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Electron Tomography to Study the Three-dimensional Structure of Plasmodesmata in Plant Tissues–from High Pressure Freezing Preparation to Ultrathin Section Collection
采用电子断层摄影术研究植物组织中的胞间连丝的三维结构 — 从高压冷冻制备到超薄切片采集   

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参见作者原研究论文

本实验方案简略版
Nature Plants
May 2017

Abstract

Plasmodesmata (PD) are nanometric (~20 nm wide) membrane lined pores encased in the cell walls of the adjacent plant cells. They allow the cells to exchange all types of molecules ranging from nutrients like sugar, hormones, to RNAs and various proteins. Unfortunately, they are also hijacked by phyto-viruses, enabling them to spread from cell-to-cell and then systematically throughout the whole plant. Their central position in plant biology makes it crucial to understand their physiology and especially link their function to their structure. Over the past 50 years, electron microscopists have observed them and attempted to ultrastructurally characterize them. They laid the foundation of what is known about these pores (Tilney et al., 1991; Ding et al., 1992; Oparka and Roberts, 2001; Nicolas et al., 2017a).

Despite the explosion of three-dimensional electron microscopy (3D-EM), PD ultrastructure remained recalcitrant to such technique. The first technical difficulty is to process them in such a way where they are as close to their native state as possible. Secondly, plant samples reveal themselves as being difficult to process due to the poor staining/fixating reagents penetration rates, their increased size, their high water content and the presence of an acidic vacuole. On top of this, their very unique position in the cell wall and their nanometric size make them difficult to conveniently stain in order to see the inner-workings of these pores.

Here we describe in detail the protocol used in Nicolas et al. (2017b) to image PD in fine detail and produce high-resolution tomograms.

Keywords: Plasmodesmata (胞间连丝), Plant (植物), Cell wall (细胞壁), Electron tomography (电子断层摄影术), Electron microscopy (电子显微镜检查), Cryofixation (冷冻固定)

Background

High Pressure Freezing (HPF) relies on the vitrification of the water present in the sample. By cooling down the sample at a high enough freezing rate (104-105 °C/sec), its contained water molecules cannot reorganize in a crystal-fashion and remain vitrified in an amorphous state (see Dubochet [2007] for further reading on the physics behind water crystallization). The sample is then said to be ‘cryoimmobilized’ or ‘vitrified’. This is generally achieved by the use of liquid nitrogen (-195 °C) or liquid ethane (-188 °C). At ambient pressure, this phenomenon can only be achieved on a few microns (< 5 microns), however by raising the pressure to approximately 2,000 bars (~2,000 atmospheres), this depth can reach at least 200 microns (up to 500 microns in certain conditions). This allows the vitrification of thick biological tissues without any osmotic artefacts.

Classically, HPF is followed by Freeze Substitution (FS). This step comprises staining, dehydration and resin embedding of the sample. The good success of the FS is critical for the final sub-cellular preservation state of the sample and will be dependent upon the staining agents used, their concentration and also the temperature kinetic followed during the different stages of FS. These parameters are key for adequate fixation and cross-linking of cellular components, the proper staining (not too little nor too much) and embedding, and heavily impact the final resolution reached. Although this protocol is a good starting point, depending on the samples (tissues, developmental stage, plant species) and organelles of interest, it will need to be adapted to the reader’s specific task.

Despite published FS protocols custom tailored for plant samples have yielded spectacular results over the past decades (Donohoe et al., 2006; Kang et al., 2011), in our hands, it was not suitable for the study of PD ultrastructure for two reasons: i) The epoxy resin used in such protocols is highly electron-scattering and generates a bad signal to noise ratio when imaged with a 120 kV electron microscope, subsequently preventing us from clearly imaging the internal details of the nanometric pores (Figure 1A). ii) The elements inside the pores are so tightly packed in such a small space (< 10 nm) that heavy staining activity during FS prevents clear imaging of these elements (Figure 1A).

In this context, we opted for HPF followed by a modified FS course to prepare our samples (Figure 1C and Table 1). This led to the production of high resolution tomograms, enabling the appreciation of PD ultrastructure in all dimensions of space. We hope our readers find this protocol useful and will eventually improve it for better visualization of nanometric membrane details in thick plant samples.

Table 1. Comparative cryofixation procedures between Donohoe et al. (2006) and Nicolas et al. (2017b). Left table recapitulates dehydration + fixation steps and right table the resin-embedding step. Cells marked in blue represent temperatures below 0 °C and cells marked in orange represent temperatures above 0 °C.



Figure 1. Comparison between freeze-substitution protocols. A. Root tip plasmodesma micrograph acquired from a 90 nm thick section prepared with the regular cryosubstitution protocol (Donohoe et al., 2006). The central element (white arrows) is barely visible, the membrane bilayers are not as lightly stained as in (B) and the overall signal-to-noise ratio is low. B. 2D micrograph of a plasmodesma situated in the root tip acquired at a 0° tilt from a 180 nm thick section prepared with our improved cryosubstitution protocol. Despite the thickness of the section, details in the vicinity of the pore are discernible, notably the central element (white arrows). Dense material can be seen in the cytoplasmic sleeve space. Insets show a close-up view of the red-boxed region ns. C. This temperature versus time curve depicts the commonly used freeze substitution schedule and the one we developed. Brown sections of the curve correspond to when the sample is submerged in the cryosubstitution cocktail containing the highly reactive staining agents. Green zone corresponds to the ideal area for the frozen hydrated samples, below which hexagonal ice cannot form and acetone won’t freeze at atmospheric pressure (between -95 °C and -80 °C). In Donohoe et al., 2006 (green curve) the samples remain in the cocktail for a prolonged amount of time, until the temperature reaches 20 °C. In our protocol (red curve, Nicolas et al., 2017b) the cocktail is removed from the sample very early on in the process when the temperature is at -50 °C.

Materials and Reagents

  1. Plant material
    1. Square plastic culture plates for Arabidopsis seedlings vertical culture (VWR, catalog number: 391-0444 )
    2. MS medium + vitamins (Duchefa Biochemie, catalog number: M0222.0050 ) for seedlings and cell cultures
    3. Sucrose (Sigma-Aldrich, catalog number: 84100 )
    4. 2-(N-morpholino)ethanesulfonic acid (MES) (Euromedex, catalog number: EU0033-A )
    5. Naphthaleneacetic acid (NAA) stock solution (10 mg/ml aliquoted at -20 °C) (Sigma-Aldrich, catalog number: N064025G )
    6. Kinetin hormone stock solution (1 mg/ml aliquoted at -20 °C) (Sigma-Aldrich, catalog number: K3253 )
    7. 1 N KOH (for pH)
    8. MES (Euromedex)
    9. Plant agar (Duchefa Biochemie, catalog number: P1001.1000 )
    10. Culture mediums for cells and seedlings (see Recipe 1)
      1. Murashige and Skoog medium for liquid cultured cells
      2. Murashige and Skoog medium for seedlings

  2. High pressure freezing (Figure 2A)
    1. Aclar sheet, 51 µm thick (Electron Microscopy Sciences, catalog number: 50426 )
    2. Cryotubes (VWR, catalog number: 479-1207 )
    3. Eppendorf 1.5 ml tubes (SARSTEDT, catalog number: 72.708 )
    4. 15 ml Falcon tube (SARSTEDT, catalog number: 62.553.542 )
    5. Tooth picks
    6. BSA fraction V (Sigma-Aldrich, old catalog number: 85040C, now catalog number: 05482 )
    7. Liquid MS medium
    8. MethylCycloHexane (MCH) (Merck Schuchardt OHG 85662 Hohenbrunn, Germany)
    9. BSA solution for cryoprotection during HPF (see Recipe 2)


      Figure 2. High Pressure Freezing procedure. A. 1. Insulated tweezers for manipulation in liquid nitrogen bath. 2. Leica loading system (see Video 1 for loading procedure) with its associated platelet holder rod. 3. 200 µm deep, 1.5 mm diameter, copper platelets. 4. 100 µm deep, 1.5 mm membrane carriers, copper membrane carriers. 5. Sample transfer metal containers. 6. Pod holder. 7. Pods. 8. Harris puncher for production of the 1.5 mm diameter aclar disks to be optionally laid at the bottom of the 200 µm deep platelets to facilitate the later separation of the sample. 9. Torque screwdriver for tightening the membrane carrier in the pods (#3, 4 and 5 respectively). B. View of the EMPACT1 touchscreen displaying the status of the machine. (10) is the valve controlling liquid nitrogen flux from the main tank (11) to the liquid nitrogen bath (12). C. View of the display after processing a sample. Curve dropping down is temperature and curve rising up is pressure.

  3. Freeze substitution (Figure 3)
    1. Screw top 2 ml tubes (SARDTEDT, catalog number: 72.693 )
    2. Disposable regular pipets 1.5, 2 and 3 ml and FS specific 1 ml with thin tips (Ratiolab, catalog number: 2600155 )
    3. Wheaton glass sample vials with snap-cap for cryomix preparation (DWK Life Sciences, WHEATON, catalog number: 225536 .)
    4. Personal cartridge half mask 6100 (Honeywell International, catalog number: 1029471 )
    5. Plastic coffins or pills Leica molds (AFS2 consumable, Leica, catalog numbers: 16707155 and 16707157 respectively)
    6. Plastic solvent containers with screw tops (Leica, catalog number: 16707158 )
    7. Tinfoil
    8. Uranyl acetate powder (Merck, catalog number: 8473 )
    9. Pure methanol
    10. Glutaraldehyde 10% EM-grade anhydrous solutions in acetone (Electron Microscopy Sciences, catalog number: 16530 )
    11. Ultra pure 100% ethanol and acetone (VWR, catalog numbers: 83813.440 and 20066.558 , respectively)
    12. Osmium tetroxide vials 0.1 g (Electron Microscopy Sciences, catalog number: 19134 )
    13. Nail polish (color does not matter)
    14. Cryosubstitution Uranyl-acetate stock solution (20%) (see Recipe 3)
    15. Cryosubstitution mix (see Recipe 4)


      Figure 3. Freeze substitution procedure. 1. AFS tube holders. The one on the right being made according to Heinz Schwarzes blueprints (heinz.schwarz@tuebingen.mpg.de). 2. Metal cups for holding cryomix tubes. 3. Metal socket for mould containers for resin polymerization. 4. Screwdriver used to transfer tube holders 1 and 2 and sample transfer containers shown in Figure 1 (#5). 5. EMS micro-needle and Ted Pella Inc. ultra-micro-needle tools. 6. Ted Pella Inc. micro-tool adapter. 7. Hand-made tool holder. 8. Leica plastic ‘pill’ mould. 9. Leica plastic ‘coffin’ mould. 10. Plastic solvent container with screw top. 11. Leica plastic mould container.

  4. Resin embedding
    1. HM20 resin (Electron Microscopy Sciences, catalog number: 14345 )
    2. HM20% solutions (see Recipe 5)

  5. Ultramicrotomy
    1. Crystalizing dish (Fisher Scientific, catalog number: 11766582 )
      Manufacturer: DWK Life Sciences, DURAN, catalog number: 213133408 .
    2. Fast absorbent paper filters (Whatman, Filter papers 41) (GE Healthcare, catalog number: 1441-070 )
    3. Glass wand (Fisher Scientific, catalog number: 12441627 )
      Manufacturer: MBL, catalog number: SRF380 .
    4. Grids with different meshes (200 L/inch and 300 L/inch Delta Microscopy) and slot grids (EMS Formvar Carbon Film 2 x 0.5 mm copper grids lot, Electron Microscopy Sciences, catalog number: EMS200-Cu )
    5. Paraffin films (Bemis, Parafilm ‘M’)
    6. Pasteur pipet (VWR, catalog number: 612-1720 )
    7. 0.2 µm filter
    8. Syringe
    9. 0.5 ml Eppendorf tubes
    10. 5 nm colloidal gold solution (BBI solution, catalog number: EM-GC5 , www.bbisolutions.com)
    11. Solid parlodion (Electron Microscopy Sciences, catalog number: 19220 )
    12. Isoamyl-acetate (Sigma-Aldrich, catalog number: W205532 )
    13. Toluidine blue powder (Sigma-Aldrich, catalog number: T3260 )
    14. Sodium borate (Sigma-Aldrich, catalog number: B3545 )
    15. Ultrapure (MilliQ) water
    16. 2% Parlodion solution for grid filming (see Recipe 6)
    17. Toluidine blue solution (see Recipe 7) for screening block sections on table top microscope during resin block milling
    18. Fiducial marker solution (see Recipe 8)

Equipment

  1. Plant material
    1. 200 ml glass flasks for liquid cell culture (Dutscher, catalog number: 232522 )
    2. Flask shaker to keep liquid cultured cells moving (Digital Shaker, Southwest Science)
    3. Green room for in vitro cultivation of seedlings and liquid cultured cells (see Recipe 1 for growth conditions)

  2. High pressure freezing (Figure 2A)
    1. Liquid nitrogen, liquid nitrogen container and adapted personal protection gear
    2. Air compressor (JUN-AIR)
    3. Leica EMPACT1 machine (Leica Microsystems)
    4. Leica loading system
    5. Pod holders
    6. Pods
    7. Puncher for aclar disks (Harri’s Micro Punch 1.20 mm)
    8. Regular biomolecular pipets from 2 µl to 1,000 µl range
    9. 200 ml flask
    10. Binocular for root dissection (Nikon, model: SMZ-10A )
    11. Heating surface for quick drying of Pods and pod holders during the session
    12. Insulated tweezers for manipulation in N2 (VOMM Germany, 22 SA ESD)
    13. Little metal scooper for sampling liquid cultured cells
    14. Membrane carriers (100 µm or 200 µm deep) (Leica Microsystems, catalog numbers: 16707898 and 16706898 respectively)
    15. Metal containers for frozen sample transfer and associated screw driver for lifting procedures (provided with EMPACT1)
    16. Torque screwdriver (TOHNICHI, Torque driver RTD60CN) set to 30Ncm

  3. Freeze Substitution and resin embedding (Figure 3)
    1. Leica EM Freeze Substitution Processor (FSP) equipped with UV light (Leica Microsystems)
    2. Leica Automatic Freeze Substitution 2 (EM AFS2) machine (Leica Microsystems, model: Leica EM AFS2 )
    3. Hand made tool holders (out of paper clips) for better disposal of the various tools in AFS2 well
    4. AFS tube holders Metal cups
    5. Metal socket for mould containers
    6. Microtools for separating the membrane carriers from frozen samples and disposing samples in the moulds correctly (micro-needle #1 0.025 mm, Electron Microscopy Sciences, catalog number: 62091-01 and tungsten 5 µm ultra-micro needle, Ted Pella, catalog number: 13625 )
    7. Ventilated hood

  4. Ultramicrotomy (Figure 4)
    1. Petri dishes of various sizes (Dutscher, catalog numbers: 068515 , 068516 , 068517 )
    2. Various EM grade precision tweezers (EMS style 2, 5, 5X, style 7 https://www.emsdiasum.com/ https://www.dumonttweezers.com)
    3. Block holders
    4. Carbon coater (optional) (SPI-MODULE Carbon Coater)
    5. Diamond knives (DIATOME Trim20 dry, Ultra wet and HISTO wet)
    6. ELMO Glow discharger (optional) (CORDOUAN Technologies)
    7. Glass knife maker (LKB BROMMA, model: 7800 Knifemaker )
    8. Glass knives for first milling steps to reach the sample
    9. Grid carriers for grid storage
    10. Leica Ultracut UC7 (Leica Microsystems, model: Leica Ultracut UC7 )
    11. Razor blades for coarse block preparation
    12. Table top microscope (Olympus, model: CX-41 )


      Figure 4. Ultramicrotomy procedure. 1. Sharp razor blades. 2. Tweezers (shapes n°3, 7, 5 and 5X, EMS). 3. Grid holders. 4. Block holders (sold with ultramicrotome, Leica Microsystems). 5. Diamond knives Histo, ultra 45° for section collection and trim 20 dry for precise trimming (from left to right). 6. Glass knife for coarse trimming. 7. Cat whisker mounted on a wooden stick for section gathering during on-grid collection step. Circular inset shows an enlarged view of the cat whisker mounted on the stick using nail polish.

Software

  1. Tecnai imaging and analysis (TIA) software (https://www.fei.com/software/)
    TEM User Interface was used in conjunction with Tecnai Imaging and Analysis (TIA) software to control and acquire micrographies with the EM (https://www.fei.com/software/)
  2. Xplore3D (FEI) was used for automated tilt series acquisitions (https://www.fei.com/software/)
  3. ImageJ (https://imagej.nih.gov/ij/download.html) with the Input/Output plugin (http://www.cmib.fr/en/download/softwares/input-output.html) were used to readout the .mrc tilt series files and tomograms
  4. IMOD suite (http://bio3d.colorado.edu/imod/)

Note: All processing was done using the IMOD suite (Kremer et al., 1996) (http://bio3d.colorado.edu/imod/), from the alignment of the raw tilt series to tomogram reconstruction, segmentation and data analysis.

Procedure

Note: This procedure involves the manipulation of dangerous compounds, namely liquid nitrogen, acetone, osmium tetroxide, glutaraldehyde, uranyl-acetate, Lowicryl resin. If you are not familiar with how to handle such compounds, please refer to the official guidelines and our Notes 1 and 2.

  1. High pressure freezing
    Steps A1 through A13 should be carried out as fast as possible to limit deterioration of sampled live tissue. In our case, this period is less than a minute. A thorough description on how to use the EMPACT1 is provided in user manual and also in the proof of concept paper (Studer et al., 2001).
    1. Turn on Leica EMPACT and air compressor hooked up to it.
    2. Fill up the chamber with MCH and evacuate air in the circuit by gently pushing on the vertical rod.
    3. Fill in liquid nitrogen tank up to ¾ (keep liquid nitrogen source handy for eventual mid-way top-ups during session).
    4. Switch on the EMPACT1 and wait until working pressure reaches between 4.8 and 5 bars.
    5. On the touchscreen tap on the corresponding valve to allow filling of the bath with liquid nitrogen (Figure 2B).
    6. Load an empty pod and tap on prepare lock start to shoot in order to clear out all the air out of the system.
    7. Charge membrane carrier on the Leica loading system (Figure 2A, #2 and Video 1).

      Video 1. Loading the biological sample in the HPF pods. (1) 200 µm deep membrane carriers (Leica Microsystems); (2) Vertically grown Arabidopsis seedlings; (3) Becker filled liquid MS medium; (4) 20% BSA diluted in liquid MS medium; (5) 0.5-2 µl and 20-200 µl range pipets (Sartorius); (6) Torque screwdriver set to 30Ncm (TOHNICHI, Torque driver RTD60CN); (7) Disposable razor blades; (8) Style 7 EMS tweezers; (9) Disposable tooth picks; (10) Leica loading system (with rod), pods, pod holder.

    8. If using the 200 µm membrane carriers with a hole on the bottom, wedge an aclar disk generated with the puncher. Disks can be generated in batches the day before cryofixation, incubated in 95% ethanol and let to evaporate.
    9. With a 0.5-2 µl pipet, fill the carrier with 20% BSA (Recipe 2) in MS (Murashige and Skoog medium). It must form a clean dome in the socket without bleeding on the edges and with no air bubbles (Video 1).
    10. Handling of biological material: i) Roots are quickly dissected under binocular in MS medium on a glass slide with a sharp razor blade and then carefully picked up with a toothpick. ii) Cells are aliquoted in a 1.5 ml Eppendorf tube from the main 200 ml flask and are let to sediment down for a few minutes. Then, the cells are picked up by scratching from the green cell pellet visible at the bottom with a toothpick or a little metal spoon shaped scooper, Cells are then carefully placed in a membrane carrier. Every 2 to 3 HPF trials (~3 min time frame when two experimenters are implicated), replace the cells in the Eppendorf tube by fresh cells.
    11. Dispose the sample in the BSA bubble in the membrane carrier. It is best when the sample is completely submerged by the BSA (Video 1).
    12. Push the membrane carrier in the pod positioned in its special socket on the Leica loading system (Video 1).
    13. Screw the sapphire tight on the carrier using the TORX screwdriver (set on 2.5 cN m-1) (Video 1).
    14. Screw the white pod holder (Video 1).
    15. Load it in the Leica EMPACT machine and the loading stage and push the piston towards the left (Video 2).

      Video 2. Loading the pod containing the sample in the HPF machine

    16. On the touch screen tap on preparelockstart. The system automatically flushes out the pod holder, making the pod loaded with the sample fall in a liquid N2 bath.
      At this stage the sample must never come out of the liquid N2, risking sudden devitrification and the waste of the sample (Video 2).
    17. Check the freezing rate and pressure rise on the touchscreen. It should look like in Figure 2C, where maximum pressure is reached ~20 msec before reaching final temperature.
    18. Place pod holder in the specially designed socket bathing in the liquid nitrogen (Video 2).
    19. Unscrew the pod holder (Video 2).
    20. Carefully release the clamping from the sapphire by unscrewing and then gently tug out the membrane carrier by tapping the pod against the bottom of the bath. Use insulated tweezers to manipulate the frozen membrane carrier (Video 2).
    21. Place the membrane carrier in the sample carrier dedicated to this usage and keep in liquid N2 (Video 2).
    22. When all samples are ready to be transferred for subsequent steps, place metal lids on the metal containers and quickly transfer the containers from the EMPACT bath to an intermediate liquid nitrogen container. Then transfer to AFS2 machine, already running, filled up with liquid N2 and pre-cooled to -90 °C.

  2. Freeze substitution
    During the freeze substitution, it is important to always maintain the frozen hydrated samples in the cold, below -80 °C to avoid too much hexagonal ice formation (Dubochet, 2007). Therefore, never pick up the tubes out of the well and always use instruments, containers and FS solutions that have been precooled in the well for a good 15-20 min.
    Because Steps B7 to B9 are tedious, we would recommend at the beginning to devote a full day to it.
    1. Under a vented hood, dispatch cryomix (see Recipe 4) immediately after having added osmium into the screw top tubes (numbered with a Sharpie and wrapped in scotch tape to avoid washing out of the ink) and then dip the tubes in liquid nitrogen to freeze the cryomix (less volatile when frozen).
    2. Insert frozen cryomix containing tubes in the AFS well, inside the metal cups.
    3. While cryomix is still solid, carefully dispatch the frozen sample containing membrane carriers inside the cryomix tubes (maximum of 2 carriers/tube).
    4. During the thawing process of the cryomix from the -195 °C to -90 °C transition, the carriers will sink in the mix and freeze-substitution will start.
    5. Set the AFS as following:

    6. (OPTIONAL but highly encouraged) At day +1 after the start of the FS, open the cryomix tubes and redispose the carriers at the bottom in such a way that the carriers do not lay on top of each other in order to maximise the exchanges between the mix and the samples. Also, a gentle shake can be given to the tubes.
    7. After completion of the -90 °C to -50 °C transition, the machine is on hold at -50 °C.
    8. Wash thoroughly every tube 3 times with ultra-pure acetone and then 3 times with ultrapure ethanol (Note 3).
    9. During the last wash, transfer the samples into a plastic mould container containing 100% ethanol.
    10. Carefully separate the samples from the membrane carriers in a plastic mould container using the microtools (Figure 3, #5 and #6). Use them to scrape the rim of the carriers to loosen the frozen BSA. If using 200 µm deep carriers (Leica Microsystems), the hole at the bottom can be used to push out the frozen sample by fitting the ultra-micro needle tool (Figure 3, #5, right one).
    11. Prior to the Transfer of the detached sample in the moulds with a 3 ml pipette, fill the moulds with cold ultrapure ethanol and let them cool down to -50 °C (Note 4).
    Note: During Steps B3 to B8, cryotubes should always be closed in between the baths in order to avoid sublimation of residual osmium tetroxide.

  3. Resin embedding
    1. When all samples are in place in the adequate moulds, gently pipet out the excess ethanol from the moulds (see Note 3 for pipetting technique) and replace with 25% HM20 (see Recipe 5).
    2. The following incubation kinetic is just a guideline. Adapt according to your work days:

    3. On the last 100% HM20 bath, place the mould containers in the metal piece (Figure 3, #3) that has been cooled down to -50 °C before hand.
    4. Mount the FSP module and set the AFS as following:

    5. Take out hardened resin blocks from their casts by either slicing the casts carefully with razor blades or, if using the pill-shape moulds, the blocks can be punched out using a hexagonal screwdriver placed on the bottom (Video 3).

      Video 3. Puncturing out the pill-shaped blocks. It requires a Wiha 353/SW 3.0x75 screwdriver for puncturing.

  4. Ultramicrotomy
    1. Grid filming
      Because we embed our samples in HM20 Lowicryl, a more fragile resin than EPON, the sections need to be deposited on grids that have been coated with parlodion or formvar.
      Formvar-coated slot grids were purchased and useful because no grid bars can potentially block the imaging in high tilts. However, they are more fragile and very sensitive to shifts induced by resin retraction. For parlodion-filmed mesh grids, they are home-made as follows (Video 4):
      1. Fill a crystalizing dish, placed in a large Petri dish, with distilled water to the top.
      2. Rack the top of the crystalizing dish with a glass wand to flatten out the surface of the water.
      3. Pipet the parlodion solution with a Pasteur pipet and place the pipet vertically over the crystalizing dish. Let a drop fall on the surface of the water.
      4. Let the parlodion spread at the surface of the water.
      5. With clamps, remove this first film (used to eliminate potential dust and particles on the water surface).
      6. Repeat Steps D1b and D1c.
      7. Carefully place the grids on the floating parlodion film (opaque side face down on the film). Use the mesh desired (200 mesh/µm2 is optimal for better overviews and tilt series acquisition).
      8. With tweezers, gently place a Whatman type 41 absorbent paper (or equivalent absorption speed) on the floating grids and let it soak.
      9. Carefully remove excess parlodion film on the sides of the absorbent paper, either by tearing it away or folding it on top of the floating paper. This is to prevent excess film to fold onto the grids, resulting in a double layer of film on the filmed grids.
      10. To avoid rippling and/or sliding of the film, firmly grip the absorbent paper by the sides with 2 tweezers type 5X or like (which close automatically) and transfer the absorbent paper by flipping it over into a glass/plastic Petri dish.
      11. Let the Petri dish open for a day under ventilated hood, in order to let the filmed grids dry out.
      Quality check: Under the binocular of the ultramicrotome, pick up a filmed grid and by reflection look for a homogenous purple glare on the grid. Under the TEM, the grids may display a few little holes, which is fine. However, when a lot of holes occur, the parlodion solution may be contaminated with water and may need to be replaced.

      Video 4. Grid filming procedure. 1- Glass Pasteur pipet and its pump; 2- Petri dish; 3- Glass wand; 4- Large Petri dish lid; 5- Whatman filter paper type 41; 6- Crystallizer; 7- EM grids. 8- 2% parlodion solution; 9- Type 5X tweezers.

    2. Section collection
      1. Since the aim of this protocol is more focused on how to process the samples as this is the critical factor for the final images, we will not go into details on how to properly use an ultramicrotome, prepare the block-face for cutting etc. Instead please refer to Hagler (2007). The following is some comments for properly performing these steps.
      2. For the study of PD, 90 nm or 180 nm thick sections were collected with the Leica Ultracut UC7 (Leica Microsystems).
      3. Producing serial sections can be useful for two main reasons: i) with one grid, more objects of interest, ii) serial tomography can be performed where the same structure is reconstructed on serial sections, making it possible to recover 3D volumes of structures a lot bigger than the section thickness (Kang et al., 2011).
      4. To do so, the block face needs to be reasonably small enough so that multiple sections can be placed on a single grid. Carving the block face in a trapeze shape will allow the sections to stick together as they are generated, creating a contiguous ribbon of serial sections that can be easily deposited on a single grid simultaneously.
      5. Although it is usually not a problem when using non-carbonated parlodion-filmed copper grids, if sections are repelled by the grids due to static charge during the collection step, grids can be glow-discharged prior to section collection.
      6. Depending on the EM voltage (more voltage equals more kinetic energy of the electrons, and therefore a better ability to image thick samples well) and the size of the structures of interest, thickness of the sections can vary from 90 nm to 180 nm. Finer sections, closer to 90 nm in thickness will yield slightly better x, y resolution, especially in medium range EM voltages as in our case. 180 nm and greater will allow better volume recovery at the expense of x, y resolution when imaged at 120 kV. In the case of PD study, simple ones have a diameter range from 20 to 40 nm thick, so 90 nm thick sections are appropriate. However, it has to be noted that PD can be of various lengths and sizes, especially when it comes to branched complex PD. Therefore they can extend to several hundreds of nm across the cell wall in all directions. In this case, increasing the section thickness grants better chances to fully recover the volume of the pores.
    3. Coating grids with fiducial markers (gold particles)
      Gold particles are used for the subsequent alignment steps required prior to tomogram reconstruction. Because pure colloidal gold tends to aggregate due to its inherent negative charge, it is often used diluted with BSA, making the spread of the gold more homogenous across the section.
      1. On a sheet of paraffin film, lay drops of gold solution (see Recipe 8) and water (Figure 5).
      2. Gently lay the grids on the gold drops for 20 sec approximately.
      3. (Optional) Gently pipet up and down the mixture to favor good contact between the grid and the gold.
      4. Sequentially lay the grids for a few seconds on two water drops to remove excess gold solution.
      5. Absorb the remaining liquid by approaching absorbent paper (Whatman paper Grade 5) on the side of the grid.
      6. Repeat Steps D3a to D3e on the other side of the grid in order to have gold particles on both sides.
      7. Let the grids dry prior to introduction in the EM (damages the ionic pump system otherwise).


        Figure 5. Laying the fiducial markers on the grids. Typical setting for fiducial marker deposition on grid prior to tomography. Grids are first laid on top of the red drops (fiducial marker + BSA solution) for 15-20 sec then dragged consecutively in the water drops and carefully dried by the rim of the grid with absorbent paper. This process needs to be repeated on both sides of the grids.

Data analysis

  1. With this high-pressure freezing–freeze substitution protocol, morphological study of nanometric details inside the cells is rendered possible. Combined with the use of Lowicryl HM-20 resin, more fragile but more electro-lucent and able to polymerize at low temperature (with UV light), this protocol allows efficient tomographic reconstruction of details around 10 nm in dimension. Low magnification images show well-preserved clumps of cells (Figure 6A). At the interfaces between these cells, PD are seen bridging the cells (Figure 6B). Tilt series acquisition of a typical type I pore (Video 5) renders tomograms where the whole volume of the pore can be appreciated (Figures 6C-6E and Video 6).

    Video 5. Tilt series of type I PD. Full range tilt series (-65° to +65°) of the type I plasmodesma (in the centre) found in 4 days old cultured cells, processed with this protocol and shown in Figure 5. Dense black spots are the fiducial markers used for alignment with Etomo.

    Video 6. Tomogram of type I PD. Reconstructed tomogram and its manual segmentation of the type I plasmodesma showed in Figure 5.


    Figure 6. Representative data obtained after HPF and FS. A. Overview of 4 days old cells processed with the procedure described in this article and used in (Nicolas et al., 2017b). B. 0° tilt of the tilt series of a type I plasmodesma (red arrow) found in the red boxed region in (A). Section thickness: 180 nm. C. 0.56 nm thick tomographic slices of the plasmodesma showed in (B). Left and right panel show the two extremities of the pore where the ER can be seen entering the pore (red arrows). D. Segmentation, realized with IMOD, of the same plasmodesma. E. Resliced tomographic slice of the plasmodesma in (C) to have the whole length on one plane. Done with the slicer tool of 3DMOD.

  2. This protocol has also succeeded in delivering clear snapshots of plant organelles such as lipid droplets and Golgi apparatuses (Wattelet-Boyer et al., 2016; Brocard et al., 2017)
  3. Biological replicates and independent experiments:
    PD counting and PD tomography were done on at least two biological replicates, three if possible. For cultured cells, one biological replicate constituted one independent high pressure freezing session on a particular date. Indeed, because the cultured cells were usually sampled in the same flask, we did not consider different blocks from the same HPF as biological replicates.
    For roots, each seedling was considered an individual, therefore were considered biological replicates. Nevertheless, data from roots was also collected on multiple HPF session.
  4. PD screening method:
    In (Nicolas et al., 2017b), counting type I versus type II pores followed basic principles of stereology (Lucocq, 1993). These are: i) avoid double counting the pores and ii) establishing a precise rule on when a PD can be included in data or not. It is important to stress out that this general method can be applied to counting other features on PD (simple versus branched for example) and also other organelles.
    i) When counting on serial sections, there is an inherent risk of double counting structures that span on multiple sections. This risk increases with the size of the structures of interest. Accordingly, a precise counting protocol adapted to the size of the object of interest is required.
    First, the ‘chance’ of encountering a given structure of interest on multiple contiguous sections is assessed. The structures are spotted on a ‘reference’ section (n), then we assess if these same structures are still present on a ‘look-up’ section (n + 1). If the structure is not there anymore, it is said to be ‘resolved’ (Figure 6). The fraction of resolved features can then be computed for section n + 1, n + 2 etc., depending on the size of object of interest relative to the section thickness.
    In the case of PD, which are at the most 40 nm wide, they are largely inferior to section thickness. Hence, at the n + 1 section in cultured cells (Nicolas et al., 2017b Figure 3g), 80% of the pores screened were resolved at section n + 1. Therefore, by counting every 2 sections (n, n + 2, n + 4 etc.), we can reasonably avoid the double counting of PD.
    ii) PD were counted as ‘valid’ and included in the data when both of the plasma membranes were visible and the PD connected at least one of the two adjacent cells (Figure 7).


    Figure 7. PD counting on serial sections. A. Cartoon depicts 4 serial sections on a grid. Structures of interest are spotted on the reference section n and then followed on look-up sections n + 1, n + 2 etc. When structure cannot be seen anymore, it is said to be resolved. (B-C) Micrographies of 90 nm thick sections of a type I PD (left) and type II (PD) typically collected for quantification. Membranes of the pores are visible, and they connect with both sides. In the latter, heterogeneous densities inside the pore allow the visualization of cytoplasmic sleeve space (white arrows). Although not as resolutive as tomography, the central element (desmotubule) can be seen spanning the pore in the center.

Notes

  1. Liquid nitrogen manipulation
    Because liquid nitrogen can burn, always wear adapted eye/face protection, hand protection and a lab coat during manipulation of liquid nitrogen containers.
    Because this gas can cause asphyxiation, always ensure the room is properly ventilated.
  2. Cryosubsitution reagents and resin
    Glutaraldehyde and uranyl acetate solutions must be stored at -20 °C in a separate container.
    Osmium tetroxide should be stored apart, preferably under a vented hood and always manipulated with special lab-gloves (SHIELDskin Orange nitrile 250). When cryomix is being manipulated outside a ventilated hood, i.e., in the AFS during extraction of the cryomix, experimenter (and others present in the same room) should have complete protection on, including lab-gloves, lab-coat, cartridge mask (SPERIAN half mask and T48-ABEK1 P3 dual cartridges) and protective eyewear to avoid any contact with osmium tetroxide fumes.
    HM20 and any other kind of resin should also be manipulated with gloves and cartridge mask as well. For convenience, we wear complete protective gear during all steps from starting of the FS to resin polymerization.
    Overall, all these compounds, including acetone, should be manipulated using lab-gloves at all time. Gloves should be changed every 20 min and changed immediately when contaminated.
  3. Bath changing advice during cryosubstitution
    Changing baths during FS must be done with care and patience. Gently pipet out the previous medium with the thin tips 1 ml pipets (prevents accidental sucking in of the sample). During this step avoid pipetting in and out; turbulences may detach the samples from the carriers. Always leave remaining medium at the bottom to prevent the samples from air-drying. Then add the next medium gently with a regular plastic pipet (either size) by letting it drip on the plastic tube wall.
  4. Unambiguous tracing of samples in moulds
    Keep precise track of what sample/condition is in which mould to avoid any ambiguity. The moulds can be marked with colored nail polish with multiple shapes/dots to distinguish them one-another.

Recipes

  1. Culture mediums for cells and seedlings
    1. Murashige and Skoog medium for liquid cultured cells (1 L)
      Murashige and Skoog medium + vitamins (Duchefa Biochemi): 4.41g
      Sucrose: 30 g
      2-(N-morpholino)ethanesulfonic acid (MES) (Euromedex): 0.5 g
      NAA: 50 µl
      Kinetin stock solution: 50 µl
      Adjust pH with 1 N KOH solution to 5.8
      Autoclave flasks 110 °C for 30 min
      Note: Cells are cultivated under constant light (20 µE/m/sec) and constant agitation at 22 °C.
    2. Murashige and Skoog medium for seedlings (1 L)
      Murashige and Skoog medium + vits (Duchefa Biochemi): 4.4 g
      MES (Euromedex): 0.5 g
      Plant agar (Duchefa Biochemi): 7 g
      Adjust pH with 1 N KOH solution to 5.8
      Autoclave flasks 110 °C for 30 min
      Note: Seedlings are cultivated in vertically placed plates in order for the root to stay on the surface of medium in a greenhouse at 22 °C set on a long day photoperiod (16 h, 100 µE/m/sec).
  2. BSA solution for cryoprotection during HPF (2 ml)
    Weigh 0.4 g of BSA and put it in a 15 ml Falcon tube
    Complete at the 2 ml mark with liquid MS medium for cells (see Recipe 1)
    Vortex thoroughly until BSA is dissolved entirely
    Store at -20 °C
  3. Cryosubstitution Uranyl-acetate stock solution (20%, 500 µl)
    Weigh 0.1g of uranyl acetate powder (gloves and dust mask required) and put in 2 ml screwtop tube
    Add pure methanol (under ventilated hood) up to 500 µl
    Wrap the tube in tinfoil and store in the dark, at -20 °C in dedicated cryosubstitution box
  4. Cryosubstitution mix

  5. HM20% solutions at different concentrations (4 ml)
    Make HM20 solutions in the FS Leica plastic solvent containers (Figure 3, #10). Make the solutions in advance and pre-cool in AFS well at -50 °C before use

  6. 2% Parlodion solution for grid filming (20 ml)
    Weigh 0.4 g of solid parlodion (Electron Microscopy Sciences)
    Add isoamyl-acetate (store in a vented solvent cupboard and keep away from humidity) up to 20 ml
    Agitate until the solid parlodion is dissolved
    Wrap the flask in tinfoil and seal the lid with Parafilm
  7. Toluidine blue solution
    Make mother solution: 1 g of Toluidine blue powder (Sigma-Aldrich) and 1 g of sodium borate (Sigma-Aldrich) in 20 ml of distilled water
    Working solution is diluted 25 times in distilled water
  8. Fiducial marker solution
    Make a solution of 0.5% BSA in MilliQ water
    Filtrate solution with a 0.2 µm filter connected to a syringe
    Mix colloidal gold solution in 0.5% BSA (1:1 ratio)
    Aliquot in 0.5 ml Eppendorf tubes and store at -20 °C

Acknowledgments

This work was supported by the Region Aquitaine (to E.M.B) and PEPS (Initial Support for Exploratory Projects to E.M.B) and the National Agency of Research (Grant ANR-14-CE19-0006-01 to E.M.B). All sample preparation and imaging was done on the Pôle Imagerie du Végétale, appended to the Bordeaux Imaging Centre (http://www.bic.u-bordeaux.fr/). The Region Aquitaine also supported the acquisition of the electron microscope (grant No. 2011 13 04 007 PFM). Thanks to Clément Chambaud that assisted in making the explanatory videos. The authors declare having no conflicts of interests.

References

  1. Brocard, L., Immel, F., Coulon, D., Esnay, N., Tuphile, K., Pascal, S., Claverol, S., Fouillen, L., Bessoule, J. J. and Brehelin, C. (2017). Proteomic analysis of lipid droplets from Arabidopsis aging leaves brings new insight into their biogenesis and functions. Front Plant Sci 8: 894.
  2. Ding, B., Turgeon, R. and Parthasarathy, M. V. (1992). Substructure of freeze-substituted plasmodesmata. Protoplasma 169: 28-41.
  3. Donohoe, B. S., Mogelsvang, S. and Staehelin, L. A. (2006). Electron tomography of ER, Golgi and related membrane systems. Methods 39(2): 154-162.
  4. Dubochet, J. (2007). The physics of rapid cooling and its implications for cryoimmobilization of cells. Methods Cell Biol 79: 7-21.
  5. Hagler, H. K. (2007). Ultramicrotomy for biological electron microscopy. In: Kuo, J. (Ed.). Electron Microscopy: Methods and Protocols. Humana Press pp: 67-96.
  6. Kang, B. H., Nielsen, E., Preuss, M. L., Mastronarde, D. and Staehelin, L. A. (2011). Electron tomography of RabA4b- and PI-4Kβ1-labeled trans Golgi network compartments in Arabidopsis. Traffic 12(3): 313-329.
  7. Kremer, J. R., Mastronarde, D. N. and McIntosh, J. R. (1996). Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116(1): 71-76.
  8. Lucocq, J. (1993). Unbiased 3-D quantitation of ultrastructure in cell biology. Trends Cell Biol 3(10): 354-358.
  9. Nicolas, W. J., Grison, M. S. and Bayer, E. M. (2017a). Shaping intercellular channels of plasmodesmata: the structure-to-function missing link. J Exp Bot.
  10. Nicolas, W. J., Grison, M. S., Trepout, S., Gaston, A., Fouche, M., Cordelieres, F. P., Oparka, K., Tilsner, J., Brocard, L. and Bayer, E. M. (2017b). Architecture and permeability of post-cytokinesis plasmodesmata lacking cytoplasmic sleeves. Nat Plants 3: 17082.
  11. Oparka, K. J. and Roberts, A. G. (2001). Plasmodesmata. A not so open-and-shut case. Plant Physiol 125(1): 123-126.
  12. Studer, D., Graber, W., Al-Amoudi, A. and Eggli, P. (2001). A new approach for cryofixation by high-pressure freezing. J Microsc 203(Pt 3): 285-294.
  13. Tilney, L. G., Cooke, T. J., Connelly, P. S. and Tilney, M. S. (1991). The structure of plasmodesmata as revealed by plasmolysis, detergent extraction, and protease digestion. J Cell Biol 112(4): 739-747.
  14. Wattelet-Boyer, V., Brocard, L., Jonsson, K., Esnay, N., Joubes, J., Domergue, F., Mongrand, S., Raikhel, N., Bhalerao, R. P., Moreau, P. and Boutte, Y. (2016). Enrichment of hydroxylated C24- and C26-acyl-chain sphingolipids mediates PIN2 apical sorting at trans-Golgi network subdomains. Nat Commun 7: 12788.

简介

Plasmodesmata(PD)是包裹在相邻植物细胞的细胞壁中的纳米(〜20nm宽)膜衬里的孔。它们允许细胞交换从糖,激素,RNA到各种蛋白质等营养物质的所有类型的分子。不幸的是,它们也被植物病毒劫持,使它们从细胞间传播,然后在整个植物体系中传播。它们在植物生物学中的核心地位使得理解其生理机制,尤其是将其功能与其结构联系起来至关重要。在过去的50年中,电子显微镜观察家们已经观察到了这些现象,并试图用超微结构来表征它们。他们为已知的这些毛孔奠定了基础(Tilney等人,1991; Ding等人,1992; Oparka和Roberts,2001; Nicolas等人, et al。,2017a)。

尽管三维电子显微镜(3D-EM)爆炸,PD超微结构仍然不支持这种技术。第一个技术难点是在尽可能接近原生状态的情况下处理它们。其次,由于染色/固定试剂穿透率差,其体积增大,含水量高以及存在酸性液泡,植物样品显示自己难以加工。最重要的是,它们在细胞壁上的独特位置和它们的纳米尺寸使得难以方便地染色以便看到这些孔隙的内部运作。

这里我们详细描述Nicolas et al。(2017b)中使用的协议,对PD进行细节化处理,并生成高分辨率的X线断层图。


【背景】高压冻结(HPF)依赖于样品中存在的水的玻璃化。通过以足够高的冷冻速度(10 4 -10 -5℃/秒)冷却样品,其包含的水分子不能以晶体方式重新组织并保持在无定形状态下玻璃化(参见Dubochet [2007],以进一步阅读水结晶背后的物理学)。然后样品被称为“冷冻固定”或“玻璃化”。这通常通过使用液氮(-195°C)或液态乙烷(-188°C)来实现。在环境压力下,这种现象只能在几微米(<5微米)的范围内实现,但是通过将压力提高到约2000巴(〜2000个大气压),该深度可以达到至少200微米(最高达500微米条件)。这允许厚的生物组织的玻璃化而没有任何渗透性人工制品。

经典,HPF后面是冻结替换(FS)。该步骤包括样品的染色,脱水和树脂包埋。 FS的良好成功对于样品的最终亚细胞保存状态是关键的,将取决于所使用的染色剂,其浓度以及在FS不同阶段的温度动力学。这些参数对于细胞组分的充分固定和交联,适当的染色(不太少或不太多)和嵌入,以及对所达到的最终分辨率有重大影响。虽然这个方案是一个很好的起点,但是根据样本(组织,发育阶段,植物种类)和感兴趣的细胞器,这需要根据读者的具体任务进行调整。

尽管已出版的FS规程在过去的几十年中为植物样品量身定做已经产生了惊人的结果(Donohoe等人,2006; Kang等人,2011),在我们的手中,由于两个原因,不适用于PD超微结构的研究:i)在这种方案中使用的环氧树脂是高度电子散射的并且在用120kV电子显微镜成像时产生不良的信噪比,从而阻止我们清晰地成像纳米孔的内部细节(图1A)。 ii)孔内的元素在如此小的空间(<10nm)中紧密堆积以致FS中的重度染色活性阻止了这些元素的清晰成像(图1A)。

在这种情况下,我们选择了HPF,然后修改了FS课程来准备我们的样本(图1C和表1)。这导致了高分辨率的X线断层图的产生,使得能够在所有空间维度上观察局部放电超微结构。我们希望我们的读者能够发现这个协议是有用的,并且最终将会改进它以更好地观察厚植物样品中的纳米膜细节。

表1. Donohoe等人(2006)和Nicolas等人(2017b)之间的比较cryofixation程序。左表概括脱水+固定步骤和右桌树脂嵌入步骤。标记为蓝色的细胞表示温度低于0℃,标记为橙色的细胞表示温度高于0℃。


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图1.冷冻置换方案之间的比较A.根据常规冷冻取代方案(Donohoe等人,2006)制备的从90nm厚的切片获得的根尖等离子体显微照片)。中心元件(白色箭头)几乎不可见,膜双层不像(B)那样轻微染色,整体信噪比低。 B.根据我们的改进的冷冻取代方案制备的位于根尖处的180°厚度切片的0°倾角处的胞间连丝的2D显微照片。尽管该部分的厚度,毛孔附近的细节是明显的,特别是中央元素(白色箭头)。在细胞质套管空间可以看到致密物质。插图显示了红框区域ns的特写视图。 C.这个温度对时间的曲线描述了常用的冻结替代计划和我们开发的计划。曲线的棕色部分对应于何时样品浸没在含有高活性染色剂的冷冻置换混合物中。绿色区域对应于冷冻水合样品的理想区域,在该区域下方不能形成六角形冰,并且丙酮在大气压下(-95℃和-80℃之间)不会冻结。在Donohoe等人(2006年)(绿色曲线)中,样品保持在混合物中持续较长的时间,直到温度达到20℃。在我们的方案(红色曲线,Nicolas等人,2017b)中,当温度在-50℃时,在过程的早期将鸡尾酒从样品中除去。


关键字:胞间连丝, 植物, 细胞壁, 电子断层摄影术, 电子显微镜检查, 冷冻固定

材料和试剂

  1. 植物材料
    1. 用于拟南芥幼苗垂直培养的方形塑料培养板(VWR,目录号:391-0444)
    2. MS培养基+维生素(Duchefa Biochemie,目录号:M0222.0050),用于幼苗和细胞培养
    3. 蔗糖(Sigma-Aldrich,目录号:84100)
    4. 2-(N-吗啉代)乙磺酸(MES)(Euromedex,目录号:EU0033-A)
    5. 萘乙酸(NAA)储备溶液(10毫克/毫升等分于-20°C)(西格玛奥德里奇,目录号:N064025G)
    6. 激动素储备溶液(1mg / ml等分于-20℃)(Sigma-Aldrich,目录号:K3253)
    7. 1 N KOH(用于pH)
    8. MES(Euromedex)
    9. 植物琼脂(Duchefa Biochemie,目录号:P1001.1000)
    10. 细胞和幼苗的培养基(见方法1)
      1. 液体培养细胞的Murashige和Skoog培养基
      2. Murashige和Skoog苗种

  2. 高压冷冻(图2A)
    1. Aclar sheet,51微米厚(Electron Microscopy Sciences,目录号:50426)
    2. 低温管(VWR,目录号:479-1207)
    3. Eppendorf 1.5毫升管(SARSTEDT,目录号:72.708)

    4. 15毫升猎鹰管(SARSTEDT,目录号:62.553.542)
    5. 牙齿选择
    6. BSA部分V(Sigma-Aldrich,旧目录号:85040C,现在目录号:05482)
    7. 液体MS培养基
    8. 甲基环己烷(MCH)(Merck Schuchardt OHG 85662 Hohenbrunn,德国)
    9. BSA在HPF期间的低温保护溶液(见方法2)


      图2.高压冻结过程A. 1.液氮浴中操作的绝缘镊子。 2. Leica加载系统(参见视频1了解加载步骤)及其相关的血小板保持杆。 3. 200微米深,1.5毫米直径的铜片。 4. 100微米深,1.5毫米薄膜载体,铜膜载体。 5.样品转移金属容器。 6.豆荚持有人。 7.豆荚。 8.哈里斯冲床用于生产1.5毫米直径的圆盘,可选择地放置在200微米深的血小板的底部以便于样品随后的分离。 9.扭矩螺丝刀用于拧紧吊舱中的膜载体(分别为#3,4和5)。 B.显示机器状态的EMPACT1触摸屏的视图。 (10)是控制从主罐(11)到液氮浴(12)的液氮流量的阀门。 C.处理样品后显示的视图。曲线下降是温度,曲线上升是压力。

  3. 冻结替代(图3)
    1. 螺杆顶部2毫升管(SARDTEDT,目录号:72.693)
    2. 一次性常规移液管1.5,2和3毫升和FS特定1毫升薄尖(Ratiolab,目录号:2600155)
    3. Wheaton玻璃样品瓶,带有用于cryomix制备的snap-cap(DWK Life Sciences,WHEATON,目录号:225536)。
    4. 个人墨盒半面罩6100(Honeywell International,目录号:1029471)
    5. 塑料棺材或药丸徕卡模具(AFS2消耗品,徕卡,目录号分别为16707155和16707157)
    6. 塑料溶剂容器与螺丝顶部(徕卡,目录号:16707158)
    7. 锡箔
    8. 乙酸双氧铀粉(Merck,目录号:8473)
    9. 纯甲醇
    10. 戊二醛10%EM级无水丙酮溶液(Electron Microscopy Sciences,目录号:16530)
    11. 超纯100%乙醇和丙酮(VWR,目录号分别为83813.440和20066.558)
    12. 四氧化锇小瓶0.1克(电子显微镜科学,目录号:19134)
    13. 指甲油(颜色无所谓)
    14. 低温取代醋酸铀酰储备溶液(20%)(见方法3)
    15. 低温置换组合(见第4部分)


      图3.冻结替代程序 1. AFS管架。右边的是根据Heinz Schwarzes蓝图制作的( heinz.schwarz@tuebingen.mpg.de ) 。 2.用于容纳cryomix管的金属杯。 3.用于树脂聚合的模具容器的金属插座。 4.用于转移图1(#5)所示管架1和2以及样品传输容器的螺丝刀。 EMS微针和泰德派拉公司的超微针工具。 6.泰德佩拉公司的微型工具适配器。 7.手工制作的刀架。 8.徕卡塑料“药丸”模具。 9.徕卡塑料“棺材”模具。 10.带螺纹顶部的塑料溶剂容器。 11. Leica塑料模具容器。

  4. 树脂嵌入
    1. HM20树脂(Electron Microscopy Sciences,目录号:14345)
    2. HM20%的解决方案(见第5章)

  5. 超薄切片
    1. 结晶盘(Fisher Scientific,目录号:11766582)
      制造商:DWK生命科学公司,DURAN,产品目录号:213133408。
    2. 快速吸收纸滤纸(Whatman,滤纸41)(GE Healthcare,目录号:1441-070)
    3. 玻璃棒(Fisher Scientific,目录号:12441627)
      制造商:MBL,目录号:SRF380。
    4. 具有不同网格(200L /英寸和300L / inch Delta显微镜)和槽格(EMS Formvar碳膜2×0.5mm铜网格批号,Electron Microscopy Sciences,目录号:EMS200-Cu)的网格
    5. 石蜡膜(Bemis,Parafilm'M')
    6. 巴斯德吸管(VWR,目录号:612-1720)
    7. 0.2μm过滤器
    8. 注射器
    9. 0.5毫升Eppendorf管
    10. 5纳米胶体金溶液(BBI溶液,目录号:EM-GC5, www.bbisolutions.com ) />
    11. 固体parlodion(电子显微镜科学,目录号:19220)
    12. 乙酸异戊酯(Sigma-Aldrich,目录号:W205532)
    13. 甲苯胺蓝粉末(Sigma-Aldrich,目录号:T3260)
    14. 硼酸钠(Sigma-Aldrich,目录号:B3545)
    15. 超纯水(MilliQ)水
    16. 2%Parlodion网格拍摄解决方案(见第6章)
    17. 甲苯胺蓝溶液(见方法7)用于在树脂块研磨过程中在台式显微镜上筛查块状物部分
    18. Fiducial标记解决方案(见方法8)

设备

  1. 植物材料
    1. 200毫升的液体细胞培养玻璃瓶(Dutscher,目录号:232522)
    2. 保持液体培养细胞运动的摇瓶振荡器(Digital Shaker,Southwest Science)
    3. 用于体外培养幼苗和液体培养细胞的绿色室(参见配方1的生长条件)

  2. 高压冷冻(图2A)
    1. 液氮,液氮容器和适应的个人防护装备
    2. 空压机(JUN-AIR)
    3. 徕卡EMPACT1机器(徕卡显微系统)
    4. 徕卡加载系统
    5. 豆荚持有人
    6. 豆荚
    7. 打孔机(Harri's微型打孔机1.20毫米)
    8. 常规生物分子移液器从2μl到1,000μl范围
    9. 200毫升的烧瓶
    10. 双目根解剖(尼康,型号:SMZ-10A)


    11. 加热表面可快速干燥豆荚和豆荚
    12. 在N 2(VOMM Germany,22 SA ESD)中操作的绝缘镊子
    13. 用于取样液体培养细胞的小金属筐。
    14. 膜载体(100微米或200微米深)(徕卡显微系统,目录号分别为16707898和16706898)
    15. 用于冷冻样品转移的金属容器和用于提升程序的相关螺丝刀(与EMPACT1一起提供)
    16. 扭力螺丝刀(TOHNICHI,扭力螺丝刀RTD60CN)设定为30Ncm

  3. 冻结替代和树脂嵌入(图3)
    1. 徕卡EM冷冻置换处理器(FSP)配有紫外线灯(徕卡显微系统)
    2. Leica Automatic Freeze Substitution 2(EM AFS2)机器(Leica Microsystems,型号:Leica EM AFS2)


    3. 手工制作的工具夹(用纸夹)可更好地处理AFS2井中的各种工具
    4. AFS管架金属杯
    5. 模具容器的金属插座
    6. 用于从冷冻样品中分离膜载体并将样品正确放置在模具中的微型工具(微针#1 0.025mm,Electron Microscopy Sciences,目录号:62091-01和钨5μm超微针,Ted Pella,目录号: 13625)
    7. 通风橱

  4. 超微切开术(图4)
    1. 各种大小的培养皿(Dutscher,目录号:068515,068516,068517)
    2. 各种EM级精密镊子(EMS风格2,5,5X,风格7) https://www.emsdiasum.com/ https://www.dumonttweezers.com
    3. 块持有人
    4. 碳涂层机(可选)(SPI-MODULE碳涂层机)
    5. 金刚石刀(DIATOME Trim20干,超湿和HISTO湿)
    6. ELMO辉光放电器(可选)(CORDOUAN Technologies)
    7. 玻璃刀制造商(LKB BROMMA,型号:7800刀制造商)
    8. 第一步铣削玻璃刀到达样品
    9. 电网运营商的网格存储
    10. 徕卡Ultracut UC7(徕卡显微系统,型号:徕卡Ultracut UC7)
    11. 用于粗块准备的剃刀刀片
    12. 台式显微镜(奥林巴斯,型号:CX-41)


      图4.超薄切片手术 1.尖锐的刀片。 2.镊子(形状n°3,7,5和5X,EMS)。 3.网格持有人。 4.块座(与超薄切片机一起销售,徕卡显微系统)。 5.金刚石刀Histo,超45°的部分收集和修剪20干的精确修剪(从左到右)。 6.玻璃刀切割粗糙。 7.在网格收集步骤中,将猫须安装在木棒上进行截面收集。圆形插图显示了使用指甲油在棒上安装的猫须的放大视图。

软件

  1. Tecnai成像和分析(TIA)软件( https://www.fei.com/software/
    TEM用户界面与Tecnai成像和分析(TIA)软件结合使用来控制和获取EM的显微照片( https ://www.fei.com/software/
  2. Xplore3D(FEI)用于自动倾斜系列采集( https://www.fei.com/software/
  3. ImageJ( https://imagej.nih.gov/ij/download.html )输入/输出插件( http://www.cmib.fr/en /download/softwares/input-output.html )用于读取.mrc倾斜系列文件和断层照片
  4. IMOD套件( http://bio3d.colorado.edu/imod/

注意:所有处理都是使用IMOD套件完成的(Kremer et al。,1996)( 从原始倾斜系列的对齐到断层图像重建,分割和数据分析。

程序

注意:此程序涉及操作危险化合物,即液氮,丙酮,四氧化锇,戊二醛,醋酸铀酰,Lowicryl树脂。如果您不熟悉如何处理此类化合物,请参阅官方指南和我们的注释1和2。

  1. 高压冻结
    步骤A1至A13应尽可能快地进行以限制取样的活组织的恶化。就我们而言,这个时间不到一分钟。有关如何使用EMPACT1的详细说明,请参见用户手册以及概念论证(Studer)。 > 。,2001)。
    1. 打开徕卡EMPACT和空气压缩机。

    2. 使用MCH填充腔室,轻轻推动垂直杆,排出回路中的空气。
    3. 填充液氮罐至¾(保持液氮源方便最终中途补足)。
    4. 打开EMPACT1并等待工作压力达到4.8至5巴。
    5. 在触摸屏上敲击相应的阀门,使液氮充满液体(图2B)。
    6. 加载一个空的盒子,然后点击准备→锁定→ start 以进行拍摄,以清除系统中的所有空气。 />
    7. 在徕卡加载系统(图2A,#2和视频1)上装载薄膜载体。

      shipin1

    8. 如果使用底部有孔的200μm薄膜载体,则用打孔器将生成的光盘楔入。冷冻固定前一天可以批量生产,在95%乙醇中温育,蒸发。
    9. 用0.5-2μl移液器,在MS(Murashige和Skoog培养基)中用20%BSA(配方2)填充载体。它必须在插座上形成一个干净的圆顶,边缘没有流血,没有气泡(视频1)。
    10. 处理生物材料:i)在MS培养基的双目下,用尖锐的刀片在玻璃载片上快速切下根,然后小心地用牙签捡起。 ii)将细胞等分在来自主200ml烧瓶的1.5ml Eppendorf管中,沉降数分钟。然后,通过用牙签或小金属勺形勺子从底部可见的绿色细胞颗粒刮取细胞,然后小心地将细胞置于膜载体中。每2至3次HPF试验(两名实验者牵连3分钟时间范围内),用新鲜细胞代替Eppendorf管中的细胞。
    11. 将样品置于膜载体中的BSA气泡中。当样品完全被BSA淹没(视频1)时最好。
    12. 将薄膜载体推入位于Leica加载系统(视频1)的专用插口中的盒子中。
    13. (视频1),使用TORX螺丝刀(2.5 cN m -1 )将蓝宝石紧固在托架上。
    14. 拧上白色的豆荚夹(视频1)。
    15. 将其装入Leica EMPACT机器和装载台,并将活塞向左推(视频2)。

      shipin2

    16. 在触摸屏上点击准备→锁定→开始。该系统自动冲出容器支架,使装有样品的容器落入液体N 2浴中。
      在这个阶段,样品绝不能从液态N 2中冒出来,冒着突然失透和样品浪费的危险(视频2)。
    17. 检查触摸屏上的冻结速度和压力升高。它应该如图2C所示,在达到最终温度之前最大压力达到〜20毫秒。

    18. 把豆荚盒放在专门设计的插座里,放在液氮中(视频2)
    19. 松开豆荚夹(视频2)。
    20. 小心地松开蓝宝石的夹紧,然后轻轻地将薄膜托架轻敲在浴缸底部。
      使用绝缘镊子操纵冰冻膜载体(视频2)
    21. 将膜载体放置在专用于此用途的样品载体中并保持在液体N 2(视频2)中。
    22. 当所有样品准备好后续步骤转移时,将金属盖放在金属容器上,并迅速将容器从EMPACT槽转移到中间液氮容器中。然后转移到已经运行的AFS2机器,装满液体N 2并预冷至-90℃。

  2. 冻结替代
    在冷冻取代过程中,始终保持冷冻水化样品在低温-80°C以避免过多的六角形冰(Dubochet,2007)非常重要。因此,绝对不要把管子从井中拿出来,总是使用预先在井内预冷15-20分钟的仪器,容器和FS溶液。
    因为步骤B7到步骤B9是单调乏味的,所以我们建议在开始的时候花一整天的时间。
    1. 在通风橱下,立即将锇加入螺旋管(用Sharpie编号并用透明胶带包裹以避免油墨冲洗掉)后立即调配低温混合物(见第4部分),然后将管浸入液氮中冷冻cryomix(冷冻时不易挥发)。

    2. 将金属杯内的冷冻低温混合管插入AFS井中。
    3. 当cryomix仍然是固体时,小心地将含有膜载体的冷冻样品放入cryomix管内(最多2个载体/管)。
    4. 在从-195℃到-90℃过渡期的低温混合物解冻过程中,载体将沉淀在混合物中并开始冻结替代。
    5. 设置AFS如下:


    6. (可选但强烈鼓励)在FS开始之后的第1天,打开cryomix管并重新放置底部的载体,使得载体不会彼此重叠以最大限度地交换混合物和样品。此外,可以给管道轻微的摇晃。
    7. 在-90°C至-50°C过渡完成后,机器保持在-50°C。

    8. 每个试管用超纯丙酮彻底清洗3次,然后用超纯乙醇彻底清洗(注3)
    9. 在最后一次洗涤过程中,将样品转移到含有100%乙醇的塑料模具容器中。
    10. 使用微型工具(图3,#5和#6),将塑料模具容器中的样品从膜载体上小心分离。用它们刮擦载体的边缘,以放松冷冻的BSA。如果使用200微米深的载体(徕卡显微系统),可以使用底部的孔通过安装超微型针头工具(图3,#5,右图)将冷冻样品推出。
    11. 在用3ml移液管将分离的样品转移到模具中之前,用冷的超纯乙醇填充模具并让它们冷却到-50℃(注4)。
    注意:在步骤B3到B8中,为了避免残余的四氧化锇的升华,应该在浴液之间始终关闭冷冻管。

  3. 树脂嵌入
    1. 当所有样品放置在适当的模具中时,轻轻地从模具中吸出多余的乙醇(参见注3),并用25%的HM20代替(见方法5)。
    2. 以下孵化动力只是一个指导原则。根据你的工作日适应:


    3. 在最后的100%HM20浴中,将模具容器置于已经冷却至-50℃的金属片(图3,#3)中,然后手动。
    4. 安装FSP模块并设置AFS如下:


    5. 从模具中取出硬化的树脂块,用刀片仔细切片,或者如果使用丸形模具,可以使用放置在底部的六角螺丝刀冲压块(视频3)。

      视频3

  4. 超薄切片
    1. 网格拍摄
      因为我们将样品嵌入HM20 Lowicryl,这是一种比EPON更脆弱的树脂,所以这些部分需要放在涂有parlodion或formvar的网格上。
      购买Formvar涂层的狭槽栅格并且是有用的,因为没有栅格栅可能在高倾斜度下潜在地阻挡成像。但是,它们更脆弱,对树脂回缩引起的位移非常敏感。对于parlodion拍摄的网格,他们是自制的如下(视频4):

      1. 填充一个结晶盘,放在一个大培养皿中,用蒸馏水

      2. 用玻璃棒将晶化盘的顶部架起来,使水面变平。
      3. 用巴氏吸管吸取parlodion溶液,并将移液管垂直放置在结晶皿上。让水滴落在水面上。
      4. 让parlodion散布在水面上。
      5. 使用夹子,取下第一层薄膜(用于消除潜在的灰尘和水面上的颗粒)。
      6. 重复步骤D1b和D1c。
      7. 小心地将网格放置在浮动的parlodion胶片上(不透明的一面朝下放在胶片上)。使用所需的网格(200目/μm 2对于更好的概述和倾斜系列采集是最佳的)。
      8. 用镊子,轻轻地放置一个沃特曼41型吸水纸(或相当的吸收速度)浮动网格,让它浸泡。
      9. 小心地将吸收性纸张上的多余的parlodion薄膜撕掉,或者将其折叠在浮动纸的顶部。这是为了防止多余的电影折叠到网格上,导致电影网格上的双层电影。
      10. 为了避免薄膜起皱和/或滑动,用2X5X型镊子(自动关闭)牢固地夹住吸收纸,并将吸收纸翻过来放入玻璃/塑料培养皿中。 />
      11. 让培养皿在通风橱下打开一天,以使拍摄的格栅变干。
      质量检查: 在超薄切片机的双筒望远镜下,拿起拍摄的网格,通过反射在网格上寻找均匀的紫色眩光。透射电镜下,网格可能会显示一些小孔,这很好。但是,当发生很多孔时,parlodion溶液可能被水污染,可能需要更换。

      视频4

    2. 部分收集
      1. 由于这个协议的目的是更关注如何处理样本,因为这是最终图像的关键因素,所以我们不会详细讨论如何正确使用超薄切片机,准备切割块面。等等相反,请参阅Hagler(2007)。以下是正确执行这些步骤的一些意见。
      2. 对于PD的研究,用Leica Ultracut UC7(Leica Microsystems)收集90nm或180nm厚的切片。
      3. 生成序列部分可能是有用的,主要有两个原因:1)有一个网格,更多的感兴趣的对象; 2)串行层析成像可以在连续部分重建相同的结构的情况下执行,使得有可能恢复结构的3D体积大于切片厚度(Kang等人,2011年)。
      4. 要做到这一点,块面需要足够小,以便多个部分可以放在一个网格上。以梯形形状雕刻块体表面将允许各部分在产生时粘在一起,从而形成连续的部分的连续带,其可以容易地同时沉积在单个网格上。
      5. 虽然在使用非碳酸盐化磷酸铜铜网格时通常不会出现问题,但是如果在收集步骤中由于静电导致栅格被栅格排斥,栅格可以在截面收集之前被辉光放电。
      6. 根据EM电压(更多的电压等于更多的电子动能,因此更好的成像厚样本的能力)和感兴趣的结构的大小,部分的厚度可以从90nm到180nm变化。更接近90nm厚度的更细的部分将产生稍微更好的x,y分辨率,特别是在我们的情况中,在中等范围的EM电压下。 180纳米和更大,将允许更好的体积恢复,牺牲x,y分辨率时,在120千伏的成像。在PD研究的情况下,简单的直径范围从20到40nm厚,所以90nm厚的切片是合适的。但是,必须注意的是,PD可以具有不同的长度和大小,特别是当涉及分支的复杂PD时。因此它们可以在所有方向上跨越细胞壁延伸到几百nm。在这种情况下,增加切片厚度可以更好地恢复毛孔的体积。
    3. 具有基准标记(金颗粒)的涂层网格
      金颗粒被用于在断层图像重建之前所需的后续对准步骤。由于纯胶体金由于其固有的负电荷而倾向于聚集,因此通常使用经BSA稀释的金,使金的扩散在整个部分更均匀。
      1. 在一张石蜡膜上,铺上金溶液(见方法8)和水(图5)。

      2. 轻轻地将网格放在黄金滴上,持续20秒左右
      3. (可选)轻轻地上下吸取混合物,以利于电网与金的良好接触。
      4. 在两个水滴上依次放置网格几秒钟以去除多余的金溶液。
      5. 通过接近网格侧面的吸水纸(沃特曼纸5级)吸收剩余的液体。
      6. 在网格的另一侧重复步骤D3a至D3e,以便在两侧都有金颗粒。

      7. 在电磁场引入之前让电网干燥(否则会损坏离子泵系统)

        图5.在网格上放置基准标记。 在层析成像之前,网格上的基准标记沉积的典型设置。首先将网格放在红色滴剂(基准标记+ BSA溶液)上15-20秒,然后在水滴中连续拖动,并用吸水纸在格栅边缘小心地干燥。这个过程需要在网格的两边重复。

数据分析

  1. 利用这种高压冷冻 - 冷冻取代方案,细胞内纳米细节的形态学研究成为可能。结合使用Lowicryl HM-20树脂,更脆弱,更电光,并能够在低温下(与紫外线)聚合,这个协议允许有效的层析重建细节约10纳米的维度。低倍图像显示保存良好的细胞团(图6A)。在这些细胞之间的界面处,看到PD桥接细胞(图6B)。倾斜系列采集一个典型的I型孔隙(视频5)呈现出可以欣赏到整个孔隙体积的层析图(图6C-6E和视频6)。

    视频5

    视频6


    图6.在HPF和FS后获得的代表性数据A.用本文描述的程序处理并用于(Nicolas等人, 2017b)。 (A)中的红色方框区域中发现的I型等离子体模型(红色箭头)的倾斜系列的0°倾斜。截面厚度:180 nm。 (B)中显示的0.56nm厚的胞间连丝断层切片。左侧和右侧显示了ER可以看到的毛孔的两个末端(红色箭头)。 D.用IMOD实现的分割,具有相同的胞间连丝。 E.(C)中的等离子体断层的层析切片在一个平面上具有全长。用3DMOD的切片工具完成。

  2. 该协议还成功地提供了植物细胞器如脂滴和高尔基体的清晰快照(Wattelet-Boyer等人,2016; Brocard等人,2017)
  3. 生物复制和独立实验:
    PD计数和PD断层扫描至少在两个生物学重复上进行,如果可能的话三个重复。对于培养的细胞,一个生物复制品在特定的日期构成一个独立的高压冻结阶段。事实上,因为培养的细胞通常在同一个烧瓶中进行采样,所以我们没有考虑来自相同HPF的不同块体作为生物学重复。
    对于根,每个幼苗被认为是一个个体,因此被认为是生物复制。然而,来自根源的数据也是在多个HPF会议上收集的。
  4. PD筛选方法:
    在(Nicolas等人,2017b)中,计数I型与II型毛孔遵循体视学的基本原理(Lucocq,1993)。它们是:i)避免重复计算毛孔,ii)建立关于什么时候PD可以包含在数据中的精确规则。重要的是要强调,这种一般的方法可以用来计算其他功能PD(简单与分支例如)和其他细胞器。
    ⅰ)当依序连续的部分时,重复计算结构的内在风险会跨越多个部分。这种风险随着感兴趣的结构的大小而增加。因此,需要适合感兴趣对象大小的精确计数协议。
    首先,评估在多个连续部分上遇到给定结构的“机会”。结构被发现在'参考'部分(n)上,然后我们评估这些相同的结构是否仍然存在于'查找'部分(n + 1) )。如果结构不在那里,就说它是“解决了”(图6)。然后可以根据感兴趣的对象的大小和截面厚度来计算第n + 1节,第n + 2节等等的解析特征的分数。
    在最宽40nm的PD的情况下,它们大大劣于截面厚度。因此,在培养细胞的n + 1切片(Nicolas等人,2017b图3g)中,筛选的80%孔隙在n + 1节解决。因此,通过计数每2个切片(n,n + 2,n + 4等),我们可以合理避免PD的重复计算。
    ii)当两个质膜可见并且PD连接两个相邻的细胞中的至少一个(图7)时,将PD计数为“有效”并包括在数据中。


    图7.连续部分的PD计数 A.卡通描述了网格上的4个连续部分。感兴趣的结构被发现在参考部分n上,然后在查找部分n + 1,n + 2 etc 之后。当不能再看到结构的时候,据说就解决了。 (B-C)通常收集用于定量的I型PD(左)和II型(PD)的90nm厚切片的显微照片。毛孔的膜是可见的,并且它们与两侧连接。在后者中,孔内的异质密度允许细胞质套间隙(白色箭头)的可视化。尽管不像层析成像那样具有分辨力,但可以看到中心元素(desmotubule)横跨中心的孔隙。

笔记

  1. 液氮操纵
    由于液氮可以燃烧,因此在液氮容器的操作过程中,请始终佩戴适当的眼睛/脸部保护装置,手部保护装置和实验室涂层。
    由于这种气体会导致窒息,请务必确保房间通风良好。
  2. 低温取代试剂和树脂
    戊二醛和乙酸双氧铀溶液必须在-20°C储存在一个单独的容器中。
    应将四氧化锇分开存放,最好放在通风橱中,并始终使用特殊的实验室手套(SHIELDskin Orange nitrile 250)进行操作。当cryomix在通风橱外进行操作时,在进行cryomix的过程中,实验者(和其他在同一个房间里的人)应该有完整的保护,包括实验室手套,实验室墨盒面罩(SPERIAN半面罩和T48-ABEK1 P3双墨盒)和防护眼镜,以避免与四氧化锇烟雾接触。
    HM20和任何其他种类的树脂也应该用手套和墨盒面罩进行操作。为了方便起见,我们在从FS开始到树脂聚合的所有步骤中穿着完整的防护装备。
    总的来说,所有这些化合物,包括丙酮,都应该使用实验室手套随时操作。
    手套应该每20分钟更换一次,并在受到污染时立即更换
  3. 在低温取代期间改变浴的建议
    FS期间更换浴缸必须小心谨慎,耐心。用薄吸头1毫升吸取轻轻吸出以前的培养基(防止意外吸入样品)。在此步骤中,避免吸入和吸出。湍流可能会从运营商分离样品。请始终在底部留下剩余的介质,以防止样品空气干燥。然后用一个普通的塑料吸管(无论大小)轻轻地添加下一个媒介,让它滴在塑料管壁上。
  4. 样品在模具中的明确追踪
    保持准确的跟踪什么样品/条件是在哪个模具,以避免任何歧义。
    模具可以用多种形状/点的彩色指甲油标记,以区分它们。

食谱

  1. 培养基为细胞和幼苗
    1. 液体培养细胞的Murashige和Skoog培养基(1 L)
      Murashige和Skoog中等+维生素(Duchefa Biochemi):4.41g
      蔗糖:30克
      2-(N-吗啉代)乙磺酸(MES)(Euromedex):0.5g
      NAA:50微升
      激动素储备液:50微升
      用1N KOH溶液调节pH至5.8
      高压灭菌瓶110°C 30分钟
      注:细胞在恒定光照(20μE/ m / sec)下培养,并在22°C恒定搅拌。
    2. 苗(1升)Murashige和Skoog培养基
      Murashige和Skoog中+ vits(Duchefa Biochemi):4.4克
      MES(Euromedex):0.5克
      植物琼脂(Duchefa Biochemi):7克
      用1N KOH溶液调节pH至5.8
      高压灭菌瓶110°C 30分钟
      注:在垂直放置的培养皿中培育幼苗,以便在长日照时间(16 h,100μE/ m / sec)下,在22°C的温室中使根停留在培养基表面上。 / em>
  2. 在HPF(2毫升)期间用于冷冻保护的BSA溶液
    称量0.4克BSA,并将其放入15毫升Falcon管中 用2毫升的液体MS培养基培养细胞(见方法1)
    彻底漩涡,直到BSA完全溶解
    在-20°C储存
  3. 低温取代醋酸铀酰储备液(20%,500μl)
    称取0.1g醋酸铀酰粉末(需要手套和防尘面罩)并放入2ml螺旋管中 加入纯甲醇(通风橱下)至500μl
    将管子包裹在锡箔中,置于-20℃的专用低温置换箱中避光保存
  4. Cryosubstitution混合


  5. 不同浓度的HM20%溶液(4毫升)
    在FS Leica塑料溶剂容器中制造HM20溶液(图3,#10)。在使用前在-50°C下预先制备溶液并预先在AFS中预冷


  6. 2%Parlodion网格拍摄(20毫升)
    称重0.4克固体parlodion(电子显微镜科学)
    加入乙酸异戊酯(储存在通风的溶剂柜中,远离湿气)最多20毫升
    搅拌,直到固体parlodion溶解
    将烧瓶包裹在锡箔中,并用Parafilm
    密封盖子
  7. 甲苯胺蓝溶液
    制备母液:在20ml蒸馏水中加入1g甲苯胺蓝粉末(Sigma-Aldrich)和1g硼酸钠(Sigma-Aldrich)
    工作溶液在蒸馏水中稀释25倍
  8. Fiducial标记解决方案
    在MilliQ水中制成0.5%BSA溶液
    用0.2μm过滤器连接到注射器的滤液
    混合0.5%BSA胶体金溶液(1:1比例)
    分装在0.5 ml Eppendorf管中,-20°C保存

致谢

这项工作得到了阿基坦大区(E.M.B)和PEPS(对E.M.B的初步探索性项目支持)和国家研究机构(授予E.M.B的ANR-14-CE19-0006-01)的支持。所有的样品制备和成像都是在波尔多成像中心附上的PôleImagerie duVégétale上进行的( http:/ / /www.bic.u-bordeaux.fr/ )。阿基坦大区还支持收购电子显微镜(授权号:2011 13 04 007 PFM)。感谢ClémentChambaud协助制作说明性视频。作者声明没有利益冲突。

参考

  1. Brocard,L.,Immel,F.,Coulon,D.,Esnay,N.,Tuphile,K.,Pascal,S.,Claverol,S.,Fouillen,L.,Bessoule,JJ和Brehelin, )。 来自拟南芥的脂质微滴的蛋白质组学分析老化的叶子带来了新的洞察生物发生和功能。


    正面植物科学8:894.

  2. Ding,B.,Turgeon,R.和Parthasarathy,M.V。(1992)。 冻结取代的胞间连丝的子结构原生质 169:28-
  3. Donohoe,B. S.,Mogelsvang,S.和Staehelin,L. A.(2006)。 ER,高尔基体和相关膜系统的电子断层扫描。
    39(2):154-162。
  4. Dubochet,J。(2007)。 快速冷却的物理学及其对冷冻细胞的影响 方法Cell Biol 79:7-21。
  5. Hagler,H.K。(2007)。 用于生物电子显微镜的超薄切片。在:Kuo,J.(Ed。)。电子显微镜:方法和协议。 Humana Press pp:&nbsp; 67-96。
  6. Kang,B.H.,Nielsen,E.,Preuss,M.L.,Mastronarde,D。和Staehelin,L.A。(2011)。 拟南芥中的RabA4b-和PI-4Kβ1标记的反式高尔基体网格室的电子断层扫描< / em>。 流量 12(3):313-329。
  7. Kremer,J.R。,Mastronarde,D.N。和McIntosh,J.R。(1996)。 使用IMOD进行三维图像数据的计算机可视化 J Struct Biol 116(1):71-76。
  8. Lucocq,J。(1993)。 细胞生物学超微结构的无偏三维定量趋势细胞生物学 3(10):354-358。
  9. Nicolas,W. J.,Grison,M. S.和Bayer,E. M.(2017a)。 形成胞间连丝的细胞间通道:结构与功能的缺失环节 J Exp Bot 。
  10. Nicolas,W. J.,Grison,M. S.,Trepout,S.,Gaston,A.,Fouche,M.,Cordelieres,F. P.,Oparka,K.,Tilsner,J.,Brocard,L.and Bayer,E.M。(2017b)。 胞质分裂后胞间连丝的构建和通透性缺乏细胞质套管 Nat植物 3:17082。
  11. Oparka,K.J。和Roberts,A.G。(2001)。 Plasmodesmata。一个不那么开放的案例。植物生理学125(1):123-126。
  12. Studer,D.,Graber,W.,Al-Amoudi,A。和Eggli,P。(2001)。 高压冷冻冷冻固定的新方法 J Microsc < 203(Pt 3):285-294。
  13. Tilney,L.G.,Cooke,T.J.,Connelly,P.S。和Tilney,M.S。(1991)。 通过质壁分离,洗涤剂提取和蛋白酶消化显示的胞间连丝结构 < J细胞生物学 112(4):739-747。
  14. Wattelet-Boyer,V.,Brocard,L.,Jonsson,K.,Esnay,N.,Joubes,J.,Domergue,F.,Mongrand,S.,Raikhel,N.,Bhalerao,RP,Moreau,P.和Boutte,Y.(2016)。 羟基化的C24-和C26-酰基链鞘脂的介导在跨高尔基体网络中介导PIN2顶端分选子域名。 Nat Commun 7:12788。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nicolas, W. J., Bayer, E. and Brocard, L. (2018). Electron Tomography to Study the Three-dimensional Structure of Plasmodesmata in Plant Tissues–from High Pressure Freezing Preparation to Ultrathin Section Collection. Bio-protocol 8(1): e2681. DOI: 10.21769/BioProtoc.2681.
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