Conventional TEM sample processing: ultrastructure
Biological samples like cells and tissues must be properly fixed, embedded in a resin for sectioning, and contrasted with heavy metals prior to TEM imaging. Beginning with chemically fixed specimens, this sample processing service includes washing, secondary fixation, en bloc staining, dehydration, epoxy resin infiltration, embedding and curing. The resultant sample-containing resin blocks can be further sectioned with ultramicrotome for EM imaging (sectioning is provided as a separate service).
Conventional TEM sample processing: immuno-EM
Like the fluorescent probes used for protein localization in light microscopy, antibody-conjugated colloidal gold particles are used to label the antigens in electron microscopy with higher resolution. The immunolabeling procedure is conducted on thin sections from samples embedded in LR White resin. The core processes chemically fixed samples into thin sections on Nickel grids for researchers to perform their customized immunolabelling (sectioning is provided as a separate service).
High pressure freezing-Freeze substitution: ultrastructure
Although chemicals like glutaraldehyde and paraformaldehyde have been used for decades to fix biological samples, this process is associated with artefacts such as structural swelling and distortion. Alternatively, high pressure freezing (HPF) can freeze samples in msecs and preserve them in a “life-like state”. The “frozen” water in samples is then gradually substituted by organic solutions containing fixatives and staining agents. After freeze substitution (FS), samples are gradually warmed up to room temperature and further processed for resin embedding and sectioning as the conventional sample preparation (sectioning is provided as a separate service). Results from HPF-FS show superior preservation of fine structures compared to conventional sample processing.
High pressure freezing-Freeze substitution: immuno-EM
Similar to the HPF-FS method for ultrastructure study, biological samples are cryo-fixed by HPF, and gradually substituted in organic solutions. Notably, this FS cocktail is designed for immuno-EM to better preserve the antigens. Samples are then infiltrated, embedded, and cured with Lowicryl resin at cryogenic temperatures to further improve the preservation of antigens. The cured blocks are gradually warmed up to room temperature for sectioning (sectioning is provided as a separate service).
Negative-stain TEM is a simple and rapid method to study thin specimens such as viruses, bacteria, exosomes, isolated organelles, and macromolecules. In this method, electron-dense chemicals such as uranyl acetate and phosphotungstic acid are used to stain specimen-adsorbed grids, contrasting the unstained specimen with a dark background.
SEM sample processing
Prior to SEM imaging, biological samples must be fully dehydrated and applied with a thin conductive coating. To best preserve the surface structures, a state-of-the-art method, critical point drying, is used to dehydrate biological samples. In this method, samples are immersed in liquid CO2 followed by CO2 evaporation at its critical point, where physical characteristics of liquid and gaseous are not distinguishable. Therefore, it avoids damages induced by surface tension when changing from liquid to gaseous state. Next, the dehydrated sample is coated with either carbon or metal for conductivity.
Volume EM sample processing
Volume EM allows researchers to visualize a sample slice by slice and create a 3D reconstruction with high resolution. Common ways include focused ion beam -SEM (FIB-SEM), serial block face -SEM (SBF-SEM), and array tomography. The core provides services to process the fixed biological samples to resin blocks for volume EM. The overall procedure is similar to the conventional TEM sample processing but using different protocols for contrasting and embedding. Before the volume EM, the resin blocks can be further trimmed to expose the sample cross-section or confined to a specific area.
Correlative Light and Electron Microscopy
Correlative light and electron microscopy (CLEM) is a technique that combines the information acquired from light (LM) and electron microscopy (EM) on the same area of interest from a sample. LM enables multi-color labelling for live and fixed samples in aqueous environment while its spatial resolution is limited. It can be complemented by the high-resolution ultrastructural visualization using EM when studying the same location. This superposition of LM and EM provides a more comprehensive and straightforward information for a biological process.
Thick, thin and cryogenic sectioning
Sectioning of sample-containing resin blocks is indispensable so that samples are thin enough for TEM visualization. Regular TEM resin sectioning is performed at room temperature using diamond knives to generate sections with thickness of 50-90 nm. Thick sections (>150 nm) can also be performed to accommodate specific project requests. In addition, sectioning at cryogenic temperatures is available. For example, sucrose and gelatin infiltrated samples can be sectioned at ~-110˚C for Tokuyasu method.