Electron microscopy

Specimen is fixed with either a mixture of 4 % paraformaldehyde and 1 % glutaraldehyde in 0,1 M phosphate buffer or 2 % glutaraldehyde in 0,1 M phosphate buffer, post-fixed with osmium tetroxide, dehydrated using increasing concentration of acetone or alcohol, and embedded in plastic resin (Epon). Ultrathin sections (70-80 nm) are post-stained with uranyl acetate and lead citrate before observation with TEM.

One of the best available methods of biological sample preparation for electron microscopy as regards to structural preservation is cryoimmobilization by high-pressure freezing (HPF) followed by freeze-substitution (FS). This method enables the observation of cells and cellular organelles in close-to-native state without the artefacts (extraction, coagulation, structural distortion) which are frequently observed when chemical fixation is used. A fresh cell or tissue sample is loaded together with filler or cryoprotectant on a sample holder and instantly frozen under very high pressure (2000 bar) using HPF machine. Freezing is followed by freeze substitution, which is done at temperature below -70°C, where water (in form of ice) in sample is replaced by organic solvent in presence of secondary fixative. After substitution samples can be embedded in plastic or rehydrated and processed by Tokuyasu technique for IEM.

For the localization of molecules at the ultrastructural level we apply Tokuyasu cryosectioning technique. Fresh specimen is fixed in 4 % paraformaldehyde in 0,1 M phosphate buffer, immersed in 2,3 M sucrose in PBS and frozen in liquid nitrogen. Thin cryosections are labeled with specific antibodies and conjugated gold markers are used to visualize antibody binding sites.

Simple and rapid technique to analyze detailed structure of macromolecules, isolated organelles, and microorganisms by surrounding or embedding sample particles in an electron dense material that provides high contrast. A small droplet of sample is adsorbed on a carbon coated and glow-discharged grid. After excess sample is removed, grid is rinsed few times on a droplet of distilled water and placed on a droplet of stain (1% uranyl acetate or uranyl formate). After staining excess fluid is withdrawn and grid is dried before observation with TEM. It is also possible to combine immunostaining with negative staining. In that case antibody incubations are done prior the negative stain is applied.

Electron microscopic tomography is a technique that enables the examination of three-dimensional biological structures. Thin specimen is tilted over a large angular range (typically ± 70°) around the tilt axis of goniometer stage and imaged at each tilt angle (usually at intervals of 1 or 2°). Obtained image set called tilt series is then processed to align images with each other and subsequently to reconstruct a three-dimensional volume. This volume is visualized as an image stack showing parallel slices through the volume. Direct volume visualization can be informative for some structures and analytical purposes. However, segmentation methods are often used with organelle structures for defining and dissecting components of the structure and to create a three-dimensional model which facilitates interpretation, the discovery of interrelationships, and measurement.

With this method it is possible to obtain series of images through conventionally prepared resin embedded tissue block. Aligned high resolution image series can then be used for ultrastructural volume reconstruction and 3D-analysis of cellular structures. In the dual beam electron microscope the focused gallium ion beam and electron source are arranged at an angle which allows the layer-by-layer removal of material from the specimen surface and subsequent imaging with scanning electron beam. Process is fully automated and once suitable area is located and milling and imaging parameters are set, instrument will automatically produce nearly aligned image series.

Vascular corrosion casting is an excellent tool for morphological examination of organ and tissue microcirculation under normal and pathological conditions. In short, mice or rats are perfused and blood is replaced with a low viscosity resin. After resin has cured, KOH maceration is used to remove the surrounding tissue and to reveal the replicate of vasculature. Due to polyurethane-based resin (PU4ii) casts are highly elastic and the shape and volume of organs are preserved. Casts are normally imaged using SEM but can be further processed for analysis using confocal microscopy (resin is autofluorescent), micro-computed tomography (microCT), or optical projection tomography (OPT), which allows the production of 3D reconstructions for quantitative morphometric analysis.

To register for an account:

To get started, you must register for an account:

1. Navigate to the core page: https://oulu.ilab.agilent.com/service_center/show_external/3635
2. In the upper-right-hand corner of the screen select the ‘Register using University of Oulu credentials’ option.
3. You will be directed to an authentication page where you will need to select your University and enter your HAKA credentials.
4. Once you have entered your credentials, click the ‘Login’ button
5. You will be directed to an iLab Registration page where you will need to select your PI/Lab and verify your contact information.
6. Once your registration has been submitted, your PI will receive a notification that you have requested membership to their lab in iLab. They will need to approve your membership and assign any Cost centers for your use.

To Create an Equipment Reservation:

Once you have been accepted into your PI’s lab and assigned Cost centers, you can schedule equipment time.

1. Navigate to the core page: https://oulu.ilab.agilent.com/service_center/show_external/3635
2. At the upper right hand of the page click ‘Sign in’ and then select the ‘Sign in using HAKA credentials’ option.
3. Enter your HAKA username and password, and sign in.
4. Select the Schedule Equipment tab and click on the ‘View Schedule’ button next to the instrument of interest. Click and drag on the time frame you would like to schedule your reservation for.
5. A window will pop up that will allow you to verify your reservations details and provide payment information before saving the reservation.

To Create a Service Request:

Once you have been accepted into your PI’s lab and assigned Cost centers, you can create service requests.

1. Navigate to the core page: https://oulu.ilab.agilent.com/service_center/show_external/3635
2. At the upper right hand of the page ‘Register using University of Oulu credentials’ option.
3. Enter your HAKA username and password, and sign in.
4. Select the Request Services tab and click on the ‘Request Service’ button next to the service of interest.
5. You will be asked to complete a form before submitting the request to the core.
6. Your request will be pending review by the core. The core will review your request and either Agree to the work or they will ask for more information if needed.

Instructions:

1. Click here to log-in: https://oulu.ilab.agilent.com/service_center/show_external/3635
2. You will use your HAKA credentials to log into iLab
3. Once logged in, look for the link in the left-hand menu that says 'my labs'. Hover-over and select your lab.
4. Set the auto-approval amount if you do not wish to approve service requests below a certain dollar amount. To do this, select the 'Members' panel and enter a dollar amount in the 'Auto Pre-Approval' amount and click 'save settings.’
5. To approve lab membership requests, select the ‘Membership Requests & Cost centers’ tab. New membership requests will show at the top of this page. Click “Approve” to accept a member into your lab. Click “Reject” if they are not a member of your lab.

Electron microscopes:

Tecnai G2 Spirit 120 kV TEM with Veleta and Quemesa CCD cameras (BF-funding)

Sigma HD VP FE-SEM equipped with ET-SE and In-lens SE detectors, VPSE G3 detector for low vacuum mode, and 5Q-BSD detector

Location – The Main Building of Medical Campus, Aapistie 5A

(Tecnai room 467B, Sigma FE-SEM room 320A)



tel: +358-(0)294 486144, int. 48-6144 (Tecnai)

Lab is equipped with modern instruments for the preparation of cells, organelles, and tissue samples for various EM analyses.

Equipment:

• Leica EM UC7 ultramicrotome with EM FC7 cryo attachment and EM CRION ionizer
• Leica EM UC6 ultramicrotome with EM FC6 cryo attachment
• Leica ultracut UCT ultramicrotome
• Leica ultracut E
• EM-Pact high pressure freezing device (Leica)
• Leica EM AFS2 freeze-substitution device with automatic liquid handling system (BF-funding)
• Leica EM TP automated tissue processor
• K850 Critical Point Dryer
• Q150T ES Instrument with interchangeable inserts for sputter coating or carbon evaporation equipped with film thickness monitor and glow discharge insert

Location – EM laboratory, Biocenter Oulu laboratories in the Main Building of Medical Campus, Aapistie 5A

(room 488B, 4th floor)

tel: +358-(0)294 486144, int. 48-6144

Prices for academic groups

BCO-TIC would greatly appreciate if the electron microscopy core facility is acknowledged in the publications when services are provided.

1. TEM specimen fixative: 1 % glutaraldehyde, 4 % formaldehyde in 0.1M phosphate buffer (pH 7.4).
   • Fresh fixative is available in the cold room 480B. Bring your own tube!
   • Fixation time: 12 hours at room temperature, after which samples can be stored at +4° C in the fixative.
   • When taking the sample it is IMPORTANT to
     1. Cut the tissue quickly from the organ and put it immediately into the fixative.
     2. Further cut the tissue to 2 mm^3 small pieces and transfer to the final fixative tube.
     3. Always take several samples (3-4), if possible. Pieces from same sample should be put to same tube !!
     4. The amount of fixative must be at least 10 times the volume of the tissue.
     5. Samples are stored and brought / sent to EM lab in a fixative.

2. IEM specimen fixative: 4 % paraformaldehyde in 0.1 M phosphate buffer with 2.5 % sucrose.
   • Fixation time: 2-6 hours or overnight at room temperature. Fixation time depends on the sensitivity of the antigen and needs to be determined experimentally.
   • When taking the sample it is IMPORTANT to
     1. Cut the tissue quickly from the organ and put it immediately into the fixative.
     2. Further cut the tissue to 2 mm^3 small pieces and transfer to the final fixative tube.
     3. Always take several samples (3-4), if possible.
     4. The amount of fixative must be at least 10 times the volume of the tissue.
   • After fixation tissue pieces are transferred to 2.3 M sucrose in PBS, rotated for 4 hours at +4°C and stored at +4°C.
   • Samples are brought / sent to EM lab in sucrose (preferably at +4°C).
   • Frozen fixative and sucrose can be obtained from the EM lab (488B).

PROTOCOL FOR TEM

1. TEM specimen fixative: 1 % glutaraldehyde, 4 % formaldehyde in 0.1M phosphate buffer or 2 % glutaraldehyde in 0.1M phosphate buffer

• Cells grown on plastic Petri dish (enough cells for at least 2 mm^3 pellet):
  1. Remove cell culture media and add fixative (at room temperature). NOTE! If cytoskeletal structures are studied, fixation temperature can be even +37 °C.
  2. After 10 min detach cells carefully with a cell scraper and continue fixation for 1 hour maximum.
  3. Centrifuge cells to a pellet (6000 rpm, 2 min) and leave the pellet in Eppendorf tube in a fixative.
• Cells grown in suspension culture:
  1. Add double concentrated fixative 1:1 to suspension.
  2. After 10 min centrifuge (2000 rpm) remove the fixative and add fresh fixative (normal strength).
  3. After 1 hour fixation centrifuge as above and leave the pellet in a fixative. Cell pellet can be stored in a fixative at +4 °C.
• Bring the cells to EM lab in a fixative.

PROTOCOL FOR IEM

2. IEM specimen fixative: 4 % paraformaldehyde in 0.1 M phosphate buffer with 2.5 % sucrose

• Cells grown on a culture dish (enough cells for at least 2 mm^3 pellet):
  1. Remove cell culture media and using a Pasteur pipette put the fixative gently on cells.
  2. After 10 min, detach cells carefully with cell scraper and continue fixation for 1 hour (depending on the sensitivity of the antigen).
  3. After fixation transfer cells to the Eppendorf tubes and centrifuge (6000 rpm, 2 min). At this point you can bring samples to EM lab (contact lab personnel beforehand)
  4. Remove the fixative and rinse the pellet twice with PBS.
  5. Put a drop of 12 % gelatine in PBS (37°C) on the pellet, loosen pellet very gently with a tip and incubate for 10 min at 37° C.
  6. Centrifuge at RT for 5 min (13 000 rpm).
  7. Put tubes on the ice for 30 min,
  8. Remove the gelatin pellet e.g. by cutting the tip of the Eppendorf tube and cut the pellet to small pieces.
  9. Transfer pieces to 2.3 M sucrose in PBS at 4°C and rotate for 4 hours at 4°C.
  10. Keep specimens in sucrose at 4°C and bring to EM lab.
• Frozen fixative and sucrose can be obtained from the EM lab (488B).

PROTOCOL FOR TEM

• TEM specimen fixative: 1 % glutaraldehyde, 4 % formaldehyde in 0.1M phosphate buffer (pH 7.4).
• Double concentrated fixative: 2 % glutaraldehyde, 8 % formaldehyde in 0.1M phosphate buffer (pH 7.4).
  1. Add double concentrated fixative 1:1 to suspension.
  2. After 10 min spun organelles into pellet, remove fixative and add fresh fixative (normal strength).
  3. After 1 hour fixation centrifuge as above and leave the pellet in a fixative.
  4. Samples can be stored at +4°C.
• For shipping it is best to embed samples in agarose.
  1. Wash the pellet with PBS 3 x 10 min.
  2. Wash the pellet with water 2 x 10 min.
  3. Embed pellet in 2.5 % agarose.
     • Pipet a drop of agarose (2.5% in water, temperature around 37°C) on top of pellet. Estimate the volume according to the size of your pellet.
     • Carefully dislodge the pellet so that it floats within agarose.
     • Cool agarose on ice
  4. Remove the agarose pellet e.g. by cutting the tip of the Eppendorf tube and trim agarose smaller. Organelle pellet should be surrounded by agarose.
  5. Store agarose pellet in the fixative.
• Bring / sent samples to EM lab in the fixative at RT. Make sure tubes fully filled with fixative, carefully closed, and lids are secured with parafilm.

PROTOCOL FOR IEM

• IEM specimen fixative: 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) with 2.5 % sucrose.
• Double concentrated IEM fixative: 8 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) with 2.5 % sucrose.
  1. Fix organelles in suspension by adding double concentrated fixative.
  2. After 10 min, pellet organelles, resuspend in fresh fixative (4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) with 2.5 % sucrose) and fix for 1-4 hrs (depending on the sensitivity of the antigen).
  3. After fixation pellet organelles in Eppendorf tubes, remove the fixative and rinse the pellet twice with PBS.
  4. Immerse pellet in a drop of 12 % gelatine in PBS (37°C), loosen pellet very gently with a tip and incubate for 10 min at 37°C.
  5. Centrifuge at RT for 5 min (13 000 rpm).
  6. Put tubes on the ice for 30 min.
  7. Remove the gelatin pellet e.g. by cutting the tip of the Eppendorf tube and trim gelatin smaller. Organelle pellet should be surrounded by gelatin.
  8. Transfer to 2.3 M sucrose in PBS at 4°C and rotate for 4 hours at 4°C.
  9. Keep specimens in sucrose at 4°C.
• Bring / sent samples to EM lab in sucrose at RT. Make sure tubes fully filled with sucrose, carefully closed, and lids are secured with parafilm.