Buffers and fixers
The preparation of biological material for study by electron microscopy involves two phases: isolation and fixation. These phases require the use of a specific buffer, cacodylate buffer or phosphate buffer. If the cacodylate buffer was traditionally the most used, the phosphate buffer is also effective and much less dangerous for the handler.
Cacodylate buffer 0.1M pH 7.4
- Sodium cacodylate (CH3)2AsO2Na3H2O, mw 214.05: 2.14g in 50ml distilled water, to which 2.7ml 0.2N HCl is added. The 0.2M cacodylate buffer is diluted volume by volume in distilled water to obtain the 0.1M cacodylate buffer pH 7.2
0.1M phosphate buffer pH 7.2
- Monosodium phosphate 0.2M: NaH2PO4, 2H2O: pm= 156. Weigh 7.8g for 250ml
- Disodium phosphate 0.2M: Na2HPO4, 2H2O: pm= 178. Weigh 17.8g for 500ml
- The 0.2M phosphate buffer is obtained by mixing 11ml of 0.2M monosodium phosphate and 39ml of 0.2M disodium phosphate. It is diluted volume by volume in distilled water to obtain the 0.1M phosphate buffer pH 7.2.
The treatment of the cells or the removal of the tissue to be studied must be as rapid as possible in order to avoid degradation of the morphology.
The samples must be small: at most 1mm thick.
The area to be studied must be identified and isolated from the start of the process.
Viruses and isolated structures
The viral suspension, or a solution containing the structure to be studied (lysate of mitochondria, exosomes, etc.) is deposited for a few minutes on a specially prepared grid (covered with a film of Formwar on which we have sprayed carbon). The grid is dried on the edge. After three washes the grid is stained using negative staining techniques, and ready for observation. This technique also makes it possible to carry out immunological labels.
The study of isolated cells is not a problem if you follow a few simple rules:
- A minimum quantity: 1 to 10 million cells, it will depend on the size of the cells. The pellet must imperatively be at least one millimeter at the bottom of an eppendorf to be usable by electron microscopy, and pay attention to losses during washing. Some cells may stick to the walls.
- Be careful not to activate "sensitive" cells (case of blood platelets for example)
- Fixing takes place in at least 1ml of fixer, 1 hour at room temperature.
- 3 washes are necessary, per centrifugation cycle.
- In the case of cultured cells, it is essential to wash the cells in serum-free medium before fixation. Without this there is a risk of precipitation of proteins from the culture medium.
- The pellet should NEVER be dry.
The particular case of adherent cells can pose a problem: the methods of detachment, trypsin or scraping, are likely to damage the cells, as well as to reduce the labeling of the membrane. In this case there is no real good solution, and it is necessary to test the different protocols to find the least destructive. After detachment, the protocol is the same as for isolated cells.
There is a solution, which also makes it possible to respond to the problem of cells available in small quantities: the solution is depositing the cells on glass slides, and carrying out the inclusion on this support. The sample will then be included as a resin monolayer. This technique therefore makes it possible to study the morphology of rare cells and/or cells attached to a substrate (glass, plastic, other cells, filter) and to carry out labeling before inclusion.
You should also know that there is always the risk of losing cells as the inclusion progresses. It is therefore also important for this to have a good quantity of material at the start as well as to choose tubes which do not retain the cells.
The first key factor in tissue isolation is speed. The sooner the sample is in the fixative, the better the preservation of its ultrastructure will be.
The second important factor is sample size. For reasons inherent to the technique (in particular the nature of the chemical fixatives used and the size of the cutting and observation tools), the size is necessarily reduced for electron microscopy. In one of these dimensions the tissue fragment must be less than 1mm. In the case of too thick samples, the zones located in depth will not be fixed and the morphology will be strongly degraded.
The third point is to have a prior reflection on the purpose of the electron microscopy study. Indeed, the counterpart to a magnification which goes from X3000 to X80000 is a reduced observable zone on the surface. It will be possible to see the structure of the organelles, the state of the nucleus, the membranes, even large macromolecules, but impossible to study a sample at the tissue level. It is therefore necessary to think before the sampling of identifying the interesting zone to isolate it and thus facilitate and accelerate the study. In short, it's better to look for it now than at the time of the superfine cut.
One last point: in some cases, it may be interesting to consider infusing the whole animal with the fixative. Fragile and/or difficult to access tissues will thus be better preserved. This method is, for example, essential for the study of the brain, a heterogeneous, fragile and difficult to access organ.
- Determine in advance the area to be studied
- Collect the tissue
- If possible, cut the sample to give it the ideal size
- Leave it for one hour in the fixative at room temperature
- Wash three times five minutes in 0.1M phosphate buffer
- If immediate inclusion is not possible, the sample can be left overnight at 4°C in buffer.
Chemical fixation consists of artificially reconstituting the highly hydrated protein gel of living organisms (75% to 95% water) in order to make the molecules of a sample insoluble in water as in organic solvents, and thus block the enzymatic systems to avoid any subsequent structural modification of the sample. Indeed, the very fine scale at which observations are made by electron microscopy makes it necessary to preserve, without modification, the structures corresponding to the living state.
is a dialdehyde that quickly interacts with proteins. It stabilizes structures by creating artificial bridges between proteins and free amine groups. With two aldehyde groups separated by a flexible chain, HCO-(CH2)3-CHO, it has great potential for creating bridges, which can appear thanks to the two groups and at varying distances. Some substances such as glycogen or the extracellular matrix are thus preserved. Nevertheless, glutaraldehyde alone remains insufficient and it is necessary to carry out a second fixation with osmium tetroxide to avoid the extraction of certain components during dehydration (in particular lipids). This step should however be kept to a minimum, as osmium can lead to the extraction of certain proteins.
While glutaraldehyde is fast acting, it is a fairly slow penetrating fixative, 1mm per hour maximum (due to the still relatively large size of the oligomers). For some tissues, in particular the skin, it is necessary to fix samples with a thickness of less than 0.5 mm for two hours.
It is very important for our techniques to use an "EM grade" glutaraldehyde, which is composed only of monomers, the only ones small enough to quickly penetrate the tissues (the other formulas are intended for tanning leather!).
Fixing with glutaraldehyde can lead to certain difficulties:
- There is creation of free aldehyde groups (which cannot be removed by washing) which can cause non-specific binding of antibodies.
- The bridges induced by glutaraldehyde do not allow the penetration of the paraffin, it is mandatory to use plastic polymers, or resin, to achieve the inclusion.
- This binding considerably hinders immunological labeling which requires the greatest possible number of intact amino acid chains. The majority of enzymatic reactions are also destroyed, although some are still possible after rapid fixation.
It is actually an insoluble polymer of formaldehyde, which yields formaldehyde monomers when dissolved in buffer. With formula (CH2)n, it is on the other hand a fast penetrating fixative (thanks to the small molecules of HCHO), about 10 mm per hour. The aldehyde group -CHO combines with the nitrogen atoms of proteins. However, it takes longer to stabilize the structures than glutaraldehyde. Fixation is therefore less effective and the ultrastructure less well preserved for morphological observation.
A mixture of the two fixers is the best fixation in certain cases: large samples (knowing that in this case there will still be a lower quality fixation in the center of the sample), very dense sample (case of the skin) or need for further immunostaining. Paraformaldehyde quickly penetrates the tissues and slightly stabilizes proteins which are then permanently fixed by glutaraldehyde. The mixture most used in electron microscopy is associated with the name of Morris J. Karnovsky, the technique having been published first as an abstract not referenced (Karnovsky, 1965): 2.5% glutaraldehyde, 4% paraformaldehyde in 0.1M cacodylate buffer.
Morphology: resin inclusion
Future cuts will be subjected to fairly powerful electronic radiation. They must be both very resistant to overheating while remaining thin enough to allow the flow of electrons to pass. This aim is achieved thanks to the use of synthetic resins which, after polymerization, will become very rigid, with a minimum alteration of the structure of the biological material. The most used resins are araldite and epon. It is the latter that is used by the platform.
After fixation with glutaraldehyde and/or paraformaldehyde, the sample is carefully washed in buffer. Then, it is possible to perform a staining with uranyl acetate. If necessary (see staining section), washing will be done with distilled water.
The following steps consist of the dehydration of the sample, which is carried out in successive baths of 70%, 90% and then 100% ethanol. The sample is then immersed in two baths of propylene oxide, then in a prepolymer/propylene oxide mixture. The epon prepolymer is prepared in advance and stored at -20°C. In the case of inclusion in a plastic container, propylene oxide is replaced with ethanol.
Finally the material is left in the pure prepolymer, to be included in gelatin capsules. The capsules are left to polymerize for 24 hours (minimum) in an oven at 60°C.
The difficulty in electron microscopy is to find the balance between preservation of the morphology and protection of the antigenic sites, while producing ultrathin sections which resist the flow of electrons.
After fixation, the material is taken up in 10% gelatin, then cooled to 4°C. Thus included, it is left for at least one night (more for tissues) in the PVP-sucrose. Finally, cut to size, the sample is frozen in liquid nitrogen.
Sections are made on a Reichert Ultracut S ultramicrotome. Semi-fine sections are made to control the quality and orientation of the sample. They are then stained with toluidine blue and can be stored on a slide. Ultra-thin cuts are cut with a diamond knife (Diatome). They are 60 to 90 nm thick.
The cut takes place in a chamber surrounded by a tank of liquid nitrogen, which keeps the sample at -100°C. The sections thus produced must be used the next day at the latest. A "cryo" labeling experiment therefore takes two full days.
Consult us for:
- Simple labelings
- Double labelings.
- Labelings before inclusion (pre-embedding)
Observations et acquisitions
In the current state of the platform, the observation is carried out with the engineer in charge. This is a guarantee of speed and quality of acquisitions.
The objective of the observation is to answer the question posed, and then to preserve the data. A first observation will always take some time to get used to the appearance of transmission electron microscopy sections and their interpretation. Once the eye has been educated, and if there is a significant result, comes the shooting stage. It is then a matter of selecting the most useful and significant fields of observation to answer the question posed.
The observations are carried out using a GATAN eslangshen camera mounted in a 35mm port, therefore at wide angle which allows a wide field of vision. Thus it is possible to quickly scan the grid and to find the points of interest. In some cases we need a higher resolution. We have a GATAN Orius camera which makes it possible to mount in enlargements and to acquire small objects more easily.
The acquisitions are carried out using the Digital Micrograph software.
The acquisitions are in the form of image files in dm3 format. This is the Digital Micrograph software format, which can only be read by itself and an imageJ plug-in.
We therefore carry out on the platform directly after the acquisition a conversion of the images into tiff format. It is at this stage that the user recovers his data. These images are in indexed color, are approximately 3400 pixels by 2600 at a resolution of 72 pixels/inch. They weigh between 8.9 and 10.4 MB depending on the camera used. We suggest users to keep these images that way, and work on converted grayscale, jpeg copies if needed.
Don't forget, if you want to reduce the size of the images in photoshop or the gimp, to increase the resolution at the same time or risk ending up with a few pixels at the end. For example, to send photos by e-mail, we suggest the following maneuver: Change the image to gray level, increase the resolution to 300 pixels/inch and at the same time decrease the size to 20 x 15 cm. Finally, save in jpeg format.
We ensure the conservation of all acquisitions made on the platform, for the moment for 3 years. However, due to the absence of the possibility of a network backup, we absolutely cannot guarantee this backup. Each user is therefore responsible for backing up their data once they have recovered it.