Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Podsumowanie

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

Streszczenie

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

Wprowadzenie

Mitochondria and endoplasmic reticulum (ER) are membrane-bound cellular organelles. Their connection is necessary for their function, and proteins related to their network have been described¹. The distance between the mitochondria and ER has been reported as approximately 100 nm using light microscopy²; however, recent super-resolution microscopy³ and electron microscopy (EM)⁴ studies have revealed it to be considerably smaller, at approximately 10-25 nm. The resolution achieved in super-resolution microscopy is lower than EM, and specific labeling is necessary. EM is a suitable technique to attain a sufficiently high-resolution contrast for structural studies of the connections between mitochondria and ER. However, a disadvantage is the limited z-axis information because the thin sections must be approximately 60 nm or thinner for conventional transmission electron microscopy (TEM). For sufficient EM z-axis imaging, three-dimensional electron microscopy (3DEM) can be used⁵. However, this involves the preparation of hundreds of thin serial sections of whole cells, which is very tricky work that only a few skilled technologists have mastered. These thin sections are collected on fragile formvar film-coated one-hole TEM grids. If the film breaks on one gird, serial imaging and volume reconstruction is not possible. Serial block-face scanning electron microscopy (SBEM) is a popular technique for 3DEM that uses destructive en bloc sectioning inside the scanning electron microscope (SEM) vacuum chamber with either a diamond knife (Dik-SBEM) or a focused ion beam (FIB-SEM)⁶. However, because those techniques are not available at all facilities, we suggest array tomography⁷ using serial sectioning and SEM. In array tomography, serial sections cut using an ultramicrotome are transferred to a glass coverslip instead of a TEM grid and visualized via light microscopy and SEM⁸. To enhance the signal for backscatter electron (BSE) imaging, we utilized an en bloc EM staining protocol employing osmium tetroxide (OsO₄)-fixed cells with osmiophilic thiocarbohydrazide (TCH)⁹, enabling us to obtain images without post-embedding double staining.

Additionally, the mitochondrial marker SCO1 (cytochrome c oxidase assembly protein 1)– ascorbate peroxidase 2 (APEX2)¹⁰ molecular tag was used to visualize mitochondria at the EM level. APEX2 is approximately 28 kDa and is derived from soybean ascorbate peroxidase¹¹. It was developed to show the detailed location of specific proteins at the EM level in the same way that green fluorescent protein-tagged protein is used in light microscopy. APEX2 converts 3,3' -diaminobenzidine (DAB) into an insoluble osmiophilic polymer at the site of the tag in the presence of the cofactor hydrogen peroxide (H₂O₂). APEX2 can be used as an alternative to traditional antibody labeling in EM, with a protein localization throughout the depth of the entire cell. In other words, the APEX2-tagged protein can be visualized by specific osmication¹¹ without immunogold labeling and permeabilization after ultra-cryosectioning. Horseradish peroxidase (HRP) is also a sensitive tag that catalyzes the H₂O₂-dependent polymerization of DAB into a localized precipitate, providing EM contrast after treatment with OsO₄. The ER target peptide sequence HRP-KDEL (lys-asp-glu-leu)¹² was applied to visualize ER within a whole cell. To evaluate our protocol of utilizing genetic tags and en bloc staining with reduced osmium and TCH (rOTO method), using the osmication effect at the same time, we compared the membrane contrast with and without the use of each genetic tag in rOTO en bloc staining. Although 3DEM with array tomography and DAB staining with APEX and HRP have, respectively, been utilized for other purposes, our protocol is unique because we have combined array tomography for 3DEM and DAB staining for mitochondria and ER labeling. Specifically, we showed five cells with and without APEX-tagged genes in the same section, which aided in investigating the effect of the genetic modification on cells.

Protokół

1. Cell culture with patterned grid culture dish and cell transfection with SCO1-APEX2 and HRP-KDEL plasmid vector

1. Seed 1 x 10⁵ HEK293T cells by placing them into 35-mm glass grid-bottomed culture dishes in a humidified atmosphere containing 5% CO₂ at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin.
2. The day after seeding the cells, when they have grown to 50%-60% confluency, introduce the SCO1-APEX2¹⁰ and HRP-KDEL¹² plasmid to the cells using transfection reagent according to the manufacturer’s instructions (SCO1-APEX2 cDNA 0.5 µg + HRP-KDEL plasmid DNA 0.5 µg per 3 µL transfection reagent).

2. Light microscopy of cells growing on patterned culture dishes and DAB staining for APEX2 and HRP

1. At 16-24 h after transfection, remove all the culture media and immediately add 250 µL of warm (30-37 °C) fixation solution (Table 1) by gentle pipetting. Immediately remove the fixation solution and replace it with 1.5 mL of fresh fixation solution. Incubate on ice for 60 min, and then wash three times for 10 min each in 1 mL of ice-cold 0.1 M sodium cacodylate buffer (Table 1).
   CAUTION: Aldehyde fumes are extremely toxic. Perform all work under a ventilated fume hood.
2. Add 1 mL of cold (0-4 °C) 20 mM glycine solution and incubate for 10 min on ice followed by three washes of 5 min each in 1 mL of cold 0.1 M sodium cacodylate buffer.
3. Prepare a fresh 1x DAB solution (3.33 mL of 0.3 M cacodylate solution + 10 µL of 30% H₂O₂ + 5.67 mL of cold water + 1 mL 10x DAB solution).
4. Add 500 µL of the freshly prepared 1x DAB solution (step 2.3) and incubate on ice for approximately 5-45 min until a light brown stain is visible under an inverted light microscope (Figure 1A).
5. Gently remove the DAB solution and rinse three times with 1 mL of cold 0.1 M sodium cacodylate buffer for 10 min each.
6. Use a phase-contrast inverted microscope (or a bright-field light microscope) to visualize the DAB staining at a magnification of 100x or higher. Use a marker pen to mark the bottom of the glass where the region of interest (ROI) is located (Figure 1B,C).

3. Sample preparation for the EM block

1. Perform cell culture and DAB staining as described in steps 2.1-2.6.
2. Post-fix the samples with 1 mL of 2% reduced OsO₄ for 1 h at 4 °C.
   CAUTION: OsO₄ fumes are highly toxic. Perform all work under a ventilated fume hood.
3. Prepare a new TCH solution (Table 1) during step 3.2 and pass through a 0.22-µm filter.
   CAUTION: TCH fumes are highly toxic. Perform all work under a ventilated fume hood.
4. Remove the fixative and rinse three times with 1 mL of distilled water for 5 min each at room temperature (RT).
5. Place the cells in 1 mL of previously prepared and filtered TCH solution for 20 min at RT.
6. Rinse the cells three times with 1 mL of distilled water for 5 min each at RT.
7. Expose the cells a second time to 1 mL of 2% osmium tetroxide in distilled water for 30 min at RT.
8. Remove the fixative and rinse three times with 1 mL of distilled water for 5 min each at RT. Add 1 mL of 1% uranyl acetate (aqueous), and leave overnight at 4 °C in the dark.
9. Wash the cells three times in 1 mL of distilled water for 5 min each at RT.
10. Pre-warm Walton’s lead aspartate solution (Table 1) in an oven at 60 °C for 30 min.
11. Stain the cells with Walton’s lead aspartate solution by adding 1 mL of the pre-warmed lead aspartate solution, and then place in an oven for 30 min at 60 °C.
12. Rinse the cells three times with 1 mL of distilled water for 5 min each at RT.
13. Incubate in a graded series of 2-mL ethanol aliquots (50%, 60%, 70%, 80%, 90%, 95%, 100%, 100%) for 20 min each at RT.
14. Decant the ethanol and incubate for 30 min in 1 mL of 3:1 ethanol:low-viscosity embedding mixture medium at RT.
15. Remove the medium and add 1 mL of 1:1 ethanol:low-viscosity embedding mixture medium. Incubate for 30 min at RT.
16. Remove the medium and add 1 mL of 1:3 ethanol:low-viscosity embedding mixture medium. Incubate for 30 min at RT.
17. Remove the medium and add 1 mL of 100% low-viscosity embedding medium and incubate overnight.
18. Embed the sample in 100% low-viscosity embedding mixture and incubate for 24 h at 60 °C.
19. Prepare 90-nm thick sections using an ultramicrotome.
20. Observe the grid under TEM at 200 kV.

4. Serial sectioning and mounting on indium-tin-oxide coated coverslips for SEM imaging

1. Substrate preparation
   1. Clean indium-tin-oxide (ITO)-coated glass coverslips (22 mm x 22 mm) by gentle agitation in isopropanol for 30-60 s.
   2. Remove the coverslips, drain off the excess isopropanol, and leave in a dust-free environment until dry.
   3. Treat the ITO-coated glass coverslips by glow discharge using a plasma coater for 1 min.
      NOTE: Plasma activating confers a hydrophilic property on the substrate surface. It creates a very thin film of water on the substrate to prevent wrinkle formation in the sections when the section is attached to the substrate.
   4. Insert the ITO-coated glass coverslips into the substrate holder, and place into the knife boat.
2. Trimming of the sample block and serial sectioning
   1. Insert the sample block into the sample holder of the ultramicrotome and set into the trimming block.
   2. Use a razor blade to trim away all excess resin around the target position (identified in step 2.6, Figure 1D-G). The shape of the block face should be trapezoid or rectangular. The leading edge and trailing edge must be absolutely parallel (Figure 1H,I).
   3. Insert the sample holder on the arm of the ultramicrotome and place the diamond knife in the knife holder. Insert the ITO glass coverslips into the ribbon carrier and clamp the carrier with the handle (Figure 1J). Set the ribbon carrier into the knife boat and fill the knife boat with filtered distilled water (Figure 1K).
   4. Adjust the carrier position with the slide of the knife by carefully pushing the handle of the holder to set the edge of the ITO glass close to the knife (Figure 1L).
   5. After cutting the section, stop the sectioning process, and slowly open the clamping screw of the tube and drain the water boat (flow rate of one drop of water per second).
   6. After completing the ribbon-collection process, remove the substrate with the handle of the clamping device and dry the ribbon (Figure 1M).

5. Imaging in the SEM and alignment of the SEM image stack

1. Mount the ITO-coated coverslip on aluminum stubs with sticky carbon tape. Seal the glass surface and the surface in the stub with sticky carbon tape, and then coat with a 10-nm thick carbon layer (Figure 1N).
2. Observe the ITO-coated coverslip in a field emission SEM at a low acceleration voltage of 5 kV and a suitable working distance for the efficient collection of BSEs.
3. Import the serial images into the Image J software (Fiji)¹³ using the virtual stack option. Open a new TrakEM¹⁴, and import the image stack into TrakEM. Click the right-mouse button and choose the align menu.
4. Then select the image range (from the first image to the last image). Finish the auto-alignment, save the aligned dataset, and choose export to compile a flat image from the selected image range (from the first image to the last image). Finally, save the flat image data in AVI format in the Image J main menu.
   NOTE: Supplemental Movie 1 and Supplemental Movie 2 show the SEM image stack and cropped image stack, respectively.

6. Segmentation of mitochondria and ER from serial images

1. Start the 3dmod in IMOD¹⁵ software and open image files.
2. In the ZaP window, draw the contour of the mitochondria and ER using middle-mouse button.
3. To visualize the segmented volume, open the Model View window (Supplemental Movie 3).

Wyniki

Figure 1 describes the schedule and workflow for this protocol. The protocol requires 7 days; however, depending on the time spent on SEM imaging, this may increase. For cell transfection, the confluency of the cells should be controlled so as not to cover the bottom of the entire grid plate (Figure 1A). A high cell density could prevent the identification of the cell of interest during light microscope and EM observation. We used genetically tagged plasmids that expressed APEX2 and HRP ...

Dyskusje

Determining the cellular localization of specific proteins at a nanometer resolution using EM is crucial to understand the cellular functions of proteins. Generally, there are two techniques to study the localization of a target protein via EM. One is the immunogold technique, which has been used in EM since 1960, and the other is a technique using recently developed genetically encoded tags¹⁶. Traditional immunogold techniques have employed antibody-conjugated gold particles or quantum dots to sh...

Materiały

Name	Company	Catalog Number	Comments
Glutaraldehyde	EMS	16200	Use only in fume hood
Paraformaldehyde	EMS	19210	Use only in fume hood
Sodium cacodylate	EMS	12300
Osmium tetroxide 4 % aqueous solution	EMS	16320	Use only in fume hood
Epon 812	EMS	14120	EMbed 812- 20 ml/ DDSA- 16 ml/ NMA- 8 ml/ DMP-30 - 0.8 ml
Ultra-microtome Leica ARTOS 3D	Leica	ARTOS 3D
Uranyl acetate	EMS	22400	Hazardous chemical
Lead citrate	EMS	17900
35mm Gridded coverslip dish	Mattek	P35G-1.5-14-CGRD
Glow discharger	Pelco	easiGlow
Formvar carbon coated Copper Grid	Ted Pella	01805-F
Hydrochloric acid	SIGMA	258148
Fugene HD	Promega	E2311
Glycine	SIGMA	G8898
3,3′ -diaminobenzamidine (DAB)	SIGMA	D8001	Hazardous chemical
30% Hydrogen peroxide solution	Merck	107210
Potassium hexacyanoferrate(II) trihydrate	SIGMA	P3289
0.22 um syringe filter	Sartorius	16534
Thiocarbonyldihydrazide	SIGMA	223220	Use only in fume hood
Potassium hydroxide	Fluka	10193426
L-aspartic acid	SIGMA	A9256
Ethanol	Merck	100983
Transmission electron microscopy	FEI	Tecnai G2
Indium tin oxide (ITO) coated glass coverslips	SPI	06489-AB	fragil glass
Isopropanol	Fisher Bioreagents	BP2618-1
Diamond knife	Leica	AT-4
Inveted light microscopy	Nikon	ECLipse TS100
Scanning electron microscopy	Zeiss	Auriga