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Jan 2015

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Cryo-transmission Electron Microscopy of Outer-inner Membrane Vesicles Naturally Secreted by Gram-negative Pathogenic Bacteria
革兰阴性致病菌自然分泌的外-内膜囊泡低温透射电子显微镜观察   

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Abstract

A protocol was developed to visualize and analyze the structure of membrane vesicles (MVs) from Gram-negative bacteria. It is now accepted that these micrometric spherical vesicles are commonly produced by cells from all three domains of life, so the protocol could be useful in the study of vesicles produced by eukaryotes and archaea as well as bacteria. The multiplicity of functions performed by MVs, related to cell communication, interaction with the immune system, pathogenesis, and nutrient acquisition, among others, has made MVs a hot topic of research.

Due to their small size (25-300 nm), the observation of MVs requires electron microscopy and is usually performed by transmission electron microscopy (TEM) of negatively stained MVs. Other protocols applied for their visualization include scanning electron microscopy, TEM after fixation and embedding of vesicles, or even atomic force microscopy. In some of these techniques, vesicle structure is altered by drying, while others are time-consuming and most of them can generate artifacts. Cryo-TEM after plunge freezing allows the visualization of samples embedded in a thin film of vitreous ice, which preserves their native cellular structures and provides the highest available resolution for the imaging. This is achieved by very high cooling rates that turn the intrinsic water of cells into vitreous ice, avoiding crystal formation and phase segregation between water and solutes. In addition to other types of characterization, an accurate knowledge of MV structure, which can be obtained by this protocol, is essential for MV application in different fields.

Keywords: Cryo-transmission electron microscopy (低温透射电子显微镜), Plunge freezing (快速冷冻制样), Membrane vesicles (膜囊泡), Outer-inner membrane vesicles (外-内膜囊泡), Gram-negative bacteria (革兰氏阴性菌)

Background

In recent years, many studies have been conducted on membrane vesicles (MVs) produced by Gram-negative bacteria (Kulp and Kuehn, 2010). MVs are spherical membranous structures with diameters ranging between 20 and 300 nm, and they enable a protected secretion of proteins, lipids, RNA, DNA and other effector molecules (Beveridge, 1999; Mashburn-Warren and Whiteley, 2006; Ellis and Kuehn, 2010). The ability of bacterial MVs to interact with and enter host cells has prompted their exploration for novel clinical and biotechnological applications (Chen et al., 2010; Gujrati et al., 2014; Robbins and Morelli, 2014; van der Pol et al., 2015).

Different methods have been used to characterize and quantify bacterial MVs such as protein or lipopolysaccharide quantification, nanoparticle tracking analysis, flow-cytometry or proteomic analysis among others, but none of them allow MV visualization or clarify MV structure (Kulp and Kuehn, 2010). In many studies, MV-producing cells and vesicle morphology have been visualized using conventional electron microscopy techniques such as scanning electron microscopy or thin-section transmission electron microscopy (TEM) of samples chemically fixed and dehydrated at room temperature (Fiocca et al., 1999; McBroom and Kuehn, 2007; Deatherage et al., 2009; Alves et al., 2015; Ercoli et al., 2015). However, in both methods the extracellular matter in which MVs are found has a propensity to collapse and be removed during sample preparation (Beveridge, 1999; Fiocca et al., 1999; Nevot et al., 2006; Mashburn-Warren and Whiteley, 2006; McBroom and Kuehn, 2007; Deatherage et al., 2009; Ellis and Kuehn, 2010; Chen et al., 2010; Gujrati et al., 2014; Robbins and Morelli, 2014; Alves et al., 2015; Ercoli et al., 2015; van der Pol et al., 2015). TEM observation of negatively stained MVs is the most commonly used technique to assess MV presence and morphology. In this case, MVs are dried on a grid at room temperature and most of them are visualized as irregular and wrinkled round structures (Figure 1A). Although useful for confirming the presence of MVs, this technique does not provide enough resolution to visualize their exact shape or the presence of atypical MVs or artifacts, which may arise from the preparation process or when working with new or genetically manipulated strains (Bernadac et al., 1998; Kolling and Matthews, 1999; Wai et al., 2003; Lee et al., 2007; McBroom and Kuehn, 2007; Roier et al., 2016). The variability created by these features can hamper studies evaluating the application of MVs in different fields (Gujrati et al., 2014; van der Pol et al., 2015). Furthermore, in conventional electron microscopy techniques, samples have to be observed under a high vacuum and the electron beam leads to the destruction of native biological structure (Nevot et al., 2006).

In cryo-transmission electron microscopy (cryo-TEM), biological specimens are immobilized or fixed using physical procedures based on freezing. The goal is to obtain vitreous or amorphous ice from the sample water after ultra-rapid freezing, avoiding ice crystal formation and thus preserving the cellular structures. In contrast with conventional methods, ultra-rapid freezing immobilizes all molecules in a sample within milliseconds. After this instantaneous cryo-immobilization, it is very important to maintain this state throughout the imaging acquisition in the cryo-electron microscope (Cavalier et al., 2008).

In recent years, improvements in cryo-TEM techniques have enabled the imaging of biological specimens with greatly enhanced resolution and close to their native state, which has refined our knowledge of bacterial structures, including bacterial MVs (Renelli et al., 2004; Jensen and Briegel, 2007; Studer et al., 2008; Palsdottir et al., 2009; Frias et al., 2010; Perez-Cruz et al., 2013 and 2015).
There are several methods for freezing samples. Plunging samples into a pre-cooled cryogen, such as ethane or propane, is a common technique for freezing suspensions (viruses, liposomes, bicelles, micelles...), molecular assemblies (proteins, nucleic acids...), isolated organelles and cell structures and even small cells such as bacteria (Dobro et al., 2010). This method, known as plunge freezing, has been used to visualize bacterial MVs (Renelli et al., 2004), and has revealed, for example, the existence of a new type of Gram-negative MVs produced by environmental and pathogenic bacteria, named outer-inner membrane vesicles (O-IMVs) (Figures 1B, 2C and 2) (Perez-Cruz et al., 2013 and 2015).


Figure 1. Different samples of isolated MVs observed by (cryo-)TEM. A. MVs from Shewanella vesiculosa M7T prepared by negative staining and observed by TEM. Vesicles appear collapsed and as irregular and wrinkled round structures. B and C. MVs from Shewanella vesiculosa M7T and (C) Acinetobacter baumannii AB41 prepared by plunge freezing and observed by cryo-TEM. MVs appear as regular round structures and different types can be observed: conventional bacterial outer membrane vesicles with one membrane layer and a new type called outer-inner membrane vesicles (O-IMV) with two membrane layers (black arrows). D and E. Exosomes produced by melanoma cancer cells and (E) liposomes synthesized from lipids. All types of vesicles appear as regular round structures and membrane layers are clearly visualized. Scale bars = 500 μm.

Plunge freezing of MVs allows us to observe their exact shape, size and integrity, the presence of one or more membrane layers, and attached surface-associated structures like viruses, among other features (Perez-Cruz et al., 2016).

Bacterial MVs are not the only focus of investigation. Another expanding research area is the study of extracellular vesicles (EVs), which are released by cells from all domains of life-Eukarya, Archaea and Bacteria-and are considered intercellular communicasomes, acting as a mechanism for distance delivery of active compounds between cells (Yoon et al., 2014). EVs have demonstrated great promise as natural drug delivery systems loaded with therapeutic molecules (Armstrong and Stevens, 2018). Other candidates for the improvement of drug delivery systems are liposomes and other lipidic formulations (Alavi et al., 2017), varying in size and lamellar number, and cryo-TEM observation could also be applied to advance their development. The described protocol therefore has potential application for other types of vesicles currently under active research (Figures 1D and 1E).


Figure 2. Cryo-TEM of isolated MVs, a general overview of the whole protocol

Materials and Reagents

  1. Tweezers No. 5 (Dumont, catalog number: 21974-1)
  2. Safe tweezers (Wiha, catalog number: 44518)
  3. Vitrobot forceps (Ted Pella, catalog number: 47000-500)
  4. Micropipettes 20, 100 and 1000 (Eppendorf, model: Research)
  5. Membrane filters, white, individually packed. 0.45 µm, diameter 47 mm, sterile (ME 25 ST, Whatman®, catalog number: 10401670)
  6. Disposable filter device, 0.22 µm, sterile and non-pyrogenic polyethersulfone membrane with polypropylene housing (PURADISC 25 AS, Whatman®, catalog number: 6780-2502)
  7. 250 ml centrifuge polypropylene bottles (Beckman Coulter, catalog number: 356011)
  8. 50 ml centrifuge polypropylene tubes (Nalgene, catalog number 3139-0050)
  9. 500 ml and 1000 ml screw neck glass bottles (DURAN®)
  10. Vacuum filtration unit (DURAN® , catalog number: XT09.1)
  11. Chocolate agar plates (Beckton Dickinson, catalog number: PA-254035.05)
  12. Lacey Carbon 300 mesh grids (Ted Pella, catalog number: 01895-F)
  13. Quantifoil® R 2/2 Cu 200 mesh grids (Quantifoil Micro Tools GmbH, catalog number: Q23209)
  14. Holey Carbon 300 mesh grids (Agar, catalog number: AGS147-3)
  15. Filter paper for Vitrobot (Ted Pella, catalog number: 47000-100)
  16. Cryo Grid Box base only (Ted Pella, catalog number: 160-41)
  17. Cryo Grid Box Handling Rod (Ted Pella catalog number: 160-46)
  18. Neoprene gloves (Seton, catalog number: 10STA002)
  19. Parafilm (Sigma-Aldrich, catalog number: P7793-1EA)
  20. Petri dish (Fisher Scientific, catalog number: 11812532)
  21. Yellow pipette tips (Sigma-Aldrich, catalog number: EP4925000111)
  22. Blue pipette tips (Sigma-Aldrich, catalog number: EP3124000121)
  23. Neisseria gonorrhoeae (ATCC 43069)
  24. Pseudomonas aeruginosa PAO1 (Own collection)
  25. Acinetobacter baumannii AB41 (Clinical isolate) 
  26. Ringer ¼ (Sigma-Aldrich, catalog number: 96724-100TAB), prepare the solution as per the manufacturer’s instructions (Reference 1)
  27. Trypticase soy broth (Oxoid, catalog number: CM129), prepare the solution as per the manufacturer’s instructions (Reference 10)
  28. Müeller-Hinton broth (Oxoid, catalog number: CM0405), prepare the solution as per the manufacturer’s instructions (Reference 11)
  29. HEPES (Sigma-Aldrich, catalog number: 7365-45-9), prepare the solution as per the manufacturer’s instructions (Reference 18)
  30. Modified Lowry Protein Assay kit (Thermo Scientific, catalog number: 23240)
  31. Ethanol 96% (Sigma-Aldrich, catalog number: 16368)
  32. Ethane (AIR liquide)
  33. Liquid nitrogen (AIR liquide)

Equipment

  1. Fume hood (Flores Valles, model: LVG 190)
  2. Glow Discharge unit (BALTEC, model: CTA005)
  3. Minishaker (IKA®, model MS2)
  4. Vitrification Robot (FEI, model: VitrobotTM Mark III)
  5. Cryo-holder and cryo-transfer station (Gatan, model: 626)
  6. Cryo-electron microscope (FEI, model: Tecnai F20 200 kV) equipped with a CCD camera (FEI, model: Eagle 4kx4k)
  7. Orbital shaker (Innova® 44, Incubator Shaker Series, New Brunswick Scientific)
  8. Centrifuge (Beckman Coulter, model: Avanti J-20 XP)
  9. Centrifuge (Beckman Coulter, model: Allegra 25R)

Software

  1. Vitrobot V1.05B051 (FEI) 
  2. Tecnai version 4.3 (FEI)
  3. TEM Imaging & Analysis version 4.4 (FEI)

Procedure

  1. Isolation of MVs
    1. Grow the bacterial strains.
      1. Grow Neisseria gonorrhoeae ATCC 43069 to confluence by spreading 0.1 ml of a 109 cell/ml suspension on the surface of chocolate agar plates using a sterile glass spreader and rotating the plate. Incubate the plates for 65 h, at 37 °C in a 5% CO2 incubator. Seeding 10 to 20 Petri dishes should provide enough MVs.
      2. Grow Pseudomonas aeruginosa PAO1 in 2 L flasks with 500 ml of trypticase soy broth at 37 °C for 5 h, and Acinetobacter baumannii AB41 in 2 L flasks with 500 ml of Müeller-Hinton broth at 30 °C for 15 h. Incubate both liquid cultures in an orbital shaker at 100 rpm.
      Note: This protocol can be applied to MVs produced by any Gram-negative bacteria; therefore, the first step is to establish the growth conditions of the strain under study. The growth temperature, atmosphere, culture media composition, incubation time, and agitation under which MV production is going to be studied should be established. Usually, liquid growth medium is used for strain growth and MV retrieval, but a solid medium could also be employed. This protocol describes the growth conditions in which the new O-IMVs were detected in pathogenic strains.
    2. Isolate MVs from Pseudomonas aeruginosa PAO1 and Acinetobacter baumannii AB41 from liquid media culture.
      1. Recover broth cultures of A. baumannii AB41 and P. aeruginosa PAO1 in the late logarithmic phase (OD1.8).
      2. Place cultures in 250 ml sterile polypropylene centrifuge bottles.
      3. Pellet the cells by centrifugation at 10,000 x g for 15 min at 4 °C (Allegra 25R, Beckman Coulter).
      4. Discard cells and place the supernatants in a sterile screw neck glass bottle.
      5. Filter the supernatant through 0.45-μm-pore-size membrane filters with a vacuum filtration unit to remove remaining bacterial cells.
      6. Place the filtered supernatants in 50 ml sterile polyprolylene centrifuge tubes and centrifuge at 45,000 x g for 1 h at 4 °C (Avanti J-20 centrifuge, Beckman Coulter).
      7. Carefully discard the supernatant and save the pellet (normally a very small pellet is obtained).
      8. Resuspend the pellet in 50 ml of 50 mM HEPES pH 6.8 and filter it through a 0.22-μm-pore-size disposable filter device using a syringe and saving the filtrate in another 50 ml sterile polyprolylene centrifuge tube.
      9. Pellet the vesicles again at 45,000 x g for 1 h at 4 °C and resuspend the pellet in 500 µl of H2O.
      10. Measure the concentration of MVs obtained with the Lowry method. For a correct visualization of the MVs by cryo-TEM, a final concentration of 0.1-1 mg/ml of protein is suitable.
      11. The final volume used to resuspend the MV pellet can be adjusted depending on the yield of MVs obtained with each particular strain. 20-50 µl should be enough to prepare grids for cryo-TEM observation.
    3. Isolate and purify MVs from Neisseria gonorrhoeae ATCC 43069 from confluent solid cultures.
      1. Resuspend cells and MVs from each agar plate by adding 15 ml of Ringer ¼ per plate and using a cell scraper. Place all the volumes together in a screw neck glass bottle.
      2. Place resuspended cells in 250 ml sterile polypropylene centrifuge bottles.
      3. Follow the previously described steps from A2c to A2k.

  2. Vitrification
    1. Switch on the Vitrobot by the toggle switch at the back (Figure 3A and Video 1).

      Video 1. Cryo-TEM of bacterial outer membrane vesicles

    2. Fill the humidifier with distilled water.
      Fill the syringe with 60 ml distilled water and inject through the plastic tube located at the bottom (Figure 3A and Video 1). 
    3. Switch on the chamber light using the “console” page of the Vitrobot User Interface.
    4. Change the blotting pads.
      Manually pull out the front door of the Vitrobot chamber and remove the plastic rings from the blotting pads, attach new pieces of filter paper to the plastic rings and clip them to the blotting pads (Video 1). 
    5. Set the conditions using the Vitrobot User Interface.
      1. On the “console” page, set the conditions of the chamber to 25 °C and 100% humidity (Figure 3B and Video 1).
      2. Go to the “options” page and set the blotting and freezing conditions as follows: blot offset 0, blot total 1, blot time 2.5, wait time 120 s, plunge time 0 and drain time 0 (Figure 3C and Video 1).
      3. Also set other user specifics: tick “using footpedal” and “turning off the semi-automatic grid” (Figure 3C and Video 1). 
      Note: Blotting and freezing conditions need to be adjusted to each kind of sample. The conditions described here proved suitable for bacterial MVs of different strains.
    6. Prepare the tools.
      Apply some ethanol to the tip of the forceps as well as nitrogen air at a pressure of 7 bars. Also apply nitrogen air to the metallic and plastic parts that will be in contact with liquid nitrogen to minimize dust contamination. The metallic parts consist of the container for liquid ethane, the support for the grid boxes and the spindle; the plastic parts consist of the grid box and the grid box container (in our case, modified Falcon tubes) (Figure 3D and Video 1).


      Figure 3. Vitrification. A. Vitrification Robot Vitrobot Mark III FEI. The black arrows point the entry pots to the climate chamber, the white arrow points the humidifier and the white arrowhead points the sample injection window. B. Vitrobot settings in “console page”. C. Vitrobot settings in “options page”. D. Vitrobot accessories: 1, modified Falcon tubes; 2, anticontamination ring; 3, coolant container; 4, Cryo-grid box handling rod; 5, cryo-grid box base; 6, grid-box support; 7, spindle; 8, ethane container; 9, Vitrobot tweezers. E. Vitrobot tweezers secured on the connection groove at the central axis. The black arrow points the grid and the white arrow points the connection groove. F. Vitrobot tweezers inside the climate chamber. The black arrows point the blotting pads. G. Cryo-immobilized grid inside the liquid ethane. The white arrow points the ethane container and the black arrow points the cryo-grid box base.

    7. Cool the coolant container.
      Mount the ethane container, the grid box support, the grid box, the spindle and the anti-contamination ring in the coolant container and fill the outer ring of the container with liquid nitrogen until all the components are cooled. All the components have been cooled when the liquid nitrogen stops boiling (Video 1).
    8. Condense the ethane.
      Position the exit tip of the ethane cylinder inside the ethane container, open the main valve of the gas cylinder and slowly liquefy the ethane. When the bubbles on the surface of the liquid ethane reach the bottom brim of the spindle, close the main valve of the ethane cylinder and remove the spindle (Video 1).
    9. Prepare the grids.
      Place the grids for the experiment on a piece of Parafilm fixed inside a Petri dish plate and bring the grids to the Glow Discharge unit (Figure 4A).


      Figure 4. Glow Discharge. A. Glow Discharge unit BALTEC CTA 005. The black arrow indicates the position of the grid inside the chamber. B. Glow Discharge settings.

    10. Glow-discharge the grids. 
      1. Remove the lid of the Petri dish plate and ensure the carbon faces of all the grids are facing upwards.
      2. Manually pull up the chamber door of the Glow Discharge device, place the opened Petri dish plate containing the grids inside and pull down the door (Video 1).
      3. Set the timer at 30 s and the current to a value between 2 and 6 mA (Figure 4B).
      4. Press the toggle switch at the back and wait for the vacuum chamber to reach about 10-1 mbar (the red LED indicating the pressure on the panel will change to yellow) and press “start” to switch on the UV light (Video 1).
      5. Wait for the set time and the UV light will switch off automatically (Video 1).
    11. Remove the grids from the Glow Discharge unit.
      1. Switch the toggle off and wait for the chamber to be vented (the sound will stop) (Video 1).
      2. Manually pull up the door of the chamber, take out the Petri dish plate containing the grids, close the door of the chamber and transfer the grids to the Vitrobot (Video 1).
        After the glow discharge, liquid samples will spread over the grid surface more easily. Use the grids within one hour after charging, otherwise they will need to be charged again.
    12. Press the foot pedal or select “place new grid” in the Vitrobot interface.
    13. Mount one grid on the Vitrobot tweezers (Video 1).
      Place the slider of the Vitrobot forceps in the upper position, carefully attach one of the grids by its edge and lock the slider on the nearest groove of the forceps. 
    14. Secure the tweezers on the connection groove at the central axis.
      The sample must be deposited on the carbon side of the grid. When securing the Vitrobot tweezers, the carbon side of the grid should be facing left or right, depending on which side entry port the operator is using to deposit the sample (Figure 3E and Video 1). 
    15. Move the tweezers into the climate chamber. Press the foot pedal or select “start process” (Figure 3F and Video 1). 
    16. Place the coolant container on the platform ring (Video 1).
    17. Raise the coolant container towards the climate chamber.
      Press the foot pedal or select “continue” (Video 1).
    18. Lower the Vitrobot forceps to the sample application position.
      Press the foot pedal or select “continue” (Video 1).
    19. Apply the sample to the grid.
      Open the side port of the climate chamber and apply 3 μl with a micropipette (Video 1).
    20. Activate the blotting and the plunge freezing.
      Press the foot pedal or select “continue” and wait for the coolant container and forceps to be lowered (Video 1).
    21. Carefully remove the forceps from the groove of the central axis of the Vitrobot.
      Transfer the coolant container and the forceps to the bench (Video 1). Be careful to keep the grid below the liquid ethane surface during this step. 
    22. Wait until the ethane cloud has dispersed and slowly lift the grid through the ethane surface.
      Once the grid is completely out, quickly insert it in the outer ring containing liquid nitrogen and transfer the grid to a position of the grid box (Figure 3G and Video 1).
    23. Repeat Steps B16-B26 until all the samples have been cryo-immobilized.
      When necessary, thaw the liquid ethane by covering the ethane container with another empty container for a few seconds. 
    24. Store the grids.
      1. When all frozen grids are inside the grid box, cool the handling rod and screw it to the center of the grid box (Video 1).
      2. Transfer the grid box to a precooled grid box container under liquid nitrogen and close it (Video 1).
      Note: If the samples are not going to be observed on the same day, store the grid box container in a Dewar with liquid nitrogen. 
    25. Switch off the Vitrobot.
      Place the coolant container in the fume hood for the evaporation of the liquid nitrogen and liquid ethane and select “exit” in the Vitrobot User Interface. When the lights are turned off, the central axis is in the parking position and the interface has shut down, turn off the toggle switch at the back of the device.

  3. Cryo-TEM observation
    1. Prepare the cryo-microscope.
      Transfer the grids in the Dewar with liquid nitrogen to the cryo-microscope room and cool the anti-contaminator of the microscope for at least 1 h (Video 1).
    2. Ensure the valves are closed.
      Go to the “Set up” tab. If the “Col. valves closed” button is not yellow, click on it (Video 1). 
    3. Press “Turbo on” (Video 1).
    4. Prepare all the accessories (Figure 5A) and cover the desk of the microscope with cold insulating materials (cork plates, bubble wrap…).


      Figure 5. Mounting the grid on the Cryo-holder. A. Accessories: 1, lid; 2, funnel; 3, cryo-transfer station; 4, platform; 5, clip-ring tool. B. Upper view of the Cryo-holder GATAN 656 in the cryo-transfer station. The black arrow points the cryo-transfer station and the white arrow points the cryo-holder. C. Detail of the cryo-transfer station. 1, cryo-holder tip; 2, cryo grid box base. The black arrow points the position of the grid at the tip of the cryo-holder.

    5. Cool down the cryo-holder.
      Place the cryo-holder into the transfer station and cool it down by pouring liquid nitrogen into the Dewar of the cryo-holder and into the cryo-holder station. Connect the cable of the control station to the cryo-holder and wait till the temperature is below -165 °C (Video 1).
    6. Prepare the cryo-holder tip.
      1. Transfer the grid box to its place on the platform of the transfer station and carefully unscrew and remove the rod (Figures 5B and 5C and Video 1).
      2. Open the cryo-shield by sliding back the circular piece at the end of the central axis of the cryo-holder (just behind the Dewar), remove the clip ring with its tool and keep it under liquid nitrogen (Video 1).
        Note: The clip-ring tool has two tabs at the tip that can be placed oppositely or contiguously by turning the two wheels of the handle. To remove the clip-ring from the cryo-holder tip, put the two tabs together, place them in the groove around the clip-ring, move the wheels to position the tabs oppositely and pull up.
    7. Mount the grid in the cryo-holder tip.
      Cool the tip of the forceps, transfer one of the grids to the slot in the cryo-holder tip, secure it with the clip-ring tool and close the cryo-shield over the sample to prevent ice contamination (Figure 5C and Video 1). To secure the grid with the clip ring, place the clip ring over the grid, press down the clip-ring tool, turn the wheels of the handle until the tabs are contiguous and remove the tool. 
    8. Disconnect the cable of the control station from the cryo-holder (Video 1).
    9. Prepare a polystyrene box under the compustage (Video 1).
    10. Press “Pre-pump air lock” (Video 1).
    11. Quickly remove the cryo-holder from the cryo-transfer station, and carefully introduce the tip of the cryo-holder through the hole of the microscope as far as it will go (Video 1).
    12. Rotate the cryo-holder to the right until its pivot matches the compustage groove. Press the cryo-holder lightly against the microscope and wait until the end of the count-down in the “vacuum overview” (Video 1). Be careful when rotating the cryo-holder to the right to avoid the liquid nitrogen falling onto the polystyrene box prepared in Step C9.
    13. Select “Cryo-holder” as the specimen holder.
    14. Once the count-down in the “vacuum overview” has ended, turn the cryo-holder to the left as far as it will go, and introduce it completely into the microscope (Figure 6A and Video 1).
    15. Quickly refill the Dewar of the cryo-holder with liquid nitrogen (Video 1).
    16. Connect the cable of the control station to the cryo-holder and check the temperature does not exceed -170 °C (Video 1).
    17. Click “Col. valves closed” and go to the “LowDose” tab (Video 1).
    18. Record images in low dose conditions and spot size 5.
      Search mode at x7800 and 0.10 e-2s, focus mode at x29000 and 0.80 e-2s and exposure mode at x29000 and 1.00 e-2s (Figure 6B and Video 1).


      Figure 6. Cryo-TEM observation. A. Cryo-electron microscope Tecnai F20 FEI. The black arrow indicates the cryo-holder position. B. Low Dose observation.

Data analysis

Reliable measurements of the vesicles were obtained from cryo-TEM images for statistical purposes. More than 7,000 vesicles were measured per strain in two independent experiments and the measurements were performed with ImageJ 1.47 software. MVs can measure between 20 and 250 nm in diameter (Perez-Cruz et al., 2015).

Notes

  1. A lab coat and gloves should be worn while manipulating the material (filter paper, grids, samples…) to avoid self-contamination and during the experiment to avoid skin damage from proximity to the liquefied gases.

Acknowledgments

We gratefully acknowledge the assistance of MJ Montes for the proper maintenance of the strains and the assistance of M Gimeno for her help in recording the videos.

Competing interests

The authors have declared that no competing interests exist.

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  15. Fiocca, R., Necchi, V., Sommi, P., Ricci, V., Telford, J., Cover, T. L. and Solcia, E. (1999). Release of Helicobacter pylori vacuolating cytotoxin by both a specific secretion pathway and budding of outer membrane vesicles. Uptake of released toxin and vesicles by gastric epithelium. J Pathol 188(2): 220-226.
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  17. Gujrati, V., Kim, S., Kim, S. H., Min, J. J., Choy, H. E., Kim, S. C. and Jon, S. (2014). Bioengineered bacterial outer membrane vesicles as cell-specific drug-delivery vehicles for cancer therapy. ACS Nano 8(2): 1525-1537.
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  20. Kolling, G. L. and Matthews, K. R. (1999). Export of virulence genes and Shiga toxin by membrane vesicles of Escherichia coli O157:H7. Appl Environ Microbiol 65(5): 1843-1848.
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  22. Lee, E. Y., Bang, J. Y., Park, G. W., Choi, D. S., Kang, J. S., Kim, H. J., Park, K. S., Lee, J. O., Kim, Y. K., Kwon, K. H., Kim, K. P. and Gho, Y. S. (2007). Global proteomic profiling of native outer membrane vesicles derived from Escherichia coli. Proteomics 7(17): 3143-3153.
  23. Mashburn-Warren, L. M. and Whiteley, M. (2006). Special delivery: vesicle trafficking in prokaryotes. Mol Microbiol 61(4): 839-846.
  24. McBroom, A. J. and Kuehn, M. J. (2007). Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response. Mol Microbiol 63(2): 545-558.
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  26. Palsdottir, H., Remis, J. P., Schaudinn, C., O'Toole, E., Lux, R., Shi, W., McDonald, K. L., Costerton, J. W. and Auer, M. (2009). Three-dimensional macromolecular organization of cryofixed Myxococcus xanthus biofilms as revealed by electron microscopic tomography. J Bacteriol 191(7): 2077-2082.
  27. Perez-Cruz, C., Canas, M. A., Gimenez, R., Badia, J., Mercade, E., Baldoma, L. and Aguilera, L. (2016). Membrane vesicles released by a hypervesiculating Escherichia coli Nissle 1917 tolR mutant are highly heterogeneous and show reduced capacity for epithelial cell interaction and entry. PLoS One 11(12): e0169186.
  28. Perez-Cruz, C., Carrion, O., Delgado, L., Martinez, G., Lopez-Iglesias, C. and Mercade, E. (2013). New type of outer membrane vesicle produced by the Gram-negative bacterium Shewanella vesiculosa M7T: implications for DNA content. Appl Environ Microbiol 79(6): 1874-1881.
  29. Perez-Cruz, C., Delgado, L., Lopez-Iglesias, C. and Mercade, E. (2015). Outer-inner membrane vesicles naturally secreted by gram-negative pathogenic bacteria. PLoS One 10(1): e0116896.
  30. Renelli, M., Matias, V., Lo, R. Y. and Beveridge, T. J. (2004). DNA-containing membrane vesicles of Pseudomonas aeruginosa PAO1 and their genetic transformation potential. Microbiology 150(Pt 7): 2161-2169.
  31. Robbins, P. D. and Morelli, A. E. (2014). Regulation of immune responses by extracellular vesicles. Nat Rev Immunol 14(3): 195-208.
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  35. Wai, S. N., Lindmark, B., Soderblom, T., Takade, A., Westermark, M., Oscarsson, J., Jass, J., Richter-Dahlfors, A., Mizunoe, Y. and Uhlin, B. E. (2003). Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin. Cell 115(1): 25-35.
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简介

开发了一种从Gram阴性细菌中观察和分析膜泡(MVS)结构的方案。目前人们普遍认为,这些微小的球形囊泡通常是由来自生命三个领域的细胞产生的,因此该方案可用于研究真核生物、古细菌以及细菌产生的囊泡。mvs具有多种功能,涉及细胞通讯、与免疫系统的相互作用、发病机制、营养物质获取等,已成为研究的热点。



由于微血管的尺寸很小(25-300nm),微血管的观察需要电子显微镜,通常用透射电子显微镜(tem)对微血管进行负染。其他应用于其可视化的方法包括扫描电子显微镜、囊泡固定和嵌入后的tem,甚至原子力显微镜。在一些这些技术中,囊泡结构通过干燥而改变,而其他的是耗时的,并且它们中的大多数可以产生伪影。冷冻后的低温tem可以显示嵌入在玻璃质冰薄膜中的样品,这保留了它们固有的细胞结构,并为成像提供了最高的可用分辨率。这是通过非常高的冷却速度来实现的,冷却速度可以将细胞内的水变成玻璃冰,避免了水和溶质之间的晶体形成和相分离。除了其他类型的表征,MV结构的精确知识,可以通过该协议获得,对于MV在不同领域中的应用是必不可少的。
【背景】近年来,人们对革兰氏阴性菌产生的膜泡(mvs)进行了大量的研究(kulp和kuehn,2010)。MVS是直径在20到300纳米之间的球形膜结构,它们能够保护蛋白质、脂质、RNA、DNA和其他效应分子的分泌(Beveridge,1999;Mashburn Warren和Whiteley,2006;Ellis和Kuehn,2010)。细菌MVS与宿主细胞相互作用和进入宿主细胞的能力促使他们探索新的临床和生物技术应用(Chen等人,2010年;Gujrati等人,2014年;Robbins和Morelli,2014年;van der Pol等人,2015年)。



已经使用不同的方法来表征和量化细菌微血管,如蛋白质或脂多糖定量、纳米颗粒跟踪分析、流式细胞术或蛋白质组分析等,但这些方法都不允许微血管可视化或阐明微血管结构(kulp和kuehn,2010)。在许多研究中,利用常规的电子显微镜技术,如扫描电子显微镜或在室温下化学固定和脱水(fioccaet)的样品的薄片透射电子显微镜(tem),可以观察到产生mv的细胞和囊泡的形态。1999年;McBroom和Kuehn,2007年;Deatherage等人,2009年;Alves等人,2015年;Ercoli等人,2015年)。然而,在这两种方法中,发现mvs的细胞外物质有崩塌的倾向,并且在样品制备过程中被去除(beveridge,1999;fiocca等。,1999;Nevot等人,2006;Mashburn Warren和Whiteley,2006;McBroom和Kuehn,2007;Deatherage等人,2009;Ellis和Kuehn,2010;Chen等人,2010;Gujrati等人,2014;Robbins和Morelli,2014;Alves等人,2015;Ercoli等人,2015年;van der Pol等人,2015年)。透射电镜观察负染mvs是评价mv存在和形态最常用的技术。在这种情况下,MVS在室温下在网格上干燥,大多数MVS被视为不规则和褶皱的圆形结构(图1A)。虽然这项技术有助于确认mvs的存在,但它并不能提供足够的分辨率来显示mvs的确切形状或非典型mvs或伪影的存在,这可能是由于制备过程或使用新的或基因操纵的菌株(Bernadac等人,1998年;Kolling和Matthews,1999年;Wai等人,2003年;Lee等人,2007年;McBroom和Kuehn,2007年;Roier等人,2016年)。这些特征所产生的变异性阻碍了评价MVS在不同领域中应用的研究(Gujrati 等.,2014;van der Pol 等。,2015)。此外,在传统的电子显微镜技术中,样品必须在高真空下观察,电子束会导致天然生物结构的破坏(nevot等人,2006)。



在低温透射电子显微镜(cryo-tem)中,生物样品在冷冻的基础上用物理方法固定或固定。其目的是在超快速冷冻后从样品水中获得玻璃状或无定形的冰,避免冰晶的形成,从而保持细胞结构。与传统方法不同的是,超快速冷冻能在几毫秒内将所有分子固定在样品中。在瞬间冷冻固定后,在冷冻电子显微镜的成像采集过程中保持这种状态非常重要(Cavalier等人,2008)。



近年来,低温透射电镜技术的改进使生物样品的成像分辨率大大提高,接近其原始状态,这提高了我们对细菌结构,包括细菌微血管的认识(Renelli等人,2004;Jensen和Briegel,2007年;Studer等人,2008年;Palsdottir等人,2009年;Frias等人,2010年;Perez Cruz等人,2013年和2015年)。

有几种冷冻样品的方法。将样品放入预先冷却的冷冻剂中,如乙烷或丙烷,是冷冻悬浮液(病毒、脂质体、双细胞、胶束……)、分子组装物(蛋白质、核酸……)、分离的细胞器和细胞结构,甚至小细胞如细菌(Dobro等人,2010年)。这种方法被称为浸入式冷冻法,已用于细菌mvs的可视化(Renelli等人,2004年),并揭示了环境和病原菌产生的一种新型革兰氏阴性mvs的存在,命名为外膜囊泡(O-IMV)(图1b、2c和2)(Perez Cruz等人,2013年和2015年)。

图1。用(低温)透射电镜(t em)观察不同分离株的mvs,用阴性染色法制备和透射电镜观察不同分离株的mvs。囊泡呈不规则皱褶的圆形结构。水下冷冻法制备鲍曼不动杆菌ab41的b和c.mvs。MVS呈规则圆形结构,可观察到不同类型:细菌膜外膜囊膜,一层膜外层囊泡(O-IMV),有两层膜(黑箭头)。黑色素瘤细胞产生的d和e。外体和脂类合成的(e)脂质体。所有类型的囊泡呈规则的圆形结构,膜层清晰可见。比例尺=500μm。



MVS的骤降冷冻使我们能够观察其确切的形状、大小和完整性、一层或多层膜的存在、附着的表面相关结构(如病毒)等特征(Perez Cruz等人,2016年)。



细菌性mvs并不是研究的唯一焦点。另一个不断扩展的研究领域是细胞外囊泡(evs)的研究,它是由真核生物、古生菌和细菌各个领域的细胞释放的,被认为是细胞间通讯体,作为细胞间活性化合物距离传递的机制(Yoon等,2014年)。evs作为一种装载治疗分子的天然药物递送系统,已经显示出巨大的前景(armstrong和stevens,2018)。脂质体和其他脂质体制剂(Alavi等人,2017年)是改进药物传递系统的其他候选药物,其大小和层数不同,低温透射电镜观察也可用于促进其发展。因此,所描述的方案对于目前正在积极研究的其他类型的囊泡具有潜在的应用(图1d和1e)。





图2。分离mvs的低温透射电镜,整个协议的概述

关键字:低温透射电子显微镜, 快速冷冻制样, 膜囊泡, 外-内膜囊泡, 革兰氏阴性菌

材料和试剂

  1. 5号镊子(杜蒙特,目录号:21974-1)
  2. 安全镊子(WIHA,目录号:44518)
  3. Vitrobot镊子(Ted Pella,目录号:47000-500)
  4. 微量移液管20、100和1000(Eppendorf,型号:研究)
  5. 薄膜过滤器,白色,单独包装。0.45μm,直径47 mm,无菌(Me 25 St,Whatman?,目录号:10401670)
  6. 一次性过滤装置,0.22μm,带聚丙烯外壳的无菌和非热原性聚醚砜膜(Puradisc 25 AS,Whatman?,目录号:6780-2502)
  7. 250毫升离心聚丙烯瓶(贝克曼库尔特,目录号:356011)
  8. 50毫升离心聚丙烯管(Nalgene,目录号3139-0050)
  9. 500毫升和1000毫升螺旋颈玻璃瓶(Duran?)
  10. 真空过滤装置(Duran?,目录号:XT09.1)
  11. 巧克力琼脂板(Beckton Dickinson,目录号:PA-254035.05)
  12. 蕾丝碳300网格(Ted Pella,目录号:01895-F)
  13. Quantifoil®R 2/2 CU 200网格(Quantifoil Micro Tools GmbH,目录号:Q23209)
  14. 多孔碳300目网格(琼脂,目录号:AGS147-3)
  15. Vitrobot滤纸(Ted Pella,目录号:47000-100)
  16. 仅限Cryo网格盒底座(Ted Pella,目录号:160-41)
  17. Cryo格栅箱操纵杆(Ted Pella目录号:160-46)
  18. 氯丁橡胶手套(SETON,目录号:10STA002)
  19. 副膜(Sigma-Aldrich,目录号:P7793-1EA)
  20. 培养皿(Fisher Scientific,目录号:11812532)
  21. 黄色吸管头(Sigma-Aldrich,目录号:EP4925000111)
  22. 蓝移液管尖端(Sigma-Aldrich,目录号:EP312400011)
  23. 淋病奈瑟菌(ATCC 43069)
  24. 铜绿假单胞菌pao1(自产)
  25. 鲍曼不动杆菌AB41(临床分离株)
  26. 振铃器1/4(Sigma-Aldrich,目录号:96724-100Tab),按照制造商的说明制备溶液(参考1)
  27. 胰蛋白酶大豆肉汤(oxoid,目录号:cm129),按照制造商的说明制备溶液(参考文献10)
  28. Mueller Hinton肉汤(Oxoid,目录号:CM0405),按照制造商的说明制备溶液(参考文献11)
  29. HEPES(Sigma-Aldrich,目录号:7365-45-9),按照制造商的说明制备溶液(参考文献18)
  30. 改良Lowry蛋白质测定试剂盒(Thermo Scientific,目录号:23240)
  31. 乙醇96%(Sigma-Aldrich,目录号:16368)
  32. 乙烷(液化空气)
  33. 液氮(液化空气)

设备

  1. 通风柜(Flores Valles,型号:190级)
  2. 辉光放电装置(BALTEC,型号:CTA105)
  3. 迷你客栈(IKA?,型号MS2)
  4. 玻璃化机器人(FEI,型号:VitrobotTMMark III)
  5. 冷冻柜和冷冻转运站(GATAN,型号:626)
  6. 低温电子显微镜(FEI,型号:Tecnai F20 200千伏)配有CCD摄像机(FEI,型号:Eagle 4Kx4K)
  7. 轨道振动筛(Innova?44,孵化器振动筛系列,新不伦瑞克科学)
  8. 离心机(贝克曼库尔特,型号:Avanti J-20 XP)
  9. 离心机(贝克曼库尔特,型号:Allegra 25R)

软件

  1. Vitrobot v1.05b051(FEI)
  2. Tecnai版本4.3(FEI)
  3. 透射电镜成像与分析版本4.4(FEI)

程序

  1. MVS隔离
    1. 培养细菌株。
      1. 将0.1毫升的109细胞/ml悬浮液用无菌玻璃撒布器涂在巧克力琼脂平板表面并旋转平板,将淋病奈瑟菌ATCC 43069培养至融合。将培养板在37℃的5%co2培养箱中培养65小时。接种10到20个培养皿应提供足够的MVS。
      2. 将铜绿假单胞菌pao1培养在2 L烧瓶中,在37°C下用500毫升胰蛋白酶大豆肉汤培养5小时,将鲍曼不动杆菌ab41培养在2 L烧瓶中,在30°C下用500毫升米勒-辛顿肉汤培养15小时。将两种液体培养物以100转/分的速度在眼眶摇瓶中培养。
      该方案可应用于任何革兰氏阴性菌产生的MVS,因此,第一步是建立所研究菌株的生长条件。应确定研究MV产生的生长温度、大气、培养基组成、培养时间和搅拌。通常,液体培养基用于应变生长和mv恢复,但也可以使用固体培养基。该方案描述了在致病菌株中检测到新o-imv的生长条件。
    2. 从液体培养基中分离铜绿假单胞菌pao1和鲍曼不动杆菌ab41的mvs。
      1. 在对数晚期(od1.8)恢复鲍曼a.baumaniab41和p.aeruginosa的肉汤培养。
      2. 将培养物放入250毫升无菌聚丙烯离心瓶中。
      3. 在10000x g下,在4°C下离心15分钟,使细胞颗粒化(Allegra 25R,Beckman Coulter)。
      4. 丢弃细胞并将上清液放入无菌螺旋颈玻璃瓶中。
      5. 使用真空过滤装置,通过0.45μm孔径的膜过滤器过滤上清液,去除剩余的细菌细胞。
      6. 将过滤后的上清液放入50毫升无菌聚丙烯离心管中,并在45000x g下在4°C下离心1小时(Avanti J-20离心机,Beckman Coulter)。
      7. 小心地丢弃上清液并保存颗粒(通常会得到非常小的颗粒)。< >
      8. 在50毫升50毫米的Hepes pH6.8中重新过滤颗粒,并使用注射器通过0.22μm孔径的一次性过滤装置进行过滤,并将过滤液保存在另一个50毫升的无菌聚丙烯离心管中。
      9. 在45000x g下在4°C下再次使囊泡颗粒化1h,并在500微升的h2o中重新使颗粒化。
      10. 用lowry法测定mvs浓度。为了通过低温透射电镜正确显示mvs,最终浓度为0.1-1 mg/ml的蛋白质是合适的。
      11. 根据每个特定菌株获得的mvs的产量,可调整用于再悬浮mv颗粒的最终体积。20-50微升应足以为低温透射电镜观测准备网格。
    3. 从融合固体培养物中分离纯化淋病奈瑟菌的mvs。
      1. 通过在每个琼脂板中加入15毫升Ringer 1/4并使用细胞刮刀,从每个琼脂板中重新培养细胞和MVS。把所有的卷放在一个螺旋颈玻璃瓶里。
      2. 将再悬浮细胞放入250毫升无菌聚丙烯离心瓶中。
      3. 按照前面描述的步骤从A2C到A2K。
        < >
  2. 玻璃化
    1. 通过后面的拨动开关打开Vitrobot(图3A和视频1)。
      < >
      视频1。细菌外膜囊泡的低温透射电镜 < >
    2. 将加湿器装满蒸馏水。
      用60毫升蒸馏水注满注射器,并通过底部的塑料管注射(图3A和视频1)。
    3. 使用Vitrobot用户界面的“控制台”页面打开舱灯。
    4. 更换吸墨纸。
      手动拉出Vitrobot腔室的前门,从吸墨纸垫上取下塑料环,将新的滤纸贴在塑料环上,并将其夹在吸墨纸垫上(视频1)。
    5. 使用Vitrobot用户界面设置条件。
      1. 在“控制台”页面上,将试验箱的条件设置为25°C和100%湿度(图3b和视频1)。
      2. 进入“选项”页面,设置吸墨纸和冷冻条件如下:吸墨纸偏移量0,吸墨纸总数1,吸墨纸时间2.5,等待时间120秒,插入时间0和排水时间0(图3c和视频1)。
      3. 还可以设置其他用户详细信息:勾选“使用踏板”和“关闭半自动栅格”(图3c和视频1)。
      注:每种样品均需调整吸液和冷冻条件。这里描述的条件证明适合于不同菌株的细菌mvs。
    6. 准备工具。
      将一些乙醇和氮气以7巴的压力涂抹在镊子的尖端。同时在与液氮接触的金属和塑料零件上施加氮气,以尽量减少粉尘污染。金属部件包括液态乙烷容器、格架盒支架和主轴;塑料部件包括格架盒和格架盒容器(在我们的例子中,是改进的猎鹰管)(图3d和视频1)。
      < >
      图3。玻璃化。a.玻璃化机器人Vitrobot Mark III Fei。黑色箭头指向气候室,白色箭头指向加湿器,白色箭头指向样品注入窗口。b.“控制台页面”中的Vitrobot设置。c.“选项页”中的Vitrobot设置。d.Vitrobot配件:1、改良Falcon管;2、防沾污环;3、冷却剂容器;4、Cryo格栅箱操纵杆;5、Cryo格栅箱底座;6、格栅箱支架;7、主轴;8、乙烷容器;9、Vitrobot镊子。e.固定在中心轴连接槽上的Vitrobot镊子。黑色箭头指向网格,白色箭头指向连接槽。f.气候室内的vitrobot镊子。黑色箭头指向吸墨纸。g.液体乙烷中的冷冻固定网格。白色箭头指向乙烷容器,黑色箭头指向低温栅格盒底座。
      < >
    7. 冷却冷却液容器。
      将乙烷容器、格架箱支架、格架箱、主轴和防污染环安装在冷却液容器中,并用液氮填充容器的外圈,直到所有部件冷却。当液氮停止沸腾时,所有部件都已冷却(视频1)。
    8. 冷凝乙烷。
      将乙烷气瓶的出口端放在乙烷容器内,打开气瓶主阀,缓慢液化乙烷。当液体乙烷表面的气泡到达主轴底部边缘时,关闭乙烷气缸主阀并拆下主轴(视频1)。
    9. 准备网格。
      将用于实验的网格放置在固定在培养皿板内的一块副膜上,并将网格带到辉光放电单元(图4a)。
      < >
      图4。辉光放电。a.辉光放电装置BALTEC CTA 005。黑色箭头指示腔室中栅格的位置。b.辉光放电设置。
      < >
    10. 辉光放电栅极。
      1. 取下培养皿盖,确保所有网格的碳面朝上。
      2. 手动拉起辉光放电装置的腔室门,将打开的包含网格的培养皿板放在里面,然后拉下门(视频1)。
      3. 将定时器设置为30秒,电流值在2至6毫安之间(图4b)。
      4. 按下后面的拨动开关,等待真空室达到大约10-1毫巴(指示面板上压力的红色LED将变为黄色),然后按“开始”打开紫外线灯(视频1)。< >
      5. 等待设定时间,紫外线灯将自动关闭(视频1)。
    11. 从辉光放电单元上拆下格栅。
      1. 关闭开关,等待腔室通风(声音将停止)(视频1)。
      2. 手动拉起试验室的门,取出包含网格的培养皿板,关闭试验室的门,并将网格转移到Vitrobot(视频1)。< > 辉光放电后,液体样品更容易在网格表面扩散。充电后一小时内使用电网,否则需要重新充电。
    12. 踩下脚踏板或在Vitrobot界面中选择“放置新网格”。
    13. 在Vitrobot镊子上安装一个网格(视频1)。< > 将Vitrobot镊子的滑块放在上部位置,小心地用边缘连接一个网格,并将滑块锁定在最近的镊子槽上。
    14. 将镊子固定在中心轴的连接槽上。
      样品必须沉积在网格的碳侧。当固定Vitrobot镊子时,网格的碳侧应朝向左侧或右侧,这取决于操作员使用哪侧入口来存放样本(图3e和视频1)。
    15. 把镊子搬进气候室。踩下踏板或选择“开始过程”(图3f和视频1)。
    16. 将冷却液容器放在平台环上(视频1)。
    17. 朝气候室方向升起冷却液容器。< > 踩下踏板或选择“Continue”(继续)(视频1)。
    18. 将Vitrobot镊子降到样本应用位置。< > 踩下踏板或选择“Continue”(继续)(视频1)。
    19. 将样本应用到网格。
      打开气候室的侧端口,用微量吸管(视频1)涂抹3μl。
    20. 启动吸吮和冷冻。< > 踩下踏板或选择“Continue”(继续)并等待冷却液容器和镊子降下(视频1)。
    21. 小心地从Vitrobot中心轴的凹槽中取出镊子。
      将冷却液容器和镊子转移到工作台上(视频1)。在此步骤中,请小心将网格保持在液态乙烷表面以下。
    22. 等到乙烷云散开,慢慢地将网格提升到乙烷表面。< > 一旦电网完全断开,快速将其插入含有液氮的外圈,并将电网转移到电网盒的位置(图3g和视频1)。
    23. 重复步骤b16-b26,直到所有样品都被冷冻固定。
      必要时,用另一个空容器覆盖乙烷容器几秒钟,使液体乙烷解冻。
    24. 存储网格。
      1. 当所有冻结网格都在网格框内时,冷却操纵杆并将其拧到网格框的中心(视频1)。
      2. 将网格盒转移到液氮下的预冷网格盒容器中并将其关闭(视频1)。
      注意:如果不打算在同一天观察到样品,则将格子盒容器存放在装有液氮的杜瓦瓶中。
    25. 关闭Vitrobot。
      将冷却液容器放在通风柜中,用于液氮和液态乙烷的蒸发,并在Vitrobot用户界面中选择“退出”。当灯关闭时,中轴处于停车位置且界面已关闭,请关闭设备背面的拨动开关。
      < >
  3. 低温透射电镜观察
    1. 准备冷冻显微镜。< > 将装有液氮的杜瓦中的网格转移到冷冻显微镜室,并将显微镜的防污染装置冷却至少1h(视频1)。
    2. 确保阀门关闭。
      转到“设置”选项卡。如果“Col.valves closed”(列阀关闭)按钮不是黄色,请单击它(视频1)。
    3. 按“turbo on”(视频1)。
    4. 准备所有附件(图5a),并用冷绝缘材料(软木板、气泡膜……)盖住显微镜的桌面。
      < >
      图5。将格栅安装到冷冻架上。a.附件:1、盖子;2、漏斗;3、冷冻转运站;4、平台;5、卡环工具。b.低温转运站中低温保持器GATAN656的俯视图。黑色箭头指向冷冻转运站,白色箭头指向冷冻架。c.低温转运站详情。1,冷冻架尖端;2,冷冻网格盒底座。黑色箭头指向低温保持架顶端网格的位置。< > < >
    5. 冷却冷冻柜。< > 将冷冻柜放入转运站,将液氮注入冷冻柜杜瓦和冷冻柜站进行冷却。将控制站的电缆连接到低温保持架,等待温度低于-165°C(视频1)。
    6. 准备冷冻架尖端。
      1. 将格栅箱转移到其在转运站平台上的位置,并小心地拧松并拆下杆(图5B和5C以及视频1)。
      2. 通过向后滑动低温保持架中心轴末端(杜瓦瓶正后方)的圆形件打开低温屏蔽,用工具移除卡环并将其置于液氮下(视频1)。< > 注意:卡环工具的尖端有两个凸耳,通过转动手柄的两个轮子可以将其相对或连续放置。要从低温保持架尖端拆卸卡环,将两个卡舌放在一起,将它们放在卡环周围的凹槽中,移动车轮使卡舌相对放置并向上拉。
    7. 将格栅安装在低温保持架尖端。
      冷却镊子尖端,将其中一个网格转移到冷冻架尖端的槽中,用卡环工具将其固定,并关闭样品上的冷冻屏蔽,以防止冰污染(图5c和视频1)。要用卡环固定网格,请将卡环放在网格上,按下卡环工具,转动手柄的轮子,直到卡舌相邻,然后拆下工具。
    8. 断开控制站电缆与低温保持架的连接(视频1)。
    9. 在ComputeStage下准备一个聚苯乙烯盒子(视频1)。
    10. 按“预泵气锁”(视频1)。
    11. 从低温转运站快速取出低温保持架,并小心地将低温保持架的尖端穿过显微镜的孔尽可能远地引入(视频1)。
    12. 向右旋转低温保持架,直到其枢轴与Compustage凹槽匹配。将低温保持架轻按在显微镜上,等待“真空概述”(视频1)中的倒计时结束。向右旋转低温保持架时要小心,以免液氮落到步骤c9中制备的聚苯乙烯盒上。
    13. 选择“冷冻架”作为样本架。
    14. 在“真空概述”中的倒计时结束后,将低温保持架尽量向左转动,并将其完全引入显微镜中(图6a和视频1)。
    15. 迅速用液氮重新注入低温保持器的杜瓦瓶(视频1)。
    16. 将控制站的电缆连接到低温保持架上,检查温度是否不超过-170°C(视频1)。
    17. 单击“Col.Valves Closed”(Col.Valves关闭)并转到“LowDose”(低剂量)选项卡(视频1)。
    18. 在低剂量条件下记录图像,光斑尺寸为5。
      X7800和0.10E-/_2S的搜索模式,X29000和0.80E-/_2S的聚焦模式,X29000和1.00E-/_2S的曝光模式(图6B和视频1)。
      < >
      图6。低温透射电镜观察。a.低温电子显微镜tecnai f20-fei.黑色箭头表示低温保持器的位置。b.低剂量观察。

数据分析

从低温透射电镜图像中获得了囊泡的可靠测量值,以便于统计。在两个独立的实验中,每个菌株测量了7000多个囊泡,并用imagej 1.47软件进行了测量。MVS可测量20至250纳米的直径(Perez Cruz等人,2015年)。

笔记

  1. 操作材料(滤纸、网格、样品等)时,应穿戴实验室工作服和手套,以避免自身污染,并在试验期间避免因接近液化气体而造成皮肤损伤。

致谢

我们非常感谢MJ Montes为妥善维护菌株提供的帮助,感谢M Gimeno为录制视频提供的帮助。

相互竞争的利益

提交人已宣布不存在任何相互竞争的利益。

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引用:Delgado, L., Baeza, N., Pérez-Cruz, C., López-Iglesias, C. and Mercade, E. (2019). Cryo-transmission Electron Microscopy of Outer-inner Membrane Vesicles Naturally Secreted by Gram-negative Pathogenic Bacteria. Bio-protocol 9(18): e3367. DOI: 10.21769/BioProtoc.3367.
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