Workflow 6: Room temperature EM of resin-embedded sections

To prepare biological material for room temperature imaging in the EM, samples are typically fixed, dehydrated and embedded in a resin. Biological material can either be fixed chemically or by rapid freezing. For bulk biological material, high-pressure freezing is typically the fixation method of choice as it allows rapid freezing of large sample volumes and prevents artefacts caused by chemical fixation. For rapid freezing, we utilise a Leica EM ICE high pressure freezer. Frozen samples are then freeze substituted, i.e. gradually dehydrated, embedded in resin and subsequently sectioned to produce ultrathin sections thin enough to be imaged in the TEM, by either projection imaging or tomography. For freeze substitution, we use a Leica EM AFS2 with an FSP automated reagent handling system. Resin-embedded RT-EM is a powerful method to observe large morphological changes that do not require ultra-high resolution.

Go to Editor View