitzko, J. M. and Baumeister, W. (2015). Cryo-focused Ion Beam Sample Preparation for Imaging Vitreous Cells by Cryo-electron Tomography. Bio-protocol 5(17): e1575. DOI: 10.21769/BioProtoc.1575.
  2. Engel, B. D., Schaffer, M., Kuhn Cuellar, L., Villa, E., Plitzko, J. M. and Baumeister, W. (2015). Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography. Elife 4: e04889.
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