To reveal the three-dimensional (3D) structure of thin samples, electron tomography (ET) is the method of choice. ET employs the transmission electron microscope to collect projections of an object that is tilted in multiple directions and uses these projections to reconstruct the object in its entirety.
Electron tomography became feasible with the advent of computer-controlled stages on the newer model TEMs, more reliable CCD cameras and the development of powerful analytical software for the creation and annotation of 3D tomograms. With resolutions of 50 Å and higher, 3D reconstructions obtained by ET provide precise information about the spatial organization of macromolecules in their cellular context.
References:
Electron Tomography Methods for Three-Dimensional Visualization of Structures in the Cell Editors: Frank, Joachim (Ed.) 2006. Springer
Fernandez de Castro et al., J. Cell Sci. 2017.
TEM
Transmission electron microscopy (TEM) involves a high voltage electron beam emitted by a cathode and formed by magnetic lenses. The electron beam that has been partially transmitted through the very thin (and so semitransparent for electrons) specimen carries information about the structure of the specimen. The spatial variation in this information (the "image") is then magnified by a series of magnetic lenses until it is recorded by hitting a fluorescent screen, photographic plate, or light sensitive sensor such as a CCD (charge-coupled device) camera. The image detected by the CCD may be displayed in real time on a monitor or computer. Transmission electron microscopes produce two-dimensional, black and white images.
A Transmission Electron Microscope has magnification and resolution capabilities that are over a thousand times beyond that offered by the light microscope. With the introduction of aberration-corrected electron lenses, both the spatial resolution and the image quality in transmission electron microscopy have been significantly improved and resolution below 0.5 ångströms has been demonstrated. In the life sciences, it is still mainly the specimen preparation which limits the resolution of what we can see in the electron microscope, rather than the microscope itself. TEM continues to play a critical role in discovering and describing new organisms, especially viruses.
Bozzola JJ, Russell LD (1999). Electron Biology Principles and techniques for Biologist, Sudbury, MA: Jones & Bartlett Learning.
Confocal microscopy is an optical imaging technique for increasing optical resolution and contrast by means of adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light. It provides the capacity for direct, noninvasive, serial optical sectioning of intact, thick, living specimens with a minimum of sample preparation as well as a marginal improvement in lateral resolution.
Biological samples are often treated with antibodies conjugated with fluorescent dyes to make selected molecules visible. Also, transgenic techniques can create organisms that produce their own fluorescent chimeric molecules (such as a fusion of GFP, green fluorescent protein with the protein of interest).
Three-dimensional studies are also done with this technique.
Molecular Mapping
Methods for in situ molecular mapping
With in situ molecular mapping techniques we can explore the particular functions developed by cell structures and how macromolecular complexes build organelles and cells. In the lab we mainly work with two groups of methods: immunogold labeling (on plastic resin sections or Tokuyasu cryosections) and labeling with clonable tags for electron microscopy and CLEM (correlative light and electron microscopy).
- Immunogold labeling on Tokuyasu cryosections. The Tokuyasu technique is mainly used for immunohistochemistry. This technique avoids dehydration and infiltration with a plastic resin. Samples are chemically fixed with aldehydes, cryo-protected with sucrose and frozen to harden them before thin sectioning. Thin sections are then thawed at room temperature and transferred to formvar/carbon-coated grids. The sections can be labelled with any affinity probe and a probe linked to colloidal gold before embedding in a mixture of uranyl acetate and methylcellulose that provides contrast and protection during drying. This technique yields an excellent visualization of membranes together with an optimal preservation and labelling of macromolecules (I. Hurbain & M. Sachse, Biology of the Cell, 2011).
- Clonable tags for EM and CLEM. METTEM (Metal Tagging Transmission Electron Microscopy) is a method developed in our laboratory to detect proteins in cells with high sensitivity. The procedure uses the small metal-binding protein metallothionein (MT) as a clonable tag. When fused with a protein of interest and treated in vivo with gold salts a single MT tag will build an electron-dense gold nanocluster ~1 nm in diameter, easily detectable by transmissible electron microscopy. When proteins are double tagged with green fluorescent protein (GFP) and MT, direct correlative light and electron microscopy allows visualization of the same macromolecular complexes with different spatial resolutions (I. Fernández de Castro, L. Sanz & C. Risco, Methods in Cell Biology 2014).
Immunogold Labeling
Immunogold is a labeling technique used in electron microscopy. This method follows the same patterns of the Indirect immunofluorescence. Colloidal gold particles are most often attached to secondary antibodies or protein A which are in turn attached to primary antibodies designed to bind a specific antigen or other cell component. Gold is used for its high electron density which increases electron scatter to give high contrast 'dark spots'. The labeling technique can be adapted to distinguish multiple objects by using differently-sized gold particles.
J.J.Bozzola and L.D. Russell Eletron Microscopy: Principles and Techniques for Biologist. (Ed.) 1999 Jones and Barlett Publishers
Electron Tomography
To reveal the three-dimensional (3D) structure of thin samples, electron tomography (ET) is the method of choice. ET employs the transmission electron microscope to collect projections of an object that is tilted in multiple directions and uses these projections to reconstruct the object in its entirety.
Electron tomography became feasible with the advent of computer-controlled stages on the newer model TEMs, more reliable CCD cameras and the development of powerful analytical software for the creation and annotation of 3D tomograms. With resolutions of 50 Å and higher, 3D reconstructions obtained by ET provide precise information about the spatial organization of macromolecules in their cellular context.
References:
Electron Tomography
Methods for Three-Dimensional Visualization of Structures in the Cell
Editors: Frank, Joachim (Ed.) 2006. Springer
Fernandez de Castro et al., J. Cell Sci. 2017.
Transmission electron microscopy (TEM) involves a high voltage electron beam emitted by a cathode and formed by magnetic lenses. The electron beam that has been partially transmitted through the very thin (and so semitransparent for electrons) specimen carries information about the structure of the specimen. The spatial variation in this information (the "image") is then magnified by a series of magnetic lenses until it is recorded by hitting a fluorescent screen, photographic plate, or light sensitive sensor such as a CCD (charge-coupled device) camera. The image detected by the CCD may be displayed in real time on a monitor or computer. Transmission electron microscopes produce two-dimensional, black and white images.
A Transmission Electron Microscope has magnification and resolution capabilities that are over a thousand times beyond that offered by the light microscope. With the introduction of aberration-corrected electron lenses, both the spatial resolution and the image quality in transmission electron microscopy have been significantly improved and resolution below 0.5 ångströms has been demonstrated. In the life sciences, it is still mainly the specimen preparation which limits the resolution of what we can see in the electron microscope, rather than the microscope itself. TEM continues to play a critical role in discovering and describing new organisms, especially viruses.
Bozzola JJ, Russell LD (1999). Electron Biology Principles and techniques for Biologist, Sudbury, MA: Jones & Bartlett Learning.
http://www.nature.com/nprot/journal/v2/n10/full/nprot.2007.304.html
Confocal microscopy is an optical imaging technique for increasing optical resolution and contrast by means of adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light. It provides the capacity for direct, noninvasive, serial optical sectioning of intact, thick, living specimens with a minimum of sample preparation as well as a marginal improvement in lateral resolution.
Biological samples are often treated with antibodies conjugated with fluorescent dyes to make selected molecules visible. Also, transgenic techniques can create organisms that produce their own fluorescent chimeric molecules (such as a fusion of GFP, green fluorescent protein with the protein of interest).
Three-dimensional studies are also done with this technique.
Methods for in situ molecular mapping
With in situ molecular mapping techniques we can explore the particular functions developed by cell structures and how macromolecular complexes build organelles and cells. In the lab we mainly work with two groups of methods: immunogold labeling (on plastic resin sections or Tokuyasu cryosections) and labeling with clonable tags for electron microscopy and CLEM (correlative light and electron microscopy).
- Immunogold labeling on Tokuyasu cryosections. The Tokuyasu technique is mainly used for immunohistochemistry. This technique avoids dehydration and infiltration with a plastic resin. Samples are chemically fixed with aldehydes, cryo-protected with sucrose and frozen to harden them before thin sectioning. Thin sections are then thawed at room temperature and transferred to formvar/carbon-coated grids. The sections can be labelled with any affinity probe and a probe linked to colloidal gold before embedding in a mixture of uranyl acetate and methylcellulose that provides contrast and protection during drying. This technique yields an excellent visualization of membranes together with an optimal preservation and labelling of macromolecules (I. Hurbain & M. Sachse, Biology of the Cell, 2011).
- Clonable tags for EM and CLEM. METTEM (Metal Tagging Transmission Electron Microscopy) is a method developed in our laboratory to detect proteins in cells with high sensitivity. The procedure uses the small metal-binding protein metallothionein (MT) as a clonable tag. When fused with a protein of interest and treated in vivo with gold salts a single MT tag will build an electron-dense gold nanocluster ~1 nm in diameter, easily detectable by transmissible electron microscopy. When proteins are double tagged with green fluorescent protein (GFP) and MT, direct correlative light and electron microscopy allows visualization of the same macromolecular complexes with different spatial resolutions (I. Fernández de Castro, L. Sanz & C. Risco, Methods in Cell Biology 2014).
Immunogold is a labeling technique used in electron microscopy. This method follows the same patterns of the Indirect immunofluorescence. Colloidal gold particles are most often attached to secondary antibodies or protein A which are in turn attached to primary antibodies designed to bind a specific antigen or other cell component. Gold is used for its high electron density which increases electron scatter to give high contrast 'dark spots'. The labeling technique can be adapted to distinguish multiple objects by using differently-sized gold particles.
J.J.Bozzola and L.D. Russell Eletron Microscopy: Principles and Techniques for Biologist. (Ed.) 1999 Jones and Barlett Publishers