Biological samples can be fixed chemically and embedded in the resin of choice. After initial aldehyde-fixation the specimen will be post-fixed and then dehydrated in an ethanol gradient before embedment in a resin. The polymerised samples can then be sectioned using an ultramicrotome (typically section-thickness < 100 nm) and the ultrastructure can be contrasted utilising heavy metal stains (uranyl acetate and/or lead citrate) before imaging.

Samples can be fixed upon arrival to the Bioimaging Centre to lead to embedded material typically within 4 days.

Negative staining enables to rapidly contrast nanoscale specimen (e.g. virus particles, liposomes, isolated organelles etc.) using a heavy metal stain. After adhering the sample onto an EM grid the bound particles will be stained using electron dense heavy metals (e.g. uranyl acetate or phosphotungstic acid) and can be imaged immediately after drying.

Immunogold-labelling with colloidal gold is a technique used for the localisation of antigenic sites at the ultrastructural level.

In the Tokuyasu-method for cryo-sectioning (Tokuyasu, 1973) we use sucrose-embedded biological samples which have been plunge-frozen in liquid nitrogen to create amorphous, vitreous, ice. We can obtain ultrathin sections of that frozen material using the RMC LN Ultra microtome. Thawed cryo-sections will then be labelled using a primary antibody of choice and the antibody localisation can then be visualised in a secondary step using a gold colloid which binds to the primary antibody. The electron-dense gold particles can be easily identified in the transmission electron microscope and enable the quantitation of the gold label in relation to the compartments of interest.

Alternatively, biological specimen can be embedded in a resin that allows for the preservation of the antigenicity, e.g. Lowicryl resins or LR White.

References:

Tokuyasu, K.T. (1973) A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol 57: 551–565.

This technique allows to replace water with a resin in freeze-fixed biological specimen at a temperature low enough to avoid the formation of destructive ice crystals. The idea is to preserve the ultrastructure of a biological sample close to the “native” state by avoiding chemical fixation and to circumvent the damaging effects observed after ambient temperature dehydration. In our facility we have the possibility to perform FS on samples frozen with a Jet Propane system for further EM analysis.

CLEM enables the acquisition of dynamic as well as ultrastructural information about the specimen of interest. This method allows for the selection of rare events or structural features in complex, heterogeneous samples. We are currently exploring various methods to combine light and electron microscopy. One way is to image specimens that adhere to a surface containing a finder grid. After life cell imaging the sample will be fixed and the finder grid can be used to locate the region of interest after processing the sample for electron microscopy and analysis/quantitation at the ultrastructural level can be achieved.

We use well established stereological tools to obtain unbiased estimates of cellular features like sizes, volumes or numbers of organelles and particles. In an initial step a rigorous sampling scheme ensures unbiased selection of the areas/locations of interest. In most cases this sampling scheme is carried out by a systematic uniform random placement throughout the specimen which gives every location an equal chance of being included in the sample. In a second step we apply stereological estimators in form of geometrical probes onto the sampled micrographs/locations and count “events” between those probes and the underlying ultrastructural features. Depending on the desired parameter these probes can be points, lines or planes. An example would be the estimation of the area of an organelle by point counting. A regular array of equally spaced points is used to count points that land over the areas of interest. The number of points multiplied by the area associated with one of those points on the grid lattice will be an estimate of profile area of that organelle.

For 3D quantitation we analyse consecutive serial sections to obtain data about organelle volumes and number.