"Living" samples cannot, in general, be imaged with electron microscopy. The beam of electrons used to image the sample cannot travel in air, the electrons would hit the air molecules and scatter. Electron microscopes are thus built to contain a vacuum, through which the beam is projected and focused using electro-magnets as lenses. The sample has to sit in the vacuum in order for imaging to take place. Biological tissues and vacuums do not mix at all well, the 80-90% water content would simply evaporate into the vacuum, and so we need to prepare the sample in order to prevent this by either removing the water or freezing the water in place. When removing the water, all the components of the cells and tissue are moved from the positions they would be in a living state. This is undesirable, our interest lies in understanding living and dynamic events, and so we use a process called fixation to halt the movement of the sample down to the molecular level, preserving the structure of the specimen as close to the living state as is possible.
There are two ways to fix samples, using freezing or chemical cross-linking to prevent activity. Freezing is very fast but has its own problems and is not suitable for all types of biological sample. Chemical fixation is much slower and thus some changes in the sample occur during the course of fixation.
In addition to fixation there are other preparation techniques needed before you can put the sample into the electron microscope and image it (including dehydration, embedding, sectioning and staining, for transmission electron microscopy). As a result, it can take several days of work before you can finally image your sample. This is one of the most frustrating aspects of electron microscopy.
At each step of the preparation procedure, something can go wrong. You cannot control all variables and even with very stringent attention to the details you can control, things can still go wrong very easily. It is fiddly (you are dealing with very small samples) and until you finally get a chance to look at your samples, you never really know how well it is going to turn out. Days of work and preparation finally result in a sample that does not answer your questions or just looks awful. Well, I had one of those experiences this morning, turning an exciting moment into one of mild disappointment. It is a constant challenge, even when you think you have all the techniques under your belt! So time to reset, make up new chemicals, get new samples, and spend the next few days trying again. At least we always have something to keep us busy and boredom is a rare occurrence in this line of work.