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Sample Preparation for Correlative Light and Electron Microscopy (CLEM) Analyses in Cellular Microbiology
用于细胞微生物学中关联光学和电子显微镜技术(CLEM)分析的样本制备   

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Abstract

Dynamic processes in cells are usually monitored by live cell fluorescence microscopy. Unfortunately, this method lacks the ultrastructural information about the structure of interest (SOI). Currently, electron microscopy (EM) is the best tool to achieve highest spatial resolution. In addition, correlative light and electron microscopy (CLEM) analysis of the same structure allows combining authentic live cell imaging with the resolution power of EM. Additionally the reference space of the SOI is revealed. Our CLEM analyses of HeLa cells allow tracing the morphology and dynamic behavior of intracellular micro-compartments in living cells and their ultrastructure and subcellular organization in a highly resolved manner.

Keywords: Sample preparation(样品的制备), Correlative(关联), Electron microscopy(电子显微镜), Eucaryotic cells(真核细胞), Protocol(协议)

Materials and Reagents

  1. General lab equipment: Gloves, lab coat, pipettes, 15 and 50 ml centrifuge tubes (e.g., BD Biosciences, Falcon®), microcentrifuge tubes (e.g., Eppendorf), plastic Pasteur pipettes, beakers
  2. 10 cm plastic Petri dish or a comparable vessel
  3. 3.5 cm plastic Petri dish or a comparable vessel (Acetone-resistant)
  4. Eukaryotic cells as biological sample (here: transfected HeLa cells expressing LAMP1-GFP)
  5. Cell-specific culture medium: DMEM (Biochrom AG, catalog number: FG 0445 ) + 10% Fetal Bovine Serum (FCS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270 )
  6. Cell-specific imaging medium: MEM (Biochrom AG, catalog number: F 0475 ) + 30 mM HEPES
  7. HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Carl Roth GmbH, catalog number: HN77.5 )
  8. Glutaraldehyde [CH2(CH2CHO)2], 25% in H2O (Electron Microscopy Sciences, catalog number: E16221 )
  9. Glycine, ultra pure (Biomol GmbH, catalog number: 04943.1 )
  10. Large gelatin capsules, size 13 (Electron Microscopy Sciences, catalog number: 70114 )
  11. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C 5670 )
  12. Osmium tetroxide (OsO4), 0.1 g ampules (Electron Microscopy Sciences, catalog number: 19134 )
  13. Ruthenium red [[(NH3)5RuORu(NH3)4ORu(NH3)5]Cl6] (AppliChem GmbH , catalog number: A34880001 )
  14. Potassium hexacyanoferrate(III) [K3[Fe(CN)6]] (Sigma-Aldrich, catalog number: 244023 )
  15. Ethanol p.a. grade (very pure chemical)
  16. Acetone p.a. grade (very pure chemical)
  17. EPON 812 (SERVA Electrophoresis GmbH, catalog number: 21045.02 )
  18. DDSA = Dodecenylsuccinic anhydride (SERVA Electrophoresis GmbH, catalog number: 20755 )
  19. MNA = Methylnadic anhydride (SERVA Electrophoresis GmbH, catalog number: 29452.03 )
  20. DMP-30 = 2, 4, 6-Tris(dimethylamino-methyl)phenol (SERVA Electrophoresis GmbH, catalog number: 36975 )
  21. Uranyl acetate dihydrate [UO2(CH3COO)2.2H2O] (Electron Microscopy Sciences, catalog number: 22400 )
  22. Lead(II) nitrate [Pb(NO3)2] (Sigma-Aldrich, catalog number: 31137 )
  23. Trisodium citrate dihydrate (Na3C6H5O7.2H2O) (Carl Roth, catalog number: 3580.1 )
  24. 1 N Sodium hydroxide (NaOH), carbonate-free (Electron Microscopy Sciences, catalog number: 21170-01 )
  25. 65% Nitric acid (HNO3)
  26. Autoclaved ultrapure H2O (MilliQ)
  27. Liquid nitrogen
  28. Graded ethanol series (50%, 70%, 80%, 90%, 95%, 100%)
  29. Mixes of acetone and EPON (3:1 and 1:3 mix)
  30. 1 M HEPES buffer (see Recipes)
  31. 2 M CaCl2 in H2O (see Recipes)
  32. 10% glycine in 0.2 M HEPES buffer (see Recipes)
  33. 1% ruthenium red in H2O (see Recipes)
  34. 15% potassium hexacyanoferrate(III) in H2O (see Recipes)
  35. 2x fixative (see Recipes)
  36. Post-fixative (see Recipes)
  37. EPON resin (see Recipes)
  38. 2% uranyl acetate in H2O (see Recipes)
  39. Lead citrate according to Reynolds (see Recipes)
  40. 3% nitric acid (HNO3) (see Recipes)

Equipment

  1. Cell culture equipment: Cell culture incubator (37 °C, 5% CO2, 90% humidity) and clean bench
  2. MatTek Glass Bottom Culture Dishes, 35 mm, uncoated, Glass No. 2, gridded (Glass Bottom Dishes, MatTek Corporation, catalog number: P35G-2-14-CGRD )
    Attention: Glass No. 2 may be too thick for some objectives with high numerical aperture (NA), but this is the only format available and worked in our application.
  3. Confocal laser-scanning microscope (CLSM) Leica SP5 equipped with an incubation chamber (homemade) maintaining 37 °C and humidity during live cell imaging (Attention: CO2-containing atmosphere can be omitted if using imaging medium buffered with 30 mM HEPES. For carbonate-buffered cell culture media, the atmosphere should contain 5% CO2). The microscope is operated with software package LAS-AF for setting adjustment, image acquisition and image processing
    1. Several objectives, such as 10x (HC PL FL 10x, NA 0.3, DIC, dry), 20x (HC PL APO CS 20x, NA 0.7, DIC, dry), 40x (HCX PL APO CS 40x, NA 1.25-0.75, DIC, oil immersion) and 100x objective (HCX PL APO CS 100x, NA 1.4-0.7, DIC, oil immersion)
    2. The polychroic mirror TD 488/543/633 for the three channels GFP/RFP/DIC (Leica Microsystems)
  4. Fume hood
  5. Ice or cold metal block
  6. Scalpel
  7. Oven, heatable to 60 °C
  8. Dewar vessel for liquid nitrogen
  9. Stereo microscope
  10. Dark permanent marker
  11. Vice
  12. Jigsaw
  13. Universal specimen holder for EPON blocks (Leica Microsystems, catalog number: 16701761 )
  14. Double edge carbon steel blades (Plano, catalog number: 121-9 )
  15. Ultramicrotome (Leica EM UC6)
  16. Diamant knife ultra 45 °C, 2 mm (DIATOME, catalog number: DU4520 )
  17. Formvar-coated EM cooper slit grids 2 x 1 mm (homemade coating) (Electron Microscopy Sciences, catalog number: G2010-Cu )
  18. Forceps for EM grids, type 7 stainless steel (Plano GmbH, catalog number: T5039 )
  19. Storage box for grids (Electron Microscopy Sciences, catalog number: G71138 )
  20. Large forceps for liquid nitrogen and small forceps for embedding procedure
  21. Automated staining device (nanofilm surface analysis ultrastainer)
  22. TEM (ZEISS, model: EFTEM 902 A ), operated at 80 kV and equipped with a 2K wide-angle slow-scan CCD camera (Teacher Retirement System of Texas) with software ImageSP (Teacher Retirement System of Texas, model: image SysProg)

Software

  1. ImageJ, http://rsbweb.nih.gov/ij/, Photoshop 5.5 (Adobe), or higher or similar software for stitching and overlay of images
  2. Imaris (Bitplane), ImageJ, ZEN (ZEISS), LASAF (Leica Microsystems), or similar software for processing fluorescence images

Procedure

Due to effort in preparation and limited shelf life the chemicals required have to be prepared at various times:

  1. Some chemicals can be prepared in advance and stored up to several months. These are 1 M HEPES buffer, 2 M calcium chloride, 10% glycine, 1% ruthenium red, 15% K3[Fe(CN)6], graded ethanol series and autoclaved MilliQ water.
  2. Some chemicals should be prepared more freshly and stored only up to several weeks. These are the resin components A and B, 2% uranyl acetate, lead citrate and 3% HNO3.
  3. Some chemicals should be prepared as fresh as possible. At the day of live cell imaging prepare fresh 2x and 1x fixative. One day before use start to prepare the post-fixative, since osmium tetroxide crystals need one night to solve (solved in dark at 4 °C in fume hood), and finish the preparation of the post-fixative shortly before usage by addition of additives. Also at the day of use prepare the EPON resin (stored at -20 °C, but equilibrate to RT before use) and prepare the acetone-EPON mixes shortly before use.

Scheme of procedure
In the following the CLEM work flow from seeding cells to TEM imaging is illustrated.


Figure 1. CLEM workflow. HeLa cells are seeded in a MatTek dish with a gridded coverslip. After one day, cells can be transfected or pulse-chased with fluid phase marker. The next day, cells are infected, imaged live and fixed as fast as possible on the microscope stage. The coverslip is removed from the dish and the sample is processed for TEM and flat-embedded in resin. During the removal of the coverslip the engraved coordinates are transferred to the resin surface and allows trimming around the ROI. Serial sections are transferred to EM grids and stained for contrast. The same ROI is imaged in TEM for overlaying with LM image.



  1. For the experiment HeLa cells are seeded two days prior to live cell imaging into a MatTek dish containing 2 ml cell culture medium and incubated at 37 °C, 5% CO2 and 90% humidity. The cell number per MatTek dish should be app. 4 x 105 cells at the day of imaging. This relative low cell number facilitates the relocation of the cells on the grid. Importantly, at the day of microscopy cells should possess a fluorescent marker for the structure of interest (SOI) to enable correlation of live cell images to ultrastructure. Therefore, cells have to be specifically treated before microscopy, meaning transfection with plasmids for expression of tagged proteins, pulse-chase with fluid phase markers (Krieger et al., 2014), or another labeling method.
  2. At least 1 h prior to live cell imaging switch on the microscope and the incubation chamber of the microscope to reach the desired temperature and atmosphere at imaging start. Start the imaging software and adjust all required settings to avoid delays before live cell imaging.
  3. Arrange freshly prepared 2x fixative pre-warmed to 37 °C and equipment (gloves, pipette and timer) as close as possible to the CLSM microscope.
  4. Directly before imaging of the cells replace cell culture medium by 1 ml imaging medium and transfer the MatTek dish to the pre-warmed microscope stage. Remove the lid from the MatTek dish before imaging and don’t touch the dish until the last image is done.
  5. A region of interest (ROI) is observed first with the 100x objective, a Z-stack is defined and the acquisition is started. The settings should allow rather fast acquisition (dependent on the dynamics of the SOI), since fast cellular processes, e.g., vesicle movement will complicate correlation with ultrastructure. As soon as the Z-stack is finished (less than 1 min) add 1 ml of warm 2x fixative to 1 ml imaging medium in the dish (ratio 1+1) and mix very gently by pipetting up and down, in order to fix cells as fast as possible directly on the microscope stage. Be careful not to touch the dish with the pipette tip. Start the timer for 1 h. Now acquire all remaining images, i.e., one additional Z-stack with the 100x objective directly after addition of 2x fixative as control for movement of SOI. Next generate overview images, each with 40x, 20x and 10x objectives without Z-stacks, for the localization of the ROI on the dish grid and the eventual re-localization of the ROI on the resin surface. Once the last image is acquired, close lid, remove the MatTek dish from the microscope and transfer to a fume hood. Keep the cells warm.


    Figure 2. Fluorescence microscopy and localization of the ROI on dish grid. Cells of interest are imaged live and a Z-stack is acquired by CLSM (A). An additional bright field image is acquired for visualization of shape and distribution of cells (B). Using this information, cells of interest are relocated on the dish grid using a lower magnification and engraved coordinates on the coverslip of the MatTek dish are recorded (in this case the coordinates “BK”) (C). Coordinates “BK” were highlighted (D).

  6. Under the fume hood the imaging medium-fixative mixture is replaced by 2 ml fresh pre-warmed 1x fixative and cells are incubated for the remainder of the fixation time of 1 h. At the end of fixation cells should be at room temperature (RT).
  7. Rinse cells thrice with 2 ml 0.2 M HEPES buffer.
  8. Incubate cells with 2 ml 50 mM glycine in HEPES buffer for 15 min to block unreacted glutaraldehyde.
  9. Rinse cells thrice with 2 ml 0.2 M HEPES buffer.
  10. Cells are cooled to 4 °C in a fridge or on ice, since the next steps have to be performed in the cold.
  11. This step has to be performed under a fume hood due to very toxic osmium tetroxide! First finish the preparation of cold post-fixative by addition of ruthenium red and potassium hexacyanoferrate (III) to the solved osmium tetroxide crystals. Next remove HEPES buffer from the ice-cold cells, add minimum of 1.5 ml ice-cold post-fixative and incubate for 1 h at 4 °C in the dark in fume hood. The osmium will stain dark the cells as well as the glue between MatTek dish and coverslip.
  12. Rinse cells five times with 2 ml ice-cold 0.2 M HEPES buffer for 2 min each (in fume hood).
    Note: This step may also be extended overnight.
  13. Remove the gridded square coverslip from the MatTak dish. This step can be performed during the dehydration whenever appropriate, but the incubation times of the dehydration are critical and should be adhered to. Prepare a 10 cm plastic Petri dish filled with the solution of the current step (for example, 0.2 M HEPES buffer) on ice. Place the MatTek dish inside the liquid of the 10 cm dish and gently but with continual force insert the sharp edge of a scalpel from the inside between the coverslip and the plastic dish bottom. As the scalpel penetrates deeper between the coverslip and the dish bottom, the coverslip starts to peel off and falls into the buffer-filled 10 cm dish. Using forceps, transfer the coverslip cell-side up into a smaller 3.5 cm plastic dish or comparable vessel (acetone-resistant) already filled with solution of the current step. The volume of all following solutions inside the 3.5 cm dish should be large enough to completely cover the cells. Consider that ethanol and acetone evaporate. Cells should never come in contact with air or dry completely.


    Figure 3. Removal of the coverslip from the MatTek dish. Using a scalpel, the square coverslip glued to the bottom of the dish is removed from the MatTek dish (A), then drops into a buffer-filled 10 cm dish on ice (B).

  14. Prepare mixtures of acetone and EPON (3:1 and 1:3 mix) in the fume hood. This step may be performed during the dehydration whenever appropriate, but should be finished at the latest when the sample is in acetone.
  15. Remove HEPES buffer from the cells and dehydrate sample in a cold graded ethanol series. In detail, cells are incubated with 50%, 70%, 80%, 90%, 95% and 100% ethanol for 10 min for each step. Cells are kept cold, e.g., by placing a cold metal block under the 3.5 cm dish until the 95% ethanol incubation. From the incubation with 100% ethanol, remove metal block and allow reaching RT.
  16. Incubate cells in anhydrous ethanol for 10 min at RT.
  17. Incubate cells in anhydrous acetone twice for 10 min at RT.
  18. Incubate cells in 3:1 mix of acetone:EPON for 1 h at RT (in fume hood).
  19. Incubate cells in 1:3 mix of acetone:EPON for 1 h at RT (in fume hood).
  20. Incubate cells in pure EPON overnight at RT (in fume hood).
  21. The next day, cells are incubated in fresh pure EPON for 4 h at RT (in fume hood). In the meantime, the oven for EPON polymerization is switched on adjusted to 60 °C. The oven should also be inside a fume hood.
  22. Fix one half of a gelatin capsule upright with the opening up and fill the capsule with fresh EPON. Avoid air bubbles inside the EPON. Air bubbles may be removed by shortly warming the filled capsule in the oven. With forceps, pick up the square coverslip from the last EPON incubation, hold it upright for approximately 1 min to allow EPON to run off and place the coverslip cell-side down on the filled capsule. Again, avoid air bubbles. Finally place the capsule with the coverslip on top for polymerization of EPON inside the oven heated to 60 °C for 48-72 h. In parallel all materials contaminated with EPON can be wrapped in aluminum foil and placed in the oven polymerization.


    Figure 4. EPON embedding of cells on detached coverslips. A lid of a 15 ml Falcon tube, one half of a large gelatin capsule, aluminum foil, and the coverslip containing cells is required for embedding (A). The half of the gelatin capsule is placed upright with opening facing upwards inside the lid of the Falcon tube and fixed with aluminum foil (B). The capsule is filled with EPON (C). After dehydration and infiltration of sample the coverslip is placed cell-side down on the EPON-filled capsule (D).

  23. After complete polymerization of EPON remove sample from oven and leave to cool to RT.
  24. Prepare a Dewar vessel with liquid nitrogen, a beaker with hot water and large forceps. With the forceps quickly immerse the EPON block with the coverslip-side down alternately into liquid nitrogen and hot water. The different physical properties of glass and EPON will lead to a separation of the coverslip. During the removal of the gridded coverslip from the EPON block the engraved grid is transferred from the coverslip to the EPON block surface.


    Figure 5. Removal of coverslip from polymerized EPON block. The polymerized EPON block is removed from the oven and Falcon lid for cooling (A). Coverslip is removed from EPON block using the nitrogen-hot water method (B).

  25. Inspect the EPON block surface under a stereo microscope for relocation of the ROI.
    Note that the grid pattern will be transferred mirrored. Mark the ROI with a dark permanent marker.
  26. By using a vice and a fine jigsaw roughly remove the excess EPON and form a rectangular EPON block (0.5 x 0.5 x 1.5 cm) with the ROI in the middle of the square surface. Be careful never to damage the EPON surface of the ROI, since the cells are directly under the EPON surface.
  27. Fix the rectangular EPON block in a metal specimen holder for ultramicrotomes and do not remove until sectioning is done completely.


    Figure 6. Roughly trimmed EPON blocks are fixed in metal holder. Relocated ROI are marked on the EPON surface and the EPON block is roughly trimmed (A). Trimmed EPON block is fixed in a metal specimen holder for precise trimming and sectioning (B).

  28. Under the stereo microscope manually trim the EPON block sides (not the face!) with a double-edged razor blade so that a pyramid with 45° angled sides and a trapezoidal surface with the ROI in the middle of the trapezoid is formed. The base edge of the trapezoidal surface should be approximately 500 µm and absolutely parallel to the opposite edge. Again, be careful not to damage the EPON face of the ROI.


    Figure 7. Trimming of EPON block. The EPON block is trimmed with a razorblade (A) so that a pyramid with 45° angled sides and a trapezoidal surface with the ROI in the middle of the trapezoid is formed (B).

  29. Transfer the metal specimen holder to an ultramicrotome equipped with a diamond knife. Essentially, the knife and trapezoidal EPON surface should be aligned parallel to X and Y direction as perfect as possible, since this alignment is most important for the final correlation of images acquired by CLSM and TEM. Pre-sectioning, meaning facing, of the EPON surface is not allowed. For more details on knife alignment, sectioning and collecting sections see Hagler (2007), especially part 3.3.5. Cutting Thin Sections.
  30. Acquire 70 nm thin serial sections of the trimmed EPON block with the ultramicrotome. Be careful not to lose any section since each section, already the first one, may have important information.
  31. Collect the serial sections floating on the water surface of the diamond knife reservoir with formvar-coated EM copper slit grids. The order of the sections is very important and should be recorded. Up to eight sections fit on one grid. Formvar coating of the grids may be done in house, but coated grids are also commercially available. Put the grids in the grid storage box. Air dry the grids completely before closing the box.
  32. For obtaining contrast for TEM, all loaded grids are stained with 2% uranyl acetate for 40 min and lead citrate for 30 min using an automated staining device. For this device also 3% HNO3 and autoclaved water should be at hand. Follow manufacturers manual or one can also stain the grids manually. Keep in mind that lead citrate is CO2-sensitive and follow specific protocols, e.g., Ellis (2004).
  33. Stained sections are observed with a TEM operated at 80 kV and images are acquired at different magnifications as needed for the final correlation. CLSM images should be at hand for the re-localization of the ROI on the resin section.


    Figure 8. Relocation of ROI on EPON sections. By means of the before acquired CLSM images (A) the cells are relocated according to their orientation and morphology on the EPON sections (B).

  34. Finally, the CLSM and TEM images are overlaid using Photoshop. If necessary, stitching of acquired TEM images is done manually or automated with ImageJ or Photoshop and the processing of CLSM images using microscope-specific software or Imaris. Image processing includes noise reduction, contrast enhancement, or color balance. Note that image processing should be according to proper scientific practice and a good overview on image manipulations can be found in the instructions for authors of the Journal of Cell Biology (http://www.jcb.rupress.org/site/misc/ifora.xhtml).

Representative data


Figure 9. Examples of CLEM analyses of the intracellular lifestyle of Salmonella. HeLa cells expressing LAMP1-GFP (green) were seeded in Petri dishes with a gridded coverslip and infected with Salmonella expressing mCherry (STM, red). Live cell imaging was performed 8 h p.i. to visualize LAMP1-GFP-positive SIF (A. maximum intensity projection [MIP], C. single Z plane). Subsequently, the cells were fixed and processed for CLEM to reveal the ultrastructure of selected cells. Several low magnification images were stitched to visualize the cell morphology (B). Higher magnification images were used to align LM and TEM images (D). Note the formation of double membrane LAMP1-GFP-positive SIF. Details of a LAMP1-GFP-positive SIF (E) and an SCV linked to a SIF (F, G) are shown. G. Two additional ultrathin sections show the membrane organization of the SCV and SIF in corresponding to panel F. H. The inner and outer membrane of a SIF and SCV are outlined in orange and yellow, respectively. Light and dark red arrowheads indicate inner and outer membrane of the Salmonella cell envelope, respectively. Labels: S, Salmonella; M, mitochondria; iL, inner lumen; oL, outer lumen. A cell representative for 10 biological replicates is shown (1–3 technical replicates with each 2–4 cells). Scale bars: 10 µm (A, B), 2 µm (C, D), 500 nm (E, F, G).

Notes

  1. Be careful with toxic substances such as glutaraldehyde, osmium tetroxide, EPON, uranyl acetate and lead citrate. Familiarize yourself with safety instructions for handling, personal protection, and take care about correct waste disposal.
  2. Use disposable vessels for every solution containing EPON. It is difficult to clean EPON contaminated vessels, thus use disposable ones in which EPON is neutralized by polymerization in an oven at 60 °C for at least 48 h.
  3. This protocol is adjusted to investigations in cellular microbiology, in particular intracellular activities of Salmonella in HeLa cells. Other infection models require optimization regarding incubation times, concentrations, temperatures.
  4.  Prepare critical solutions as fresh as possible prior to the experiment.

Recipes

  1. 1 M HEPES buffer (500 ml)
    1. Weigh 119.3 g HEPES
    2. Add autoclaved MilliQ water to 500 ml and mix well
    3. Adjust pH 7.4 by addition of 5 M HCl
    4. Autoclave to sterilize
    5. Buffer can be stored at RT up to several months
    6.  For 0.2 M solution dilute 1:5 with water
  2. 2 M CaCl2 in H2O (10 ml)
    1. Weigh 2.22 g CaCl2
    2. Add autoclaved MilliQ water to 10 ml and mix well
    3. Autoclave to sterilize
    4. Reagent can be stored at 4 °C up to several months
  3. 10% glycine in 0.2 M HEPES buffer (5 ml)
    1. Weigh 500 mg glycine
    2. Add 0.2 M HEPES to 5 ml and mix well
    3. Sterilize by filtration
    4. Solution can be stored at 4 °C up to several months
    5. For 50 mM add 37.6 µl of 10% solution to 1 ml 0.2 M HEPES buffer
  4. 1% ruthenium red (RR) in H2O (5 ml)
    Note: Harmful if swallowed.
    1. Weigh 50 mg RR
    2. Add autoclaved MilliQ water to 5 ml and mix well
    3. Aliquot 500 µl in Eppendorf tubes
    4. Aliquots can be stored at -20 °C up to several months
  5. 15% potassium hexacyanoferrate(III) in H2O (5 ml)
    1. Weigh 750 mg K3[Fe(CN)6]
    2. Add autoclaved MilliQ water to 5 ml and mix well
    3. Aliquot 500 µl in Eppendorf tubes
    4. Aliquots can be stored at -20 °C up to several months
  6. 2x fixative
    5% glutaraldehyde + 10 mM CaCl2 in 0.4 M HEPES buffer (12.5 ml)
    Note: Toxic! Dispose as special waste!
    1. Fill 4.938 ml autoclaved MilliQ water in a dark glass flask
    2. Add 5 ml 1 M HEPES buffer (final 0.4 M)
    3. Add 2.5 ml from 25% glutaraldehyde (final 5%)
    4. Add 62.5 µl from 2 M CaCl2 (final 10 mM)
    5. Reagent can be stored at 4 °C for few days
    6. For 1x fixative, dilute 1:2 with water
  7. Post-fixative
    2% osmium tetroxide + 0.1% ruthenium red + 1.5% potassium hexacyanoferrate(III) in 0.2 M HEPES buffer (5 ml)
    Note: Very toxic! Dispose as special waste!
    1. Transfer complete content from the 0.1 g OsO4 ampule in a dark glass flask
    2. Add 3 ml autoclaved MilliQ water
    3. Add 1 ml 1 M HEPES buffer (final: 0.2 M)
    4. Close the flask and seal with parafilm
    5. Store the flask at 4 °C in fume hood overnight to dissolve OsO4 crystals
    6. The next day add additives: 0.5 ml from 1% ruthenium red (final 0.1%) and 0.5 ml from 15% potassium hexacyanoferrate(III) (final 1.5%)
    7. Mix well and use immediately at 4 °C
  8. EPON resin
    Note: Toxic! Dispose as special waste!
    Solution A
    1. Mix 31 ml of EPON 812 with 50 ml of DDSA in a plastic container
    2. Stir with wooden stick
    3. The solution can be stored at -20 °C up to several weeks
    Solution B
    1. Mix 50 ml of EPON 812 with 42.5 ml of MNA in a plastic vessel
    2. Stir with wooden stick
    3. The solution can be stored at -20 °C up to several weeks
    A-B mix
    1. Mix Sol A + B 1:1 in plastic dish with syringe
    2. Add 1.5 Vol.-% DMP-30 (very viscous)
    3. Stir with wooden stick
    4. Reagent can be stored at -20 °C up to several days
  9. 2% uranyl acetate in H2O (100 ml)
    Note: Very toxic! Chemically toxic and radioactive! Dispose as special waste!
    1. Weigh 2 g uranyl acetate
    2. Add autoclaved MilliQ water to 100 ml and stir with magnetic stirrer
    3. Filter the solution with 0.2 µm nitrocellulose filter
    4. Transfer to dark glass flask
    5. Reagent can be stored at 4 °C up to several weeks
  10. Lead citrate according to Reynolds (100 ml)
    Note: Very toxic! Dispose as special waste!
    1. Weigh 2.66 g lead nitrate and 3.52 g sodium citrate
    2. Add 60 ml MilliQ water (autoclaved, degassed by evacuation) and stir for 10 min on magnetic stirrer
    3. Allow to stand at RT for 10 min with intermittent shaking, solution will be of milky white color
    4. Add 11 ml of 1 N NaOH (carbonate-free) and mix, solution will turn clear
    5. Measure pH and adjust exactly to pH 12.0 by drop-wise adding 1 N NaOH (carbonate-free)
    6. Fill to 100 ml with about 28 ml MilliQ water (autoclaved, degased)
    7. Reagent can be stored at 4 °C up to several weeks
  11. 3% nitric acid (HNO3) (1 L)
    Note: Acidic!
    1. To 954 ml autoclaved MilliQ water add 46 ml of 65% HNO3
    2. Filter the solution through 0.2 µm nitrocellulose filter
    3. The solution can be stored at RT up to several weeks

Acknowledgments

Work in our group was supported by the DFG through grant HE1964/18-1 and the Z project of SFB944 ‘Physiology and dynamics of cellular microcompartments’.

References

  1. Ellis, E. A. (2014). Staining sectioned biological specimens for transmission electron microscopy: conventional and en bloc stains. Methods Mol Biol 1117: 57-72.
  2. Hagler, H. K. (2007). Ultramicrotomy for biological electron microscopy. Methods Mol Biol 369: 67-96.
  3. Krieger, V., Liebl, D., Zhang, Y., Rajashekar, R., Chlanda, P., Giesker, K., Chikkaballi, D. and Hensel, M. (2014). Reorganization of the endosomal system in Salmonella-infected cells: the ultrastructure of Salmonella-induced tubular compartments. PLoS Pathog 10(9): e1004374.

简介

通常通过活细胞荧光显微镜监测细胞中的动态过程。 不幸的是,这种方法缺乏关于感兴趣的结构(SOI)的超结构信息。 目前,电子显微镜(EM)是实现最高空间分辨率的最佳工具。 此外,相同结构的相关光和电子显微镜(CLEM)分析允许组合真实活细胞成像与EM的分辨率功率。 此外,揭示了SOI的参考空间。 我们对HeLa细胞的CLEM分析允许以高度解析的方式追踪活细胞中的细胞内微隔室及其超微结构和亚细胞组织的形态和动态行为。

关键字:样品的制备, 关联, 电子显微镜, 真核细胞, 协议

材料和试剂

  1. 一般实验室设备:手套,实验室外套,移液管,15和50ml离心管(例如BD Biosciences,Falcon),微量离心管(例如 Eppendorf),塑料巴斯德移液器,烧杯
  2. 10厘米塑料培养皿或同类容器
  3. 3.5cm塑料培养皿或类似容器(耐丙酮)
  4. 真核细胞作为生物样品(此处:表达LAMP1-GFP的转染的HeLa细胞)
  5. 细胞特异性培养基:DMEM(Biochrom AG,目录号:FG 0445)+ 10%胎牛血清(FCS)(Thermo Fisher Scientific,Gibco TM,目录号:10270)
  6. 细胞特异性成像介质:MEM(Biochrom AG,目录号:F 0475)+ 30mM HEPES
  7. HEPES = 4-(2-羟乙基)-1-哌嗪乙磺酸(Carl Roth GmbH,目录号:HN77.5)
  8. 戊二醛[CH 2(CH 2 CHO)2],在H 2 O中25%(电子显微镜科学,目录号:E16221)
  9. 甘氨酸,超纯(Biomol GmbH,目录号:04943.1)
  10. 大型明胶胶囊,尺寸13(Electron Microscopy Sciences,目录号:70114)
  11. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C 5670)
  12. 四氧化锇(OsO 4),0.1g安瓿(Electron Microscopy Sciences,目录号:19134)
  13. 钌红[[(NH 3)5 RuORu(NH 3)4] ORu(NH 3) (AppliChem GmbH,目录号:A34880001)
  14. 六氰基铁酸钾(III)[K 3+ [Fe(CN)6]](Sigma-Aldrich,目录号:244023)
  15. 乙醇等级(非常纯的化学品)
  16. 丙酮等级(非常纯的化学品)
  17. EPON 812(SERVA Electrophoresis GmbH,目录号:21045.02)
  18. DDSA =十二碳烯基琥珀酸酐(SERVA Electrophoresis GmbH,目录号:20755)
  19. MNA =甲基纳迪克酸酐(SERVA Electrophoresis GmbH,目录号:29452.03)
  20. DMP-30 = 2,4,6-三(二甲基氨基甲基)苯酚(SERVA Electrophoresis GmbH,目录号:36975)
  21. 乙酸铀二水合物[UO 2(CH 3 COO)2] 2 O 2 [ O](Electron Microscopy Sciences,目录号:22400)
  22. 硝酸铅(II)[Pb(NO 3)2](Sigma-Aldrich,目录号:31137)
  23. 柠檬酸三钠二水合物(Na 3 H 6 H 6 H 5 O 7) sub> 2 O)(Carl Roth,目录号:3580.1)
  24. 1N氢氧化钠(NaOH),无碳酸盐(Electron Microscopy Sciences,目录号:21170-01)
  25. 65%硝酸(HNO 3)
  26. 高压灭菌的超纯H 2 O(MilliQ)
  27. 液氮
  28. 分级乙醇系列(50%,70%,80%,90%,95%,100%)
  29. 丙酮和EPON(3:1和1:3混合物)的混合物
  30. 1 M HEPES缓冲区(参见配方)
  31. 在H 2 O中的2 M CaCl 2(参见配方)
  32. 10%甘氨酸的0.2M HEPES缓冲液(参见配方)
  33. 1%钌红的H 2 O(参见配方)
  34. 15%六氰基铁酸钾(III)在H 2 O中(参见配方)
  35. 2x固定剂(参见配方)
  36. 后固定(见配方)
  37. EPON树脂(参见配方)
  38. 2%的H 2 O 2中的乙酸双氧铀(参见配方)
  39. 根据Reynolds的柠檬酸铅(参见配方)
  40. 3%硝酸(HNO 3)(参见配方)

设备

  1. 细胞培养设备:细胞培养培养箱(37℃,5%CO 2,90%湿度)和净化台
  2. MatTek玻璃底培养皿,35mm,未涂覆,玻璃No.2,格栅(玻璃底盘,MatTek公司,目录号:P35G-2-14-CGR??D)
    注意:对于高数值孔径(NA)的物镜,2号玻璃可能太厚,但这是我们应用中唯一可用的格式。
  3. 在活细胞成像期间(注意:CO )的共焦激光扫描显微镜(CLSM)Leica SP5,配备有保持37℃和湿度的孵育室如果使用用30mM HEPES缓冲的成像介质,则可以省略含有大气的气氛。对于碳酸盐缓冲的细胞培养基,气氛应该包含5%CO /em> )。显微镜使用软件包LAS-AF操作,用于设置调整,图像采集和图像处理
    1. 几个目标,如10x(HC PL FL 10x,NA 0.3,DIC,干),20x (HC PL APO CS 20x,NA 0.7,DIC,dry),40x(HCX PL APO CS 40x,NA 1.25-0.75,DIC,油浸)和100x物镜(HCX PL APO CS 100x, NA 1.4-0.7,DIC,油浸)
    2. 用于三个通道GFP/RFP/DIC(Leica Microsystems)的多色镜TD 488/543/633,
  4. 通风橱
  5. 冰块或冷金属块
  6. Scalpel
  7. 烤箱,可加热至60°C
  8. 用于液氮的杜瓦瓶
  9. 立体显微镜
  10. 黑色永久标记

  11. 拼图
  12. 用于EPON块的通用试样夹(Leica Microsystems,目录号:16701761)
  13. 双边碳钢刀片(Plano,目录号:121-9)
  14. 超薄切片机(Leica EM UC6)
  15. 直径超过45℃,2mm(DIATOME,目录号:DU4520)的刀具
  16. 2×1mm(自制涂层)(电子显微镜科学,目录号:G2010-Cu)的表面涂覆EM铜合缝隙栅格
  17. 用于EM网格的钳子,7型不锈钢(Plano GmbH,目录号:T5039)
  18. 网格存储盒(Electron Microscopy Sciences,目录号:G71138)
  19. 大镊子液氮和小钳子嵌入程序
  20. 自动染色装置(nanofilm surface analysis ultrastainer)
  21. 使用软件ImageSP(德克萨斯的教师退休系统,型号:image SysProg)配备有2K广角慢扫描CCD照相机(得克萨斯州的教师退休系统)的TEM(ZEISS,型号:EFTEM 902A)

软件

  1. ImageJ, http://rsbweb.nih.gov/ij/,Photoshop 5.5(Adobe)或更高或类似的软件用于拼接和叠加图像
  2. Imaris(位平面),ImageJ,ZEN(ZEISS),LASAF(Leica Microsystems)或用于处理荧光图像的类似软件

程序

由于制备的努力和有限的保质期,所需的化学品必须在不同的时间制备:

  1. 一些化学品可以提前准备并储存多达几个 个月。这些是1 M HEPES缓冲液,2M氯化钙,10%甘氨酸, 1%钌红,15%K 3 [Fe(CN)6],分级乙醇系列和高压灭菌 MilliQ水。
  2. 一些化学品应该更新鲜和 储存只有几个星期。这些是树脂组分A和B, ?2%的乙酸双氧铀,柠檬酸铅和3%的HNO 3
  3. 一些化学品 应尽可能新鲜。在活细胞成像当天 ?准备新鲜2x和1x固定剂。使用前一天开始准备 后固定剂,因为四氧化锇晶体需要一晚 解决(在通风橱中4℃下黑暗处理),并完成制备 的后固定剂在使用前不久通过添加添加剂。也 ?在使用当天制备EPON树脂(储存在-20℃, 在使用前平衡至RT),并立即制备丙酮-EPON混合物 ?使用前。

计划程序
下面说明从播种细胞到TEM成像的CLEM工作流程。


图1. CLEM工作流。将HeLa细胞接种在具有格栅盖玻片的MatTek培养皿中。一天后,可以用流体相标记物转染或脉冲追踪细胞。第二天,细胞被感染,成像并在显微镜载物台上尽可能快地固定。从培养皿中取出盖玻片,将样品加工成TEM并平坦地包埋在树脂中。在去除盖玻片期间,雕刻的坐标被转移到树脂表面并允许围绕ROI进行修整。将连续切片转移到EM网格并染色以获得对比度。相同的ROI在TEM中成像以覆盖LM图像
  1. 对于实验,将HeLa细胞在活细胞成像前两天接种到含有2ml细胞培养基的MatTek培养皿中,并在37℃,5%CO 2和90%湿度下温育。每个MatTek培养皿的细胞数应该是app。 4×10 5个细胞。这个相对低的单元号有利于网格上的单元的重新定位。重要的是,在显微镜的一天,细胞应该拥有感兴趣的结构(SOI)的荧光标记,以使活细胞图像与超微结构的相关性。因此,在显微镜检查之前必须对细胞进行特异性处理,这意味着用质粒转染用于表达标记的蛋白质,用流体相标记物脉冲追踪(Krieger等人,2014)或另一种标记方法。 br />
  2. 至少1小时前活细胞成像开关在显微镜和显微镜的孵化室达到所需的温度和大气在成像开始。启动成像软件并调整所有必需的设置,以避免活细胞成像之前的延迟。
  3. 将预先温热至37°C的新鲜制备的2x固定剂和设备(手套,移液管和定时器)尽可能靠近CLSM显微镜。
  4. 直接在细胞成像之前,用1ml成像介质代替细胞培养基,并将MatTek培养皿转移到预热的显微镜阶段。在成像前从MatTek皿中取出盖子,在完成最后一张图像之前不要触摸碟子
  5. 首先用100x物镜观察感兴趣区域(ROI),定义Z堆叠并开始采集。这些设置应该允许相当快的采集(取决于SOI的动态特性),因为快速的蜂窝过程,例如, 囊泡运动会使超微结构的相关性复杂化。一旦Z-堆栈完成(小于1分钟)添加1毫升温暖的2x固定剂到1毫升成像介质在盘中(比率1 + 1)和非常轻轻地通过吹吸上下混合,以固定细胞尽可能快地直接在显微镜载物台上。小心不要使用移液器吸头接触培养皿。启动定时器1小时。现在,在添加2x固定剂作为SOI的移动的控制之后,立即用100x物镜获得所有剩余的图像,即一个额外的Z堆叠。接下来生成概述图像,每个具有40×,20×和10×物镜,没有Z堆叠,用于在盘格上的ROI的定位以及树脂表面上的ROI的最终重新定位。一旦获得最后一个图像,关闭盖子,从显微镜中取出MatTek皿,并转移到通风橱。保持细胞温暖。


    图2.荧光显微镜和在盘网格上的ROI的定位。感兴趣的细胞被活体成像,并且通过CLSM获得Z-堆叠(A)。获取附加明场图像以用于可视化细胞(B)的形状和分布。使用该信息,感兴趣的细胞使用较低的放大倍数在餐具网格上重新定位,并记录MatTek培养皿的盖玻片上的雕刻坐标(在这种情况下是坐标"BK")(C)。高亮显示坐标"BK"(D)。

  6. 在通风橱下,将成像介质固定剂混合物用2ml新鲜的预热的1x固定剂代替,并将细胞在1小时的固定时间的剩余时间中孵育。在固定结束时,细胞应处于室温(RT)
  7. 用2ml 0.2M HEPES缓冲液冲洗细胞三次
  8. 孵育细胞与2毫升50mM甘氨酸的HEPES缓冲液15分钟,以阻止未反应的戊二醛。
  9. 用2ml 0.2M HEPES缓冲液冲洗细胞三次
  10. 将细胞在冰箱或冰上冷却至4℃,因为接下来的步骤必须在冷的条件下进行。
  11. 该步骤必须在通风橱下进行,因为非常有毒的四氧化锇!首先通过将钌红和六氰基铁酸钾(III)加入到溶解的四氧化锇晶体中来完成冷后固定剂的制备。接下来从冰冷的细胞中去除HEPES缓冲液,加入最少量的1.5 ml冰冷的后固定剂,并在通风橱中在黑暗中4°C孵育1小时。锇将使细胞变黑,以及MatTek培养皿和盖玻片之间的胶
  12. 用2ml冰冷的0.2M HEPES缓冲液冲洗细胞5次,每次2分钟(在通风橱中)。
    注意:此步骤也可能延长一晚。
  13. 从MatTak盘中取出带格栅的方形盖玻片。该步骤可以在脱水期间任何时候进行,但是脱水的温育时间是关键的并且应当遵守。准备填充了当前步骤的溶液(例如,0.2 M HEPES缓冲液)在冰上的10厘米塑料培养皿。将MatTek培养皿放在10厘米培养皿的液体中,轻轻地,但持续不断地插入手术刀锋利的边缘从盖玻片和塑料培养皿底部之间的内侧。随着手术刀在盖玻片和盘底之间更深地渗透,盖玻片开始剥落并落入缓冲液填充的10cm盘中。使用镊子,将盖玻片电池侧向上转移到一个较小的3.5厘米的塑料皿或类似的容器(丙酮抗性)已经充满当前步骤的溶液。在3.5cm皿内的所有以下溶液的体积应足够大以完全覆盖细胞。考虑乙醇和丙酮蒸发。细胞不应与空气接触或完全干燥。


    图3.从MatTek培养皿中取出盖玻片。使用手术刀,将粘在培养皿底部的方形盖玻片从MatTek培养皿(A)中取出,然后滴入缓冲液在冰上10厘米的盘子(B)
  14. 在通风橱中制备丙酮和EPON(3:1和1:3混合物)的混合物。该步骤可以在脱水期间在适当时进行,但应当最晚在样品处于丙酮中时完成。
  15. 从细胞中取出HEPES缓冲液,并在冷分级乙醇系列中脱水样品。详细地,对于每个步骤,将细胞与50%,70%,80%,90%,95%和100%乙醇孵育10分钟。通过将冷的金属块置于3.5cm培养皿下直至95%乙醇温育,使细胞保持冷却。从用100%乙醇孵育,除去金属块,并允许达到RT
  16. 在室温下将细胞在无水乙醇中孵育10分钟
  17. 在室温下将细胞在无水丙酮中孵育两次,每次10分钟。
  18. 孵育细胞在丙酮:EPON的3:1混合物在室温下1小时(在通风橱)
  19. 在室温下(在通风橱中)将细胞在丙酮:EPON的1:3混合物中孵育1小时。
  20. 孵育细胞在纯EPON过夜在室温(通风橱)。
  21. 第二天,将细胞在新鲜的纯EPON中在室温(通风橱中)温育4小时。同时,将用于EPON聚合的烘箱调节至60℃。烤箱也应在通风橱内。
  22. 固定一半明胶胶囊直立与开口和填充胶囊新鲜EPON。避免在EPON内部产生气泡。可以通过在炉中短时加热填充的胶囊来移除气泡。用钳子,从最后的EPON孵化,拿起方形盖玻片,保持直立大约1分钟,以允许EPON跑掉,将盖玻片单元格面朝下放在填充的胶囊。再次,避免气泡。最后将胶囊与盖玻片放在顶部用于聚合EPON在加热至60℃的烘箱中48-72小时。同时,所有被EPON污染的材料可以包裹在铝箔中,并放置在烘箱聚合中

    图4.EPON将细胞包埋在分离的盖玻片上。需要一个15ml Falcon管的盖子,一个大的明胶胶囊的一半,铝箔和包含细胞的盖玻片用于包埋(A) 。将明胶胶囊的一半直立放置,其开口面向上在Falcon管的盖内,并用铝箔(B)固定。胶囊填充有EPON(C)。在样品脱水和浸润后,将盖玻片以细胞侧向下放置在填充EPON的胶囊(D)上。

  23. EPON完全聚合后,将样品从烘箱中取出并放置冷却至室温
  24. 准备一个杜瓦瓶用液氮,烧杯用热水和大镊子。用镊子快速地将EPON块与盖玻片侧向下交替地浸入液氮和热水。玻璃和EPON的不同物理性能将导致盖玻片的分离。在从EPON块移除网格盖玻片期间,雕刻的栅格从盖玻片转移到EPON块表面。


    图5.从聚合的EPON块中去除盖玻片。将聚合的EPON块从烘箱和Falcon盖中取出用于冷却(A)。使用氮 - 热水法(B)从EPON块除去盖片
  25. 在立体显微镜下检查EPON块表面,以重新定位ROI。
    请注意,网格图案将被镜像传输。使用黑色永久标记标记投资回报率。
  26. 通过使用虎钳和精细的拼图粗略地去除过量的EPON并形成矩形EPON块(0.5×0.5×1.5cm),ROI在正方形表面的中间。小心不要损坏ROI的EPON表面,因为细胞直接在EPON表面下。
  27. 将矩形EPON块固定在超薄切片机的金属样品支架中,直到切片完全完成后才能取出。


    图6.粗修整的EPON块固定在金属支架中。重新定位的ROI在EPON表面上标记,EPON块被粗略修剪(A)。修剪的EPON块固定在金属样品架中,用于精确的修剪和切片(B)
  28. 在立体显微镜下,用双刃刀片手动修剪EPON块侧面(而不是表面),使得形成具有45°成角度侧面的棱锥和梯形中间具有ROI的梯形表面。梯形表面的底边应为大约500μm,并且绝对平行于相对边缘。同样,注意不要损坏ROI的EPON面。


    图7.修剪EPON块。用剃刀刀片(A)修剪EPON块,以便形成45°角的棱锥和梯形中间的ROI梯形表面(B)。

  29. 将金属样品架转移到配有金刚石刀的超薄切片机上。本质上,刀和梯形EPON表面应该在X和Y方向上尽可能完美地平行对准,因为这种对准对于由CLSM和TEM获得的图像的最终相关性是最重要的。不允许对EPON表面进行预切片,意味着面对。有关刀片对齐,切片和收集部分的更多详细信息,请参阅Hagler(2007),特别是第3.3.5部分。切割薄切片。
  30. 用超薄切片机获得修整的EPON块的70nm薄连续部分。注意不要丢失任何部分,因为每个部分,已经是第一部分,可能有重要的信息
  31. 收集浮动在金刚石刀储存器的水面上的具有形成为涂层的EM铜缝隙栅格的串联部分。节的顺序非常重要,应该记录。最多八个部分适合一个网格。格栅的格式涂覆可以在内部进行,但是涂覆的格栅也是可商购的。将网格放在网格存储框中。在关闭盒子之前,完全空气干燥网格。
  32. 为了获得TEM的对比度,使用自动染色装置用2%乙酸铀酰染色40分钟和柠檬酸铅30分钟染色所有负载的网格。对于该装置,也应该有3%的HNO 3和高压灭菌的水。按照制造商手册或一个也可以手动染色网格。记住,柠檬酸铅是CO 2敏感的,并遵循具体的方案,例如。 Ellis(2004)。
  33. 用在80kV下操作的TEM观察染色的部分,并且根据最终相关性需要在不同的放大倍率下获取图像。 CLSM图像应该在树脂部分的ROI的重新定位

    图8.EPON部分上的ROI的重新定位通过之前获得的CLSM图像(A),根据它们在EPON部分(B)上的取向和形态来重新定位细胞。

  34. 最后,使用Photoshop覆盖CLSM和TEM图像。如果需要,手动进行获得的TEM图像的缝合或使用ImageJ或Photoshop自动化,并使用显微镜专用软件或Imaris处理CLSM图像。图像处理包括降噪,对比度增强或颜色平衡。注意,图像处理应该根据适当的科学实践,并且对图像操作的良好概述可以在细胞生物学杂志( http://www.jcb.rupress.org/site/misc/ifora.xhtml )。

代表数据


图9.沙门氏菌的细胞内生活方式的CLEM分析的实施例将表达LAMP1-GFP(绿色)的HeLa细胞接种在具有格栅盖玻片的培养皿中, em>沙门氏菌表达mCherry(STM,红色)。在8小时后进行活细胞成像以显现LAMP1-GFP阳性SIF(A.最大强度投影[MIP],C.单Z平面)。随后,固定细胞并处理CLEM以显示所选细胞的超微结构。缝合几个低放大率图像以显现细胞形态(B)。使用更高放大率的图像来对准LM和TEM图像(D)。注意形成双膜LAMP1-GFP阳性SIF。显示了LAMP1-GFP阳性SIF(E)和与SIF(F,G)连接的SCV的细节。两个额外的超薄切片显示对应于图F的SCV和SIF的膜组织。H.SIF和SCV的内膜和外膜分别以橙色和黄色勾画。浅红色和深红色箭头分别表示沙门氏菌细胞包膜的内膜和外膜。标签:S,<沙门氏菌; M,线粒体; iL,内腔; oL,外腔。显示了代表10个生物重复的细胞(1-3个技术重复,每个2-4个细胞)。比例尺:10μm(A,B),2μm(C,D),500nm(E,F,G)。

笔记

  1. 小心有毒物质,如戊二醛,四氧化锇,EPON,乙酸双氧铀和柠檬酸铅。熟悉有关处理,个人防护以及正确处理废物的安全说明。
  2. 对于含有EPON的每种溶液,使用一次性容器。难以清洁受EPON污染的容器,因此使用一次性容器,其中EPON在60℃的烘箱中通过聚合中和至少48小时。
  3. 将该方案调整到在细胞微生物学中的研究,特别是HeLa细胞中沙门氏菌的细胞内活性。其他感染模型需要关于孵育时间,浓度,温度的优化
  4.  在实验之前准备尽可能新的关键解决方案

食谱

  1. 1 M HEPES缓冲液(500ml)
    1. 称量119.3g HEPES
    2. 加入高压灭菌MilliQ水至500 ml,并混匀
    3. 通过加入5M HCl调节pH 7.4
    4. 高压灭菌
    5. 缓冲液可在室温下储存几个月。
    6.  对于0.2 M溶液,用水稀释1:5
  2. 在H 2 O(10ml)中的2M CaCl 2溶液
    1. 称重2.22g CaCl 2
    2. 加入高压灭菌MilliQ水至10 ml,并混匀
    3. 高压灭菌
    4. 试剂可以在4°C储存几个月
  3. 10%甘氨酸的0.2M HEPES缓冲液(5ml)中
    1. 称量500mg甘氨酸
    2. 加入0.2 M HEPES至5 ml,并充分混匀
    3. 过滤灭菌
    4. 溶液可在4℃至多个月存储
    5. 对于50mM,将37.6μl10%溶液加入到1ml 0.2M HEPES缓冲液中
  4. 1%钌红(RR)在H 2 O(5ml)中的溶液 注意:吞咽有害。
    1. 称量50 mg RR
    2. 加入高压灭菌MilliQ水至5ml,并混匀
    3. 在Eppendorf管中等分500μl
    4. 等分试样可以储存在-20°C至多个月
  5. 15%六氰基铁酸钾(III)在H 2 O(5ml)中的溶液
    1. 称重750mg K 3 [Fe(CN)6]
    2. 加入高压灭菌MilliQ水至5ml,并混匀
    3. 在Eppendorf管中等分500μl
    4. 等分试样可以储存在-20°C至多个月
  6. 2x固定剂
    5%戊二醛+ 10mM CaCl 2在0.4M HEPES缓冲液(12.5ml)中的溶液。
    注意:有毒!作为特殊废物处理!
    1. 在暗玻璃烧瓶中装入4.938ml高压灭菌的MilliQ水,
    2. 加入5ml 1 M HEPES缓冲液(最终0.4M)
    3. 加入2.5ml的25%戊二醛(最终5%)
    4. 加入62.5μl来自2M CaCl 2(最终10mM)
    5. 试剂可以在4℃保存几天
    6. 对于1x固定剂,用水稀释1:2
  7. 后固定剂
    2%四氧化锇+ 0.1%钌红+ 1.5%六氰基铁酸钾(III)在0.2M HEPES缓冲液(5ml)中的溶液。
    注意:很有毒!作为特殊废物处理!
    1. 在暗玻璃烧瓶中转移来自0.1g OsO 4安瓿的完全内容物
    2. 加入3ml高压灭菌MilliQ水
    3. 加入1ml 1M HEPES缓冲液(终浓度:0.2M)
    4. 关闭烧瓶并用密封垫密封
    5. 将烧瓶在4℃在通风橱中储存过夜以溶解OsO 4晶体
    6. 第二天添加添加剂:0.5ml从1%钌红(最终 0.1%)和0.5ml来自15%的氰铁酸铁(III)(最终1.5%)
    7. 混合均匀,并立即在4°C使用
  8. EPON树脂
    注意:有毒!作为特殊废物处理!
    解决方案A
    1. 将31ml EPON 812与50ml DDSA在塑料容器中混合
    2. 用木棍搅拌
    3. 该溶液可以在-20℃下储存多达数周
    解决方案B
    1. 在塑料容器中混合50ml EPON 812与42.5ml MNA
    2. 用木棍搅拌
    3. 该溶液可以在-20℃下储存多达数周
    A-B混合
    1. 使用注射器
      将Sol A + B 1:1混合在塑料盘中
    2. 加入1.5vol .-%DMP-30(非常粘稠)
    3. 用木棍搅拌
    4. 试剂可以在-20°C储存多达几天
  9. 2%的H 2 O(100ml)中的乙酸双氧铀 注意:很有毒!化学毒性和放射性!作为特殊废物处理!
    1. 称量2g乙酸铀酰酯
    2. 将高压灭菌的MilliQ水加至100ml并用磁力搅拌器搅拌
    3. 用0.2μm硝化纤维素滤膜过滤溶液
    4. 转移到深色玻璃烧瓶中
    5. 试剂可以在4°C储存几个星期
  10. 根据Reynolds(100ml),柠檬酸铅 注意:很有毒!作为特殊废物处理!
    1. 称量2.66g硝酸铅和3.52g柠檬酸钠
    2. 加入60ml MilliQ水(高压灭菌,通过抽真空脱气),并在磁力搅拌器上搅拌10分钟
    3. 允许在室温下放置10分钟,间歇摇动,溶液将呈乳白色
    4. 加入11ml 1N NaOH(无碳酸盐)并混合,溶液将变澄清
    5. 通过滴加1N NaOH(无碳酸盐),测量pH并精确调节至pH 12.0
    6. 用约28ml MilliQ水填充至100ml(高压灭菌,脱气)
    7. 试剂可以在4°C储存几个星期
  11. 3%硝酸(HNO 3)(1L) 注意:酸性!
    1. 向954ml高压灭菌的MilliQ水中加入46ml 65%HNO 3
    2. 通过0.2μm硝化纤维素滤膜过滤溶液
    3. 该溶液可以在RT下储存多达几周

致谢

我们组的工作由DFG通过授权HE1964/18-1和SFB944"生理和动力学的细胞微隔室"的Z项目支持。

参考文献

  1. Ellis,E.A。(2014)。 透射电子显微镜的染色切片生物标本:常规和整体染色。 Methods Mol Biol 1117:57-72。
  2. Hagler,H.K。(2007)。 生物电子显微镜的超薄切片术 方法 369 :67-96。
  3. Krieger,V.,Liebl,D.,Zhang,Y.,Rajashekar,R.,Chlanda,P.,Giesker,K.,Chikkaballi,D.and Hensel,M.(2014)。 在沙门氏菌感染的细胞中重组内体系统:超微结构 10
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Liss, V. and Hensel, M. (2015). Sample Preparation for Correlative Light and Electron Microscopy (CLEM) Analyses in Cellular Microbiology. Bio-protocol 5(19): e1612. DOI: 10.21769/BioProtoc.1612.
  2. Krieger, V., Liebl, D., Zhang, Y., Rajashekar, R., Chlanda, P., Giesker, K., Chikkaballi, D. and Hensel, M. (2014). Reorganization of the endosomal system in Salmonella-infected cells: the ultrastructure of Salmonella-induced tubular compartments. PLoS Pathog 10(9): e1004374.
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