TEM is used to give high-resolution information at molecular, cell, and tissue levels e.g. interactions between intracellular organelles, changes in cellular architecture after genetic mutation, localization of nanoparticles after intracellular uptake. Specimen preparation for TEM involves chemical fixation, dehydration embedding and cutting of 70 nm thin sections that are contrasted with heavy metals before viewing in the electron microscope.
Transmission Electron Microscopy (TEM)
Transmission electron micrograph of Zebra fish muscle (specimen courtesy of Dr J Leslie)
High pressure freezing/freeze substitution (HPF/FS)
Specimens that contain a thick cell wall are not amenable to chemical fixation instead a process of high pressure freezing is used to fix and immobilise the material, followed by substitution of the specimen ice at low temperatures and embedding either at room or at low temperatures. These procedures are typically used for C elegans, yeasts and plant material but are increasingly used for cell biology studies since they provide material closest to the living state.
Section showing nerve tissue in C.Elegans (specimen courtesy Of Dr Q Ch’ng)
Small, very thin specimens can be viewed with minimal preparation using the technique of negative staining in which small droplets of the sample are placed on an EM grid and exposed to a heavy metal stain that outlines the structure of interest thus avoiding extensive preparation procedures. This method is used for studying the structure of isolated molecules and virus preparations.
Virus particles imaged using negative staining (specimen Courtesy Professor M Linden)
Electron Energy loss spectroscopy/Energy Filtered Transmission Electron Microscopy (EELS/EFTEM)
Electrons that have interacted with the specimen to generate X-rays lose energy dependent on the atomic number of the atoms involved. In the technique of EELs the transmitted electrons are collected to give a spectrum that show ‘edges’ where energy loss has occurred that give information about the element content of the specimen. In EFTEM, the image is formed by collecting only those electrons that have interacted with a specific element. EELs/ EFTEM / and EPXMA are complementary techniques used for detecting and mapping the presence of nanoparticles within cells and for the study of cell physiology.
Publications: see e.g Morris et al (2011) J Neurochemistry 116, 617-667
EFTEM image of iron oxide nanoparticles within lysosomes
TEM micrographs give a 2-D representation of a 3-D specimen and even in a thin section detail is lost in the z-direction. Using TEM tomography a series of images is taken at different tilt angles and realigned to give the equivalent of a confocal stack from which 3D information about structural interrelations are reconstructed. This technique is increasingly being used to study 3D interrelationships beween intracellular organelles.
A 3-D computer model showing part of the intercalated disc of heart tissue. Courtesy Amanda Wilson and Pauline Bennet.
Scanning Electron Microscopy (SEM)
In SEM a focused electron beam is scanned across the surface of the specimen eliciting the emission of secondary electrons that give an image showing topographical detail. SEM is used to provide images from low to very high magnifications, the using a wide range of different specimens e.g individual cells, pharmaceutical compounds, dental materials. EPXMA is also available in the SEM allowing elemental characterisation of area of interest.
Mouse bronchial epithelium imaged by SEMv
Environmental Scanning Electron Microscopy (ESEM)
In SEM specimens are usually prepared by fixation followed by critical point drying, and coating with gold, these procedures however are unsuitable for delicate surface structures. Instead the technique of ESEM in which the specimen is examined at a pressure close to ambient and in the presence of water vapour can be used, allowing examination of the specimen in a close to native state. This technique is used for the study of cell/matrix interactions in tissue engineering, pharmaceutical compounds, watery structures such as mucins, and for the study of botanical specimens.
Image of the surface of a flower
Cryo-scanning electron microscopy (cryoSEM)
In cryoSEM specimens are fixed by rapid freezing followed by fracturing before examination in the SEM. This method reveals the details of internal structures e.g. the starch granules in the sea kale root, without the shrinkage caused by normal dehydration steps. This technique is used for the study of intracellular structures, lipid micelles, mucins, cell matrix interactions in tissue engineering.
Cellular components in fractured sea kale root
Immunogold labelling uses colloidal gold coupled to a secondary antibody as a highly dense reporter system to detect the localisation of specific epitopes at the EM level. This technique complements fluorescent labelling by localising immune reactions to specific intracellular structures at a resolution that cannot be achieved with fluorescence and is mandatory to prove co-localisation of antigens. The technique is also used at SEM level to provide details about the surface distribution of antigens.
Publications: Smeele MS et al. Circulation Research 2011; 108: 1165-1169
Labelling of KATP channels in mitochondria