The Transmission Electron Microscope

Presented by SK. MOSIUR RAHAMAN Guided by Dr. Vandana Soni Dept. Pharmaceutical Sciences

Dr. H S Gour University, Sagar

Over View of TEM

Contents

Introduction Basic Systems Making Up a Transmission Electron Microscope

Illuminating System Specimen Manipulation System Imaging System

High Contrast High Resolution

Major Operational Modes of the Transmission Electron Microscope

MAGNIFICATION
Comparison of Light Microscope to TEM & SEM

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. 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 4 resolutions of 0.1 nm

Basic Systems Making Up a TEM

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. 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..

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. Electron Gun. Within the electron gun the filament serves as the source of electrons. The standard filament, or cathode is composed of a Vshaped tungsten wire approximately 0.1 mm in diameter (about the thickness of a human hair). Being a metal, tungsten contains positive ions and free electrons that are strongly attracted to the positive ions.

(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).

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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Illuminating System

The so-called saturation point of the gun is the point where the number of electrons emitted from the gun no longer increases as the filament is heated. It is important that the operator realize that increasing the heat of the filament beyond the saturation point will not increase the brightness of the gun but will considerably shorten the filament life.

On the other hand, undersaturation of the filament may lead to instabilities in the illumination of the specimen and cause problems
Moving the filament closer to the shield aperture will permit more electrons to pass through to the condenser lenses. However, if the filament is placed too close to the aperture, the bias control by the shield will be lost, and the emission will become excessive. Filaments placed too far away from the shield aperture, on the other hand, may never yield sufficient numbers of electrons from the gun. 11

Illuminating System
The choice of kV should be considered carefully. Lower kVs such as 50 kV will generate an image with higher contrast but lower resolution, while higher kVs (100125 kV) improve resolution but lower overall contrast. Less chances of specimen damage will result at the higher kVs since the speedier electrons interact for a shorter period of time with the specimen.

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Illuminating System: LaB6

Besides being made of tungsten, filaments may also be constructed of lanthanum hexaboride, which has a lower work function. Typically, these filaments operate at temperatures 1,000 K lower than tungsten and have a brightness several times greater than a standard tungsten source. The lifetime of such filaments ranges from 700 to 2,000 hours. This type of filament may be made from a single LaB6 crystal with one end having a point measuring only several micrometers across. LaB6 filaments are useful when small beam crossover sizes containing large numbers of electrons are necessary as in high magnification/resolution studies, for elemental analysis, or in high resolution scanning electron microscopy.

Lanthanum hexaboride cathode. The crystal (C) is held in place by means of pyrolytic graphite (G) blocks with compressive force generated by molybdenum (M) alloy posts designed to withstand extremely high 13 temperatures.

Illuminating System:

cold field emission gun

A totally different gun, nearly a thousand times brighter than the standard gun, may also be used under certain conditions.

Electrons are not generated by thermionic emission (heating), but are actually drawn out from the tungsten crystal by a series of positive high voltage anodes that act as electrostatic lenses to focus the gun crossover to a spot size of 10 nm

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Illuminating System:

cold field emission gun

A major disadvantage of the cold field emission gun is the ultrahigh vacuum required (greater than 10-8 Pa) and the extreme susceptibility of the filament to contaminants. Cold field emission guns are very useful in high resolution scanning and scanning transmission electron microscopes and are now being incorporated into high resolution transmission electron microscopes.
Comparison of the Three Major Filaments in Terms of Brightness, Size of the Source Crossover, Energy Spread, Service Life, and Vacuum Required Cold Field Emission Brightness (A/cm2 __ sr) Source Diameter (nm) Energy Spread (eV) Service Life (hours) Vacuum Required (Pa) 109 <10 0.20.3 >2,000 10-8 Lanthanum Hexaboride 107 104 1.02.0 1,0002,000 10-5
Tungsten Filament

106 >104 1.02.0 40100 10-3

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Illuminating System-

Condenser Lenses

Condenser Lenses. This second major part of the illuminating system gathers the electrons of the first crossover image from the gun and

focuses electrons onto the specimen.

Modern transmission electron microscopes have two condenser lenses. The first condenser lens (designated C1) is a demagnifying lens that decreases the size of the 50 m gun crossover to generate a range of spot sizes from 20 m to 1 m down.

The second condenser lens (C2), on the other hand, enlarges the C1 spot. The overall effect of both lenses is to control precisely the amount of electron irradiation or illumination striking the specimen.

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The condenser lens system. (A) In this mode, the 50 m gun crossover is reduced to 5 m by condenser lens 1, C1, and then slightly enlarged by condenser lens 2, C2, to yield a 10 m spot on the specimen that is five times brighter than the initial gun crossover. (B) At higher magnifications, the 50 m gun crossover is reduced to 1.5 m by a highly energized C1. This refracts the peripheral electrons to such a great angle that they cannot enter C2 and are therefore lost. After C2 slightly enlarges the C1 spot, the resulting 2 m spot is rather dim.

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Illuminating System

Condenser Lenses

Suppose one is working at a magnification of 50,000X. At this high magnification, the C1 lens should be highly energized to demagnify the 50 m illumination spot from the gun down to 1 to 2 m. Next, the C2 lens should be used to adjust the size of the C1 illumination spot to cover only the specimen area being viewed. Since the average viewing screen is about 100 mm across, a 2 m spot of illumination enlarged 50,000X would just cover the screen (2 m X 50,000 = 100 mm).
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Illuminating System

Condenser Lenses

on the design of the transmission electron microscope, one or both condenser lenses may have apertures of variable sizes. Generally, the C1 aperture is an internal aperture of a fixed size, while the C2 aperture is variable by inserting into the electron beam pathway apertures of different sizes attached to the end of a shaft. A popular method is to use a molybdenum foil strip containing 3 or 4 holes of 500, 300, 200, and 100 m in diameter.
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Apertures in Condenser Lenses. Depending

Variable aperture holder from a TEM. The rod contains a molybdenum strip (m) with apertures of various sizes. Positioning screws (s) permit the precise alignment of the apertures in the electron beam. An O-ring seal (o) permits the aperture to be sealed off inside the vacuum of the microscope column. Insert shows enlargement of the molybdenum aperture strip held in place by a brass retainer clip. Arrows point to apertures in the strip.
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Illuminating System

Condenser Lenses

Larger condenser apertures permit most of the electrons to pass through the lens and, therefore, yield a brighter spot on the specimen. Smaller apertures cut out more peripheral electrons and, hence, reduce the illumination on the specimen.

However, since spherical aberration is concomitantly reduced, greater resolution is possible using smaller condenser apertures. The operational principle to remember is larger

condenser apertures give more illumination but with more spherical aberration.
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Specimen Manipulation System

Most biological specimens are mounted on a copper meshwork or

grid.

Grids are placed into a specimen holder and, after insertion into an air lock, the chamber is evacuated and the specimen holder is inserted into the stage of the microscope. The specimen stage is a micromanipulator for moving the specimen in x and y directions in increments as small as 10 nm, the width of a cell membrane. Depending on the design of the specimen holder and stage, it may also be possible to tilt and rotate the specimen inside the column of the electron microscope. Some of the newer micro-processor-controlled TEMs have automated stage controls that permit motorized and precise movement of the specimen. An important feature of such computer-controlled stages is the ability to memorize specified coordinates and to be able to return to these locations on command.
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Specimen Manipulation System

Side-entry stages provide much more versatile manipulation of the specimen. Besides the standard x and y horizontal movements, the specimen holder may permit tilting, rotation, a second axis of tilt (double-tilt stage), and special modifications. Since it is also necessary to accurately set the specimen in the correct focal plane of the objective lens, a z-axis or vertical movement is always provided to allow accurate eucentric positioning. Modern side-entry stages offer high resolution capabilities nearly comparable to top-entry stages and permit more versatility for specimen manipulation and orientation for analytical purposes. For these reasons, the side-entry stage is currently favored over the top-entry stage in the latest generation of TEMs.
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Special Stages

It is possible to manipulate the specimen in the electron microscope in a number of ways using special specimen stages or holders. For instance, the specimen may be subjected to stretching and compression in a tensile stage, and heating or cooling in specially modified thermal stages. Of particular interest to biologists is the cold stage, since it permits the examination of rapidly frozen specimens (such as live virus preparations) that are still hydrated and have not been exposed to chemical fixation or staining. Besides examination of fluid specimens, it is also possible to study ultrathin frozen, hydrated sections of unprocessed biological materials for elemental analysis. Although specimen preparatory techniques are still being refined, cold stages offer tremendous potential when combined with the analytical capabilities of the TEM.
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Imaging System
This part of the microscope includes the objective, intermediate, and projector lenses. It is involved in the generation of the image and the magnification and projection of the final image onto a viewing screen or camera system of the microscope.

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Objective Lens.

By far, this is the single most important lens in the transmission electron microscope, since it forms the initial image that is further magnified by the other imaging lenses. In order to achieve such high resolutions, the lens must be highly energized to obtain the short, 1 to 2 mm focal lengths necessary. The objective lens is used primarily to focus the image. The objective lens also initially magnifies the image whereas other lenses are used to magnify the image further. Of all of the lenses used in the magnification of an image, the objective lens is the least variable so that it can maintain the very short focal lengths necessary for high resolution and still be convenient to focus Currently, as magnifications are changed, the adjustments to the objective lens needed to bring the image into focus are not excessive.
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The major function of the aperture is to help remove peripherally deflected electrons to enhance image contrast. Consequently, when using smaller apertures in both the objective and condenser lenses to generate narrow aperture angles, the entire depth of the specimen is in focus. This is in contrast to the light microscope, where larger aperture angles result in rather narrow depths of field, making it necessary to focus through the various levels to view the entire depth in the specimen.
Depth of field (Dfi) occurs in the object plane, Depth of focus (Dfo) refers to the depth in the image plane that is in focus. In the bottom figure, note that an aperture increases both the depth of field 29 and depth of focus.

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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Major Operational Modes of the Transmission Electron Microscope

High Contrast High Resolution

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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. 2. Lower accelerating voltages are used.

3. Smaller objective apertures should be utilized.

4. Photographic procedures may be employed.

5. The specimen may be prepared to enhance contrast.

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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.

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Magnification

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.
Equation. 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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Comparison of Light Microscope to TEM & SEM

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Thank you
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