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COLLEGE OF VETERINARY & ANIMAL SCIENCES,BIKANER DEPARTMENT OF VETERINARY MICROBIOLOGY TRANSMISSION ELECTRON MICROPSCOPY

SUBMITTED TO :Dr. Sunil Maherchandani By:- Tara Chand Nayak

INTRODUCTION
A TRANSMISSION ELECTRON MICROSCOPE, or TEM, has magnification and resolution capabilities that are over a thousand times beyond that offered by the light microscope. It is an instrument that is used to reveal the ultrastructure of plant and animal cells as well as viruses and may provide an image of the very macromolecules that make up these biological entities.

INTRODUCTION
The TEM is a complex viewing system equipped with a set of electromagnetic lenses used to control the imaging electrons in order to generate the extremely fine structural details that are usually recorded on photographic film. Since the illuminating electrons pass through the specimens, the information is said to be a transmitted image. The modern TEM can achieve magnifications of one million times with resolutions of 0.1 nm.

Basic Systems Making Up a Transmission Electron Microscope

The transmission electron microscope (Figures 6.20A, B and C) is made up of a number of different systems that are integrated to form one functional unit capable of orienting and imaging extremely thin specimens. The illuminating system consists of the electron gun and condenser lenses that give rise to and control the amount of radiation striking the specimen. A specimen manipulation system composed of the specimen stage, specimen holders, and related hardware is necessary for orienting the thin specimen outside and inside the microscope.
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Basic Systems Making Up a Transmission Electron Microscope

The imaging system includes the objective, intermediate, and projector lenses that are involved in forming, focusing, and magnifying the image on the viewing screen as well as the camera that is used to record the image. A vacuum system is necessary to remove interfering air molecules from the column of the electron microscope. In the descriptions that follow, the systems will be considered from the top of the microscope to the bottom. See Tables 6.3 and 6.4.

TABLE 6.3 Major Column Components of the TEM* Component Illumination System Electron Gun Gun, Source Generates electrons and provides first coherent crossover of electron beam Synonyms Function of Components
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Condenser Lens 1
Condenser Lens 2

C1, Spot Size


C2, Brightness

Determines smallest illumination spot size on specimen (see Spot Size in Table 6.4)
Varies amount of illumination on specimen in combination with C1 (see Brightness in Table 6.4) Reduces spherical aberration, helps control amount of illumination striking specimen

Condenser Aperture

C2 Aperture

Specimen Manipulation System

Specimen Exchanger
Specimen Stage Imaging System Objective Lens Objective Aperture Intermediate Lens

Specimen Air Lock


Stage

Chamber and mechanism for inserting specimen holder


Mechanism for moving specimen inside column of microscope

Diffraction Lens

Forms, magnifies, and focuses first image (see Focus in Table 6.4) Controls contrast and spherical aberration Normally used to help magnify image from objective lens and to focus diffraction pattern Selects area to be diffracted

Intermediate Aperture Diffraction Aperture, Field Limiting Aperture

Projector Lens 1
Projector Lens 2

P1
P2

Helps magnify image, possibly used in some diffraction work


Same as P1
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Observation and Camera Systems Viewing Chamber Binocular Microscope Camera Focusing Scope Contains viewing screen for final image Magnifies image on viewing screen for accurate focusing Contains film for recording

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Illuminating System
TABLE 6.4 Major Components on Control Panels of the TEM*

Component
Filament Bias High Voltage Reset High Voltage Select Magnification Control Brightness Gun Tilt Gun Horizontal Spot Size

Synonyms
Emission HV, kV Reset HV, kV Select MAG C2 C1

Functions of Component
Effects emission of electrons upon heating Adjusts voltage differential between filament and shield to regulate yield of electrons Activates high voltage to gun Selects amount of high voltage applied to gun Controls final magnification of image by activating combinations of imaging lenses Controls current to second condenser lens Electronically tilts electron beam beneath gun Electronically translates electron beam beneath gun Controls final illumination spot size on specimen

Objective Stigmator

OBJ STIG

Corrects astigmatism
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Focus Wobbler Exposure Meter Vacuum Meter Focusing Control

Focus Aid VAC Focusfine, medium, coarse

Helps focus accurately at low magnifications Monitors illumination for accurate exposures Monitors vacuum levels in various parts of scope Controls current to objective lens for accurate focusing of image

Brightness Center
Condenser Stigmator

Illumination Centration
COND STIG

Translates entire illumination system onto screen center


Corrects astigmatism in condenser lenses

Intermediate Stigmator
Bright/Dark

INT STIG

Corrects astigmatism in intermediate lens


Selects brightfield or darkfield operating mode

Main
Main Evac HV Wobbler Objective Wobbler

EVAC HV Modulate OBJ MODUL

Main power switch to console


Main switch to vacuum system Wobbles high voltage to locate voltage center for alignment Wobbles current to objective lens for alignment
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Illuminating System

This system is situated at the top of the microscope column and consists of the electron gun (composed of the filament, shield, and anode) and the condenser lenses.

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Figure 6.21 (A) Diagram of an electron gun showing filament, shield, and anode. The shield is connected directly to the high voltage, whereas the high voltage leading to the filament has a variable resistor (VR) to vary the amount of high voltage. The output from the variable resistor is then passed through two balancing resistors (BR) which are attached to the filament. (B) Actual electron gun from TEM showing filament (f), shield (s), and anode (a). Compare to line drawing in 6.21(A).
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Figure 6.22 Standard V-shaped tungsten filament (f) used in most electron microscopes. The filament is spotwelded to the larger supporting arms, which pass through the ceramic (c) insulator and plug into the electrical leads of the gun.
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Figure 6.21 (A) Diagram of an electron gun Showing filament, shield, and anode. The shield is connected directly to the high voltage, whereas the high voltage leading to the filament has a variable resistor (VR) to vary the amount of high voltage. The output from the variable resistor is then passed through two balancing resistors (BR) Which are attached to the filament. (B) Actual electron gun from TEM showing filament (f), shield (s), and anode (a). Compare to line drawing in 6.21(A).

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Figure 6.24 The self-biased electron gun. The shield (Wehnelt cylinder) is slightly more negative than the filament to control the release of electrons from the gun.
A variable bias resistor (see Figure 6.21A) regulates the degree of negativity of the filament. The anode serves as a positive attracting force and serves as an electrostatic lens (in combination with the shield) to help focus the electrons into a crossover spot approximately 50 m across.

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Viewing System and Camera

The final image is projected onto a viewing screen coated with a phosphorescent zinc-activated cadmium sulfide powder attached to the screen with a binder such as cellulose nitrate. Most electron microscopes provide for an inclination of the viewing screen so that the image may be conveniently examined either with the unaided eye or with a stereomicroscope called the binoculars. With the stereomicroscope, although the image may appear to be rough due to the 100 m-sized grains of phosphorescent particles making up the screen, it is necessary to view a magnified image in order to focus accurately.
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Viewing System and Camera


A shutter is provided to time the exposure so that the proper negative density (as determined by the previous calibration) may be obtained. Most electron microscopes have timers that vary from a fraction of a second to "hold" positions in which a timer may be used for very long manual exposures. Electron micrographs are exposed for 0.5 to 2 seconds in order to record all density levels and to minimize image shift or drift (i.e., slow movement of the image after exposure to the beam).

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Viewing System and Camera

Once the time has been selected, the illumination level is adjusted with the C1 and C2 lens controls until the exposure meter reaches the calibration point. The film is then advanced under the viewing screen, and the screen is moved to permit electrons to pass onto the film. As one begins to raise the viewing screen, the beam is blocked by the shutter until the screen is totally raised. The shutter is then opened for the proper interval, after which the beam is again blocked until the screen is repositioned.
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High Contrast High Resolution Dark Field Diffraction

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Major Operational Modes of the Transmission Electron Microscope


During the alignment procedure, one should be aware that the conventional transmission electron microscope may be set up for operation in several different operational modes. Depending on the design of the microscope, this may involve relatively few or many mutually exclusive adjustments. In addition, certain specimen preparation techniques may be utilized to further enhance these operational modes.

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High Contrast
A constant problem with biological specimens is their low contrast. In the high contrast mode, the instrument is adjusted to give contrast at the expense of high resolution. As a result, this mode is generally used at magnifications under 50,000 X. The conditions that may be changed to enhance contrast are summarized below

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How to Obtain High Contrast


1. The focal length of the objective lens is increased. This necessitates using shorter specimen holder cartridges (Figure 6.30, left) in a top entry stage to position the specimen higher in the objective lens. In a side entry stage, adjustment of the zaxis or specimen positioning may also be needed if a special holder is not provided. It may be recalled that longer focal lengths result in narrower aperture angles, a worsening of chromatic aberration, and a loss of resolution.

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Figure 6.30 (left) Short, top-entry grid holder for high contrast, low-magnification work. Resolution is not as good with this type of grid holder since the specimen is placed higher in the objective lens, necessitating a longer focal length of the lens. (right) Standard top-entry specimen grid holder for high resolution work. The specimen grid is placed on the end of the grid holder shaft and held in place with a sleeve that is slipped over the shaft (arrow). These holders are placed in the specimen stage with the grids in the downward position in the polepiece.

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How to Obtain High Contrast

2. Lower accelerating voltages are used. The resulting lower energy electrons are more readily affected by differences in specimen density and thickness, and contrast will be thereby increased. Unfortunately, this interaction with the specimen generates a population of imaging electrons with a wide range of energies, resulting in an increase in chromatic aberration. Lower accelerating voltages are also more damaging to the specimen, since the electrons are slowed down more and transfer more energy to the specimen, resulting in excessive heating.

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How to Obtain High Contrast 2.


Lower accelerating voltages are used
Lower energy electrons are more susceptible to poor vacuum conditions, with the exacerbation of chromatic aberration. Clean, high vacuums are needed to minimize electron energy losses, and the microscope itself should be clean, since these electrons are more easily affected by astigmatism. Lastly, it will be recalled that lower energy electrons have longer wavelengths, so that the resolving power will be degraded.

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How to Obtain High Contrast


3. Smaller objective apertures should be utilized. These apertures will remove more of the peripherally deflected electrons from the specimen, so that the subtractive image from the objective lens will be accentuated in contrast (i.e., the signal-to-noise ratio is increased). Small apertures are more prone to astigmatism problems, making clean vacuums and specimen anticontaminators essential.

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How to Obtain High Contrast


4. Photographic procedures may be employed. Most images generated in the transmission electron microscope are enhanced for contrast using photographic techniques. During exposure of the electron micrograph, the sensitivity of the exposure meter may be adjusted to slightly overexpose the film. Underdevelopment will then enhance the contrast range in the final negative. Details will necessarily be lost in the intermediate density ranges. Of course, during the printing of the negative, one may use higher contrast photographic papers (see Chapter 8).

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How to Obtain High Contrast


5. The specimen may be prepared to enhance contrast. Standard fixation and staining techniques will increase density by depositing the heavy metals along various organelles. Certain embedding media (polyethelene glycol) that may be dissolved or etched away will help boost contrast, or one may utilize stained, frozen sections without any embedding media. The easiest approach is simply to cut thicker sections; however, the resulting chromatic aberration and superimposition of structure will degrade resolution.
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High Resolution
Most of the conditions used to achieve high resolution in the electron microscope are the opposite conditions discussed above for the high contrast mode. Since contrast will be lacking in these specimens, efforts should be made to boost contrast using appropriate specimen preparation and darkroom techniques, as described in the previous section.

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How to Obtain High Resolution


1. The objective lens should be adjusted to give the shortest possible focal length and the proper specimen holders used. In some systems, this is simply a matter of pressing a single button, whereas, in certain microscopes several lens currents must be changed concomitantly. Perhaps it may even be necessary to insert a different polepiece in the objective lens.

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How to Obtain High Resolution

2. Adjustments to the gun, such as the use of higher accelerating voltages, will result in higher resolution for the reasons already mentioned in the discussion on high contrast. Chromatic aberration may be further lessened by using field emission guns since the energy spread of electrons generated from such guns is considerably narrower. (The energy spread for tungsten = 2 eV while field emission = 0.20.5 eV.) In an electron microscope equipped with a conventional gun, a pointed tungsten filament will generate a more coherent, point source of electrons with better resolution capabilities.
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How to Obtain High Resolution

3. Use apertures of appropriate size.


For most specimens, larger objective lens apertures should be

used to minimize diffraction effects. If contrast is too low due to the larger objective aperture, smaller apertures may be used but resolution will be diminished. In addition, they must be kept clean since dirt will have a more pronounced effect on astigmatism. Small condenser lens apertures will diminish spherical aberration, but this will be at the expense of overall illumination. The illumination levels may be improved by altering the bias to effect greater gun emissions; however, this may thermally damage the specimen.

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How to Obtain High Resolution


4. Specimen preparation techniques may also enhance the resolution capability. Extremely thin sections, for instance, will diminish chromatic aberration. Whenever possible, no supporting substrates should be used on the grid. To achieve adequate support, this may require the use of holey films with a larger than normal number of holes (holey nets, see Chapter 4). The areas viewed are limited to those over the holes.
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How to Obtain High Resolution

5. Miscellaneous conditions such as shorter viewing and exposure times will minimize contamination, drift, and specimen damage, and help to preserve fine structural details. Some of the newest microscopes have special accessories for minimal electron dose observation of the specimen and may even utilize electronic image intensifiers to enhance the brightness and contrast of the image. Anticontaminators over the diffusion pumps and specimen area will diminish contamination and resolution loss. High magnifications will be necessary, so careful adjustment of the illuminating system is important.
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How to Obtain High Resolution


It may take nearly an hour for the eyes to totally adapt to the low light levels, and this adaption will be lost if one must leave the microscope room. Alignment must be well done and stigmation must be checked periodically during the viewing session. The circuitry of the microscope should be stabilized by allowing the lens currents and high voltage to warm up for 1 to 2 hours before use. Bent specimen grids should be avoided since they may place the specimen in an improper focal plane for optimum resolution. In addition, they prevent accurate magnification determination and are more prone to drift since the support films are often detached.

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Darkfield
In the normal operating mode of the transmission electron microscope, the unscattered rays of the beam are combined with some of the deflected electrons to form a brightfield image. As more of the deflected or scattered electrons are eliminated using smaller objective lens apertures, contrast will increase. If one moves the objective aperture off axis, as shown in Figure 6.50, left, the unscattered electrons are now eliminated while more of the scattered electrons enter the aperture. This is a crude form of darkfield illumination. Unfortunately, the off-axis electrons have more aberrations and the image is of poor quality.

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Figure 6.50 Schematic diagram showing two ways of setting up microscope for darkfield imaging: (left) displacement of objective aperture off-axis; (right) tilt of illumination system into on-axis objective aperture.
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Darkfield
Higher resolution darkfield may be obtained by tilting the illumination system so that the beam strikes the specimen at an angle. If the objective aperture is left normally centered, it will now accept only the scattered, on-axis electrons and the image will be of high quality (Figure 6.50, right). Most microscopes now have a dual set of beam tilt controls that will permit one to adjust the tilt for either brightfield or darkfield operation. After alignment of the tilt for brightfield followed by a darkfield alignment, one may rapidly shift from one mode to the other with the flip of a switch. Both sets of controls also provide for separate stigmation controls to correct for any astigmatism introduced by the tilting of the beam to large angles.

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Darkfield
The darkfield mode can be used to enhance contrast in certain types of unstained specimens (thin frozen sections) or in negatively stained specimens. An example of a darkfield image is shown in Figure 6.51.

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Figure 6.51 (top) Darkfield image obtained by tilting illumination system. (bottom) Same specimen viewed in standard brightfield mode. Specimen consists of inorganic salt crystals.

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Diffraction

In specimens that contain crystals of unknown composition, the diffraction technique may be used to measure the spacing of the atomic crystalline lattice and determine the composition of the crystal, since different crystals have unique spacings of their lattices. The diffraction phenomenon is based on the reflection or diffraction of the electron beam to certain angles by a crystalline lattice. Instead of focusing a conventional image of the crystal on the viewing screen using the objective lens, one uses the intermediate or diffraction lens to focus on the back focal plane to see the selected area diffraction (SAD) on the screen.
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Diffraction
Since the crystalline lattice diffracts electrons to form bright spots on the viewing screen (similar to the mirrored rotating sphere sometimes used in ballrooms to reflect a light source onto the walls), the image will consist of a central, bright spot surrounded by a series of spots, which are the reflections. The central bright spot represents nondiffracted rays while the peripheral spots represent rays diffracted at various angles.

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Diffraction

The distance of these spots from the bright central spot is inversely proportional to the spacing of the crystalline lattice. A crystal with small lattice spacings will diffract the electrons to greater angles to give spots that are spaced far from the central spot. This is unfortunate for biologists, since organic crystals, such as protein, with large lattice spacings will diffract the beam so little that the spots will be crowded around the central bright spot and engulfed by its brilliance. With organic crystals, the specialized technique of high dispersion electron diffraction must be used.
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Diffraction Practices
After the crystal is located (using the standard bright-field imaging mode), the crystal is centered on the viewing screen and the objective aperture removed. After placing the TEM in the selected area diffraction (SAD) mode, an SAD aperture of the appropriate size is inserted to select the area of the crystal one wishes to diffract. Focus sharply on the edge of the SAD aperture using the SAD (intermediate lens) control.

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Diffraction Practices

Refocus the image using the objective lens focus controls to bring the image into the same plane as the intermediate aperture. (If contrast is inadequate at this point, temporarily reinsert the objective aperture to check focus and then remove it before proceeding.) Place the TEM into the diffraction mode (usually a button labeled ''D" or "DIFF") and ensure that the second condenser (C2) lens is spread to prevent burning of the viewing screen. For photography, adjust the size of the diffraction pattern using the camera length control, readjust the C2 lens so that the pattern is very dim, and focus the central bright spot as small as possible using the intermediate lens.
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Diffraction Practices
In order to cut down on the glare from the bright central spot, a physical beam stopper is inserted to cover it. Exposures are usually made for 30 to 60 seconds in the manual mode since the illumination levels will be very low. Single crystals will generate separate spots while polycrystalline specimens will produce so many spots around the central point that they will blend to form a series of concentric rings (Figure 6.52). Some biological applications of diffraction may be to confirm that a crystal present in human lung tissue is a form of asbestos, or to identify an unknown crystal in a plant or bacterial cell. See also Chapter 15 and the reference sources at the end of this chapter.

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Figure 6.52 Diffraction pattern obtained from polycrystalline specimen showing characteristic ring pattern.

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Electrons, Waves, and Resolution


Physicists have demonstrated that, besides being discrete particles having a negative charge and a mass of 9.1X10-23 kg, electrons also have wave properties. In fact, the wavelength () of an electron is expressed by the equation of the French physicist de Broglie as shown in Equation 6.3. Equation 6.3: de Broglie Equation for Wavelength of an Electron

=h/mv
where h = Planck's constant (6.626 X 10-23 ergs/sec) m = mass of the electron v = electron velocity

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Electrons, Waves, and Resolution


After appropriate substitutions associating kinetic energy to mass, velocity, and accelerating voltage, the equation may be expressed: =1.23/(V)1/2 Where: V = accelerating voltage Therefore, if one were operating a transmission electron microscope at an accelerating voltage of 60 kV, the wavelength of the electron would be 0.005 nm, and the resolving power of the systemafter substitution of these values into Equation 6.2should be approximately 0.003 nm.

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Determine resolving power

Radius of Airy Disc: the radius of the Airy disc as measured to the first dark ring is express by Equation 6.1: r= 0.612 /n (sin )

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Electrons, Waves, and Resolution


In fact, the actual resolution of a modern high resolution transmission electron microscope is closer to 0.1 nm. The reason we are not able to achieve the nearly 100-fold better resolution of 0.003 nm is due to the extremely narrow aperture angles (about 1,000 times smaller than that of the light microscope) needed by the electron microscope lenses to overcome a major resolution limiting phenomenon called spherical aberration.

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Electrons, Waves, and Resolution


In addition, the diffraction phenomenon as well as chromatic aberration and astigmatism (to be discussed later) all degrade the resolution capabilities of the TEM. To appreciate these problems, it is necessary to understand how lenses function.

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Magnification
Besides forming images with high resolution, the lenses of the electron microscope are able to further magnify these images. Magnification refers to the degree of enlargement of the diameter of a final image compared to the original. In practice, magnification equals a distance measured between two points on an image divided by the distance measured between these same two points on the original object, or

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Magnification

Consequently, if the image distance between two points measures 25.5 mm while the distance between these same two points on the object measures 5 mm, then the magnification is

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Magnification
As will be discussed later, there are at least three magnifying lenses in an electron microscope: the objective, intermediate, and projector lenses. The final magnification is calculated as the product of the individual magnifying powers of all of the lenses in the system as shown in Equation 6.6.

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Magnification

Equation 6.6: Calculation of Total Magnification, MT, of the TEM

where: MT = total magnification or mag MO = mag of objective lens MI = mag of intermediate lens MP = mag of projector lens(es)

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Magnification
For example, if the transmission electron microscope is operating in the high magnification mode, typical values for the respective lenses might be: 200 50 20 = 200,000. If one were to operate the microscope in the low magnification mode, perhaps the values would be: 50 0.5 50 = 1.250.

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Magnification
Intermediate magnifications may be produced by varying the current to the various lenses. Sometimes, it is desirable to view as much of the specimen as possible in order to evaluate quickly the quality of the preparation or to locate a particular portion of the specimen. In this case, an extremely low magnification is obtained by placing the microscope in the scan magnification mode, which can be accomplished by shutting off the objective lens and using the next lens (the intermediate lens) as the imaging lens as follows: 1 0.5 100 = 50. Of all the lenses used to change magnification, the objective lens is used the least. Normally it is maintained at around 50 or 100 while the other lenses are varied.

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Magnification
Although it is theoretically possible to increase the magnification indefinitely, the quality of the image magnified is dependent on the resolving power of the lenses in the system. Consequently, the term useful magnification is used to define the maximum magnification that should be used for a particular optical system. It is defined by the formula in Equation 6.7.

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Magnification

Equation 6.7: Useful Magnification

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Magnification

In the case of the light microscope, a typical value would be 1,000 because the resolving power of the human eye is about 0.2 mm, while the resolving power of the light microscope is approximately 0.2 m. An electron microscope with a resolving power of 0.2 nm could be expected to have a top magnification of approximately 1,000,000, or a thousand times greater than the light microscope. In practice, due to the diminished illumination at such high magnifications, microscopists would probably take the micrograph at a magnification of 250,000 and photographically enlarge the negative to the needed magnification. However, only rarely do biologists need such high magnifications.
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Magnification

Depending on the model of TEM, the magnification changes may occur as a series of discrete steps or as an infinitely variable or ''zoom" magnification series. Modern electron microscopes have digital displays that give the approximate total magnification when one varies the magnification control. Older microscopes usually have an analog gauge that may either read the current to one lens, or they may have a magnification knob with a series of click stops that may be correlated to a particular magnification.

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Magnification
All microscopes (including light microscopes) must be calibrated in order to determine more accurately the total magnification, since a number of variables may cause the magnification to vary by as much as 20 to 30% over a short period of time. Even modern instruments with direct reading digital displays are guaranteed to be accurate to only 5 to 10% of the stated values.

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Thank you!

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