BACHELOR OF SECONDARY EDUCATIONCENTRAL BICOL STATE UNIVERSITY OF AGRICULTURE
INTRODUCTIONElectron Microscopy involves the study of different specimens by using an electron microscope. Electron microscopes are scientific instruments that use a beam of energetic electrons to examine objects on a very fine scale.Through the use of an electron microscope we can see things that we would not normally be able to see with our naked eyes and has greater magnification than light microscope. This required 10,000x plus magnification which was not possible using current optical microscopes
TYPES OF ELECTRON MICROSCOPE1. TRANSMISSION ELECTRON MICROSCOPE- form images using electrons that are transmitted through a specimen.2. SCANNING ELECTRON MICROSCOPE- utilize electron that have bounced off the surface of the specimen.
The transmission electron microscope (TEM) was the first typeof Electron Microscope to be developed and is patterned exactly onthe light transmission microscope except that a focused beam ofelectrons is used instead of light to "see through" the specimen. Itwas developed by Max Knoll and Ernst Ruska in Germany in 1931.
TRANSMISSION ELECTRON MICROSCOPY (TEM) Transmission electron microscopy (TEM) is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transmitted through the specimen; the image is magnified and focused onto an imaging device, such as a fluorescent screen, on a layer of photographic film, or to be detected by a sensor such as a CCD camera.
DIFFERENCES BETWEEN OM AND EM OPTICAL MICROSCOPE ELECTRON MICROSCOPE1. The source of light. 1. The light source is replaced by a beam of2. The specimen. very fast moving electrons.3. The lenses that makes the 2. The specimen usually has to be specially specimen seem bigger. prepared and held inside a vacuum4. The magnified image of the chamber from which the air has been specimen that you see. pumped out (because electrons do not travel very far in air). 3. The lenses are replaced by a series of coil- shaped electromagnets through which the electron beam travels. 4. The image is formed as a photograph (called an electron micrograph) or as an image on a TV screen.
DIAGRAM TO REPRESENT TEM’S WORKING Virtual Source First Condenser Lens Second Condenser Lens Condenser Aperture Sample Objective Lens Objective ApertureSelected Area Aperture First Intermediate Lens Second Intermediate Lens Projector LensMain Screen (Phosphor)
DIFFERENT COMPONENTS OF TEM 1. HIGH TENSION CABLE2. ELECTRON EMITTER 3. STEPPER MOTORS FOR CENTERING THE ELECTRON BEAM4. CONDENSER 11. VACUUM PUMP LEADS5. APERTURE CONTROLS 12. GONIOMETER 6. SPECIMEN HOLDER 13. VACUUM AND 7. OBJECTIVE LENS MAGNIFICATION CONTROL 8. PROJECTOR LENS 14. FOCUSING CONTROL 9. OPTICAL LENS 10. FLUORESCENT SCREEN
TEM SAMPLE PREPARATION Cleaning the surface of the specimen The proper cleaning of the surface of the sample is important because the surface can contain a variety of unwanted deposits, such as dust, silt, and detritus, media components, or other contaminants. The best way to clean the surface of specimen from contaminants is to carefully rinse them three times for 10 min in 0.1 M cacodylic acid buffer (pH 7.3) at room temperature.
TEM SAMPLE PREPARATION Primary fixation of the specimen Fixation can be achieved by perfusion and microinjection, immersions, or with vapours using various fixatives including aldehydes(glutaraldehyde), osmium tetroxide,, tannic acid, or thiocarbohydrazide. FIXATIVES- are chemicals that denature and precipitate cellular macromolecules.
Some common fixatives:1. GLUTARALDEHYDE- is a 5-carbon with an aldehyde group at each end of the molecule. An aldehyde groups reacts with amino groups and cross link with the proteins into an insoluble network.2. OSMIUM- heavy metal that reacts primarily with fatty acids leading to the preservation of cellular membranes.
TEM SAMPLE PREPARATION Rinsing of the specimen After the fixation step, samples must be rinsed in order to remove the excess fixative. To remove excess glutaraldehyde from the samples, the specimen should be subjected to a thorough but carefully conducted rinsing procedure. Specimens can be washed in 0.1 M cacodylic acid buffer (pH 7.3), starting with one time for 10 min, and then three times for 20 min at 4 oC.
TEM SAMPLE PREPARATION Secondary fixation of the specimen Specimen can be successfully stabilized for TEM investigation by post fixation with 1% osmium tetroxide prepared in 0.1 M cacodylic acid buffer (pH 7.3) for 1.5 hrs at room temperature (immersion fixation).
TEM SAMPLE PREPARATION Dehydrating the specimen For TEM investigation, specimen can be dehydrated in a graded series of ethanol. More specifically, the following protocol is useful: Dehydration of specimen in 50% ethanol for 5 min, 70% ethanol for 10 min, 80% ethanol for 10 min, 90% ethanol for 15 min, and 99.9% ethanol (dried with a 4-mesh molecular sieve) twice for 20 min at room temperature. This process allows the water in the samples to be slowly exchanged through liquids with lower surface tensions.
TEM SAMPLE PREPARATION Infiltration of the specimen with a transitional solvent The reason why this step is required is that the ethanol is not miscible with the plastic embedding medium I found most suitable for TEM investigation of the specimen (mollicutes). The replacement of the dehydration solution by another intermediary solvent (i.e., propylene oxide) is thus necessary [1, 23, 26]. This process is essentially an alcohol substitution. The immersion of mollicutes in propylene oxide twice for 20 min at room temperature is sufficient before attempting to embed the specimens in a resin.
TEM SAMPLE PREPARATION Infiltration with resin and embedding the specimen Mollicutes can be embedded in a variety of different media depending on the use (e.g., conventional TEM or immuno TEM). For conventional TEM of mollicutes, the epoxy resin Durcupan ACM is quite suitable. The following protocol can be used: Immersion of mollicutes in propylene-oxide/Durcupan-ACM (1:1; v/v) at room temperature overnight (use gloves and a fume hood, and leave the specimen container open for the propylene oxide to evaporate). The next day, the specimens should be immersed in a freshly prepared Durcupan ACM mixture (pure) and left for 2 hrs at room temperature. A second Durcupan ACM mixture (pure) is then prepared and used as the embedding medium (free of air bubbles!).ible with the plastic embedding medium I found most suitable for TEM investigation of mollicutes. The replacement of the dehydration solution by another intermediary solvent (i.e., propylene oxide) is thus necessary [1, 23, 26]. This process is essentially an alcohol substitution. The immersion of mollicutes in propylene oxide twice for 20 min at room temperature is sufficient before attempting to embed the specimens in a resin. Polymerization of the epoxy mixture can be achieved by placing the specimens in a drying cabinet for 2 days at 40 oC and for an additional 2 days at 60 oC. Leaving the samples after heat polymerization for an additional 1-2 weeks at room temperature can improve the subsequent cutting experience as the resin blocks continue to harden during this time.
TEM SAMPLE PREPARATION Sectioning and staining of the specimen The procedure for cutting specimens into semithin and ultrathin slices (sections) is known as microtomy and ultramicrotomy, respectively. Semithin sections (0.5 μm to 2 μm) were typically stained with toluidine blue for 1 min on a hot plate (70 oC to 90 oC), examined by LM, and used for identifying the specimen within the resin block before proceeding with ultramicrotomy. Ultrathin sections (about 70 nm to 90 nm) were typically stained with uranyl acetate followed by lead citrate.
Cryofixation and the use of Frozen specimen CRYOFIXATION-like chemical fixatives it stops metabolic processes and preserves biological structure. The method involves ultra-rapid cooling of small samples to liquid nitrogen temperature (-196 oC) or below, thus stopping all motion and metabolic activity, and preserving the internal structure by freezing all fluid phases solid.
The ultimate objective is to freeze the specimen so rapidly (at 104 to 106 K per second) that ice crystals are unable to form, or are prevented from growing sufficiently large to cause damage to the specimens ultrastructure.
NEGATIVE STAINING The main purpose of negative-staining is to surround or embed the biological object in a suitable electron dense material which provides high contrast and good preservation. This method is capable of providing information about structural details often finer than those visible in thin sections, replicas, or shadowed specimens. In addition to the possibility of obtaining a spectacular enhancement of contrast, negative- staining has the advantage of speed and simplicity.
The technique has mainly been used to examine particulate (purified) specimens - e.g.. ribosomes, enzyme molecules, viruses, bacteriophages, microtubules, actin filaments, etc. at a resolution of 1.5-2.5 nm. This technique generally allows the shape, size, and the surface structure of the object to be studied as well as provide information about subunit stoichiometries and symmetry in oligomeric complexes. Any surface of the specimen accessible to water can potentially be stained, and thus, that part of the specimen will be imaged at high contrast.
SHADOW CASTING The grid containing the specimen are placed in sealed chamber which is the evacuated by vacuum pump. The chamber contains a filament composed of a heavy metal together with carbon.
The shadow cast by the nanoparticles (NPs) on metal deposition isvisible in the high-magnification images.
FREEZE FRACTURE REPLICATIONAND FREEZE ETCHING Small pieces of tissue are placed on a small metal disk and rapidly frozen. The disk is then mounted on a cooled stage within a vacuum chamber and a frozen tissue block is struck by a knife edge. The resulting fracture plane spreads out from the point of contact, splitting the tissue into two pieces.
Freeze fracturing can separate the two phospholipid leaflets that formevery cellular membrane(a) A preparation of cells or tissues isquickly frozen in liquid nitrogen at −196°C, which instantly immobilizes cellcomponents. (b) The block of frozen cellsis fractured with a sharp blow from a coldknife. The fracture plane is irregular, oftenbetween the leaflets of the plasma or anorganelle membrane. (c) Membraneproteins and particles remain bound to oneleaflet or the other, as illustrated in theexpanded view of a fractured membrane.
shadowing coating specimen with a thin film of a heavy metal freeze-etching freeze specimen then fracture along lines of greatest weakness (e.g., membranes)
GRID IS A SIEVE WOVEN FROM A THIN METAL WIRE,USUALLYNICKEL OR COPPER GRIDS OF 3 mm DIAMETER ARECOMMERCIALLY AVAILABLE WITH DIFFERENT MESHSIZES(GENERALLY OF 100-200 µm SIZE)
Staphylococcus aureus platinum replica image shot on a TEM at 50,000X magnification
CONCLUSION and RECOMMENDATION Sample preparation for TEM involves more and some different steps than those for SEM. Like in any multi-step preparation procedure, virtually every step can affect the quality of the final electron micrograph. A single mistake in one of these steps will affect all remaining steps, and thus the outcome of the entire study. It is therefore important that the investigator plans and executes every step in great detail. These procedures involve a significant time commitment and require patience and skills that come only with practice. It is important to mention that most of the chemicals used in EM are dangerous. Investigator must be aware of potential hazards such as fire, chemical, electrical, and physical associated with these items.