Electron Microscopy Facility - Techniques
1. Bright field Image: The bright field imaging mode is the most common mode of operation for TEM where the image is formed by the direct interaction of the beam with the specimen. Mass-thickness and diffraction contrast contribute to image formation in this mode and thus thick areas in which heavy atoms are enriched, and crystalline areas appear dark.
2.Cryo TEM: Cryo TEM imaging is a technique that can be applied to unique objects such as flexible large protein complexes, irregular viruses,organells and small cells for imaging at cryogenic temperatures. Specimens are preserved in a near native, 'frozen-hydrated' state by vitrification process using a dedicated plunge freezing setup.
3. Tomography: Tomograghy technique is used to obtain detailed 3D structures of sub-cellular macro-molecular objects, on the basis of multiple 2D projection images of a 3D objects, obtained over a wide range of veiwing directions (+75° to -75°)
Secondary Electron Imaging:- This mode provides high-resolution imaging of fine surface morphology. Inelastic electron scattering caused by the interaction between the sample's electrons and the incident electrons results in the emission of low-energy electrons from near the sample's surface. The topography of surface features influences the number of electrons that reach the secondary electron detector from any point on the scanned surface.
Backscatter Electron Imaging:- This mode provides image contrast as a function of elemental composition, as well as, surface topography. Backscattered electrons are produced by the elastic interactions between the sample and the incident electron beam. These high-energy electrons can escape from much deeper than secondary electrons, so surface topography is not as accurately resolved as for secondary electron imaging.
Variable Pressure SEM:- By and large, SEM requires an electrically-conductive sample or continuous conductive surface film to allow incident electrons to be conducted away from the sample surface to ground. If electrons accumulate on a nonconductive surface, the charge buildup causes a divergence of the electron beam and degrades the SEM image. In variable-pressure SEM, imaging can be performed on a nonconductive sample when the chamber pressure is maintained at a level where most of the electrons reach the sample surface, Some air is allowed into the sample chamber, and the interaction between the electron beam and the air molecules creates a cloud of positive ions around the electron beam. These ions will neutralize the negative charge from electrons collecting on the surface of a nonconductive material to reduce the charging. Variable pressure SEM is also valuable for examination of samples that are not compatible with high vacuum.
EDAX Technique:-The EDS technique detects x-rays emitted from the sample during bombardment by an electron beam to characterize the elemental composition of the analyzed volume.
Cryo SEM imaging Technique:- Cryo-method has the great advantage wherein the material is so rapidly frozen that vulnerable biological structures are well preserved and thus when using cryo-fracture the material breaks clean along weak edges (e.g. membranes) without causing much malformation to the sample structure. Additionally this technique can be used for observing ‘difficult’ samples, such as those with greater beam sensitivity and of an unstable nature. Soluble materials are retained in this technique. There is almost no exposure to toxic reagents during sample preparation.
Inlens-duo imaging technique:- The Inlens-duo detector allow simultaneous imaging and mixing of a high contrast topography as well as clear compositional contrast .
Scanning transmission electron microscopy (STEM):- STEM combines the principles of transmission electron microscopy and scanning electron microscopy . The STEM technique scans a very finely focused beam of electrons across the sample in a raster pattern. As in the SEM, secondary or backscattered electrons can be used for imaging in STEM; but higher signal levels and better spatial resolution are available by detecting transmitted electrons.
1. Ultramicrotomy is a method for cutting specimens into ultra-thin sections, that can be studied and documented at different magnifications in a transmission electron microscope(TEM) or scanning transmission electron microscope(STEM). It is used mostly for biological specimens, but sections of plastics and soft metals can also be prepared. For best resolutions, sections should be from 30 to 70 nm.
2. Cryo ultramicrotomy, is a technique similar to room temperature ultramicrotomy but carried out at freezing temperatures between −70° and −120°C. Cryo ultramicrotomy can be used to cut ultra-thin frozen biological specimens. One of the advantages over the more "traditional" ultramicrotomy process is speed, as it is possible to prepare, freeze and section a specimen faster than resin embedded method.
3. Serial Section on Silicon Wafers for Ultra-Structural Volume Imaging of Cells and Tissues for 3D reconstruction in FESEM, High resolution, three-dimensional (3D) representations of cellular ultra structure are essential for structure function studies in all areas of cell biology. While serial section imaging using transmission electron microscopy can reveal limited subcellular volumes, complete ultra structural reconstructions of large volumes,entire cells or even tissue are difficult to achieve using TEM. We have successfully introduced a new attachment for serial sectioning of tissue for our existing ultramicrotome. Ribbons containing hundreds of 150 nm-200 nm thick sections can be generated, lined on a silicon wafer and imaged serially to obtain 3D ultrastructure of large volumes with high resolution using the combination of imaging techniques in the Zeiss FEG SEM.