Transmission Electron Microscopy (TEM) Methods
Specialised methods
Conventional resin-embedding
Biological samples can be fixed chemically and embedded in a resin of choice. After initial aldehyde-fixation, the specimen will be post-fixed and dehydrated in an ethanol gradient before embedment in 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
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
Immunogold-labelling with colloidal gold is a technique used for the localisation of antigenic sites at the ultrastructural level. Combined with the quantitation of densities of gold per cellular compartment, this allows for the molecular mapping of epitopes with nanometre resolution.
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 our cryo-ultramicrotome. Thawed cryo-sections will then be labelled using a primary antibody of choice and the antibody localisation can 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.
Negative staining
Freeze substitution is a process applied to samples fixed cryogenically, to replace water with a resin in 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 high-pressure freezer. We utilise either a quick freeze substitution method1 or a freeze substitution unit.
References:
- K.L. McDonald and R.I. Webb, Freeze substitution in 3 hours or less. (2011). Journal of Microscopy. 243(3), 227-233.
Correlative Light and Electron Microscopy (CLEM)
CLEM enables the analysis of a specimen with both, light as well as electron microscopy. This method allows for the selection of rare events or structural features in complex, heterogeneous samples by fluorescent microscopy, followed by the ultrastructural analysis of the same sample and region of interest using an electron microscope. 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. Another approach utilises protocols to sustain the fluorophores of a sample during the sample preparation for EM. The fluorescent data can then be acquired from resin sections and directly overlaid with the EM image1.
References:
- Hamied et al., Identification and Characterization of Highly Fluorescent Pigment Cells in Embryos of the Arabian Killifish (Aphanius Dispar). (2020). iScience. 23, 101674.
Stereology
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.
References:
- Hacker et al., Ultrastructural Analysis and Quantification of Peroxisome-Organelle Contacts. (2023). Methods in Molecular Biology. Volume 2643.
- Lucocq J and Hacker C., Cutting a fine figure: One the use of thin sections in electron microscopy to quantify autophagy. (2013). Autophagy. 9(9).
- Lucocq J et al., Systems biology in 3D space – enter the morpheme. (2014). Trends in Cell Biology. 25(2).
Array tomography
Array tomography (AT) is a non-destructive, 3D electron microscopy technique, providing ultrastructural details of selected region of interest. The volumetric imaging is achieved this through semi-automated SEM scanning of consecutive ultrathin sections, each approximately 100 nm thick. The resulting series of 2D images, with a pixel resolution down to 5 nm, is aligned to create a 3D digital image of the region of interest.?
Samples, including monolayers, suspensions, and tissues are prepared in the Bioimaging Centre using heavy-metal staining protocols embedded into the epoxy resins. These samples are then cut into thin sections that are collected onto the conductive coverslips. The imaging is performed in the Zeiss Gemini 500 SEM using specialised software designed for automated imaging of serial sections.?