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The Transmission ElectronMicroscope

Presented bySK. MOSIUR RAHAMAN

Guided byDr. Vandana Soni

Dept. Pharmaceutical Sciences

Dr. H S Gour University, Sagar

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Over Viewof TEM

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Contents

Introduction Basic Systems Making Up a Transmission Electron

Microscope Illuminating System Specimen Manipulation System Imaging System

Major Operational Modes of the Transmission Electron Microscope High Contrast High Resolution

MAGNIFICATION

Comparison of Light Microscope to TEM & SEM

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INTRODUCTION A TRANSMISSION ELECTRON MICROSCOPE, or TEM, has

magnification and resolution capabilities that are over a thousandtimes beyond that offered by the light microscope.

It is an instrument that is used to reveal the ultrastructureof plantand 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 ofelectromagnetic lensesused to control the imaging electronsin orderto generate the extremely fine structural detailsthat are usuallyrecorded on photographic film.

Since the illuminating electrons pass throughthe specimens, theinformation is said to be a transmittedimage.

The modern TEM can achieve magnifications of one million times withresolutions of 0.1 nm

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Basic Systems Making Up a TEM

The illuminating systemconsists of the electron gun and

condenser lensesthat give rise to and control theamount of radiation striking the specimen.

A specimen manipulation systemcomposed of thespecimen stage, specimen holders, and related hardware

is necessary for orienting the thin specimen outside andinside the microscope.

The imaging systemincludes the objective,intermediate,and projectorlenses that are involved in forming,focusing, and magnifying the image on the viewing

screen as well as the camerathat is used to record theimage.

A vacuum systemis necessary to remove interfering airmolecules from the column of the electron microscope..

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Illuminating System

This system is situated at the top of the microscope columnand consists of the electron gun(composed of thefilament, 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 aV-

shaped tungsten wire approximately 0.1 mm in diameter

(about the thickness of a human hair). Being a metal, tungsten contains positive ions and free

electronsthat are strongly attracted to the positive ions.

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(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 variableresistor (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).

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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 pointof the gun is the point where the

number of electrons emitted from the gunno longer increasesas

the filament is heated.

It is important that the operator realize that increasing the heat ofthe filament beyond the saturation pointwill not increase thebrightness of the gunbut will considerably shorten the filament life.

On the other hand, undersaturationof the filament may lead toinstabilities in the illuminationof the specimen and cause problems

Moving the filament closer to the shield aperturewill permit moreelectronsto pass through to the condenser lenses.

However, if the filament is placed too close to the aperture, the biascontrol by the shield will be lost, and the emission will becomeexcessive. Filaments placed too far away from the shield aperture,on the other hand, may never yield sufficient numbersof electrons

from the gun.

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Illuminating System

The choice of kV should be considered carefully.

Lower kVs such as 50 kV will generate an imagewith higher contrast but lower resolution, while

higher kVs (100125 kV)improve resolutionbutlower overall contrast.

Less chances of specimen damagewill result atthe higher kVs since the speedier electronsinteract for a shorter period of time with thespecimen.

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Illuminating System: LaB6 Besides being made of tungsten,

filaments may also be constructed of

lanthanum hexaboride,which has a lowerwork function.

Typically, these filaments operate attemperatures 1,000K lower thantungsten andhave a brightness severaltimes greaterthan a standard tungsten

source. The lifetime of such filaments ranges

from 700 to 2,000 hours. This type offilament may be made from a single LaB6crystalwith one end having a pointmeasuring only several micrometers

across.LaB6filaments are useful when small beamcrossover sizescontaining large numbersof electrons are necessaryas in highmagnification/resolution studies, forelemental analysis, or in high resolutionscanning electron microscopy.

Lanthanum hexaboride cathode. Thecrystal (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

temperatures.

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Illuminating System:cold field emission gun

A totally different gun, nearly athousand times brighter than thestandard gun, may also be used undercertain conditions.

Electrons are notgenerated bythermionic emission (heating), but areactually drawn out from the tungstencrystal by a series of positive highvoltage anodesthat act as electrostatic

lensesto focus the gun crossover to aspot 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 ultrahighvacuum required (greater than 10-8 Pa)and the extreme susceptibility ofthe filament to contaminants.

Cold field emission guns are very useful in high resolution scanningandscanning transmissionelectron 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 LanthanumHexaboride

Tungsten

Filament

Brightness (A/cm2__ sr) 109 107 106

Source Diameter (nm) 104

Energy Spread (eV) 0.20.3 1.02.0 1.02.0

Service Life (hours) >2,000 1,0002,000 40100

Vacuum Required (Pa) 10-8 10-5 10-3

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Illuminating System-

Condenser Lenses Condenser Lenses.This second major part of

the illuminating system gathers the electrons ofthe first crossover image from the gun andfocuses electrons onto the specimen.

Modern transmission electron microscopes havetwo condenser lenses. The first condenser lens

(designated C1) is a demagnifying lensthatdecreases the size of the 50 mgun crossoverto generate a range of spot sizesfrom 20 m to1 m down.

The second condenser lens (C2),on the other

hand, enlarges the C1 spot. The overall effect ofboth lensesis to control precisely the amount ofelectron irradiationor illumination striking thespecimen.

Th d l t

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The condenser lens system.

(A) In this mode, the 50 m guncrossover is reduced to 5 m bycondenser lens 1, C1,

and then slightly enlarged bycondenser 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 50m gun crossover is reduced to1.5 m by a highly energized C1.This refracts the peripheral

electrons to such a great angle

that they cannot enter C2 and aretherefore 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 of50,000X.

At this high magnification, the C1 lens should behighly energized to demagnify the 50 m

illumination spotfrom the gun down to 1 to 2m.

Next, the C2 lens should be used to adjust thesize of the C1 illumination spot to cover only the

specimen area being viewed. Since the averageviewing screen is about 100 mm across, a 2 mspot of illumination enlarged 50,000X would justcover the screen (2 m X 50,000 = 100 mm).

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Apertures in Condenser Lenses.Dependingon the design of the transmission electronmicroscope, one or both condenser lenses mayhave apertures of variable sizes.

Generally, the C1 aperture is an internalaperture of a fixed size, while the C2 aperture isvariableby inserting into the electron beampathway aperturesof different sizes attached tothe end of a shaft.

A popular method is to use a molybdenum foilstrip containing 3 or 4 holes of 500, 300, 200,and 100 m in diameter.

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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 electronbeam. 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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Larger condenser apertures permitmost of the electronsto pass through the lensand, therefore, yield a brighterspot on the specimen.

Smaller apertures cut out more peripheral electronsand,hence, reduce the illumination on the specimen.

However, since spherical aberration is concomitantlyreduced, greater resolution is possible using smallercondenser apertures.

The operational principle to remember is largercondenser apertures give more illumination but withmore 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 holderand, after insertion into an

air lock, the chamber is evacuated and the specimen holder isinserted into the stage of the microscope.

The specimen stageis a micromanipulator for moving the specimenin x and y directionsin 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 ofthe electron microscope.

Some of the newer micro-processor-controlled TEMs haveautomated stage controlsthat permit motorized and precisemovement of the specimen.

An important feature of such computer-controlled stages is theability to memorize specified coordinatesand to be able to return to

these locations on command.

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Specimen Manipulation System

Side-entry stages provide much more versatile manipulationof the specimen.

Besides the standard x and y horizontal movements, thespecimen holder may permit tilting, rotation, a second axis oftilt(double-tilt stage), and special modifications.

Since it is also necessary to accurately set the specimenin thecorrect focal plane of the objective lens, a z-axis or verticalmovement is always providedto allow accurate eucentricpositioning.

Modern side-entry stages offer high resolution capabilitiesnearly comparable to top-entry stages and permit moreversatility for specimen manipulation and orientation foranalytical purposes.

For these reasons, the side-entry stage is currently favoredover the top-entry stage in the latest generation of TEMs.

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Special Stages

It is possible to manipulate the specimenin the electronmicroscope in a number of ways using special specimen stagesor holders.

For instance, the specimen may be subjected to stretching andcompression 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 aslive virus preparations)that are still hydrated and have notbeen exposed to chemical fixation or staining.

Besides examination of fluid specimens, it is also possible tostudy ultrathin frozen, hydrated sections of unprocessedbiological materials for elemental analysis.

Although specimen preparatory techniques are still beingrefined, cold stages offer tremendous potentialwhen combinedwith the analytical capabilities of the TEM.

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Imaging System

This part of the microscope includes theobjective, intermediate, andprojector lenses.

It is involved in the generation of theimageand the magnification andprojection of the final imageonto a

viewing screenor camera systemof themicroscope.

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Objective Lens. By far, this is the single most important lensin 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 highlyenergized 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 magnifiesthe image whereas otherlenses are used to magnify the image further.

Of all of the lenses used in the magnification of an image, theobjective lens is the least variableso that it can maintain the very

short focal lengths necessary for high resolutionand still beconvenient to focus

Currently, as magnifications are changed, the adjustments to theobjective lensneeded to bring the image into focusare notexcessive.

The major function of the aperture is

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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

aperturesin both the objective andcondenser lensesto generate narrowaperture angles, the entire depth ofthe specimen is in focus.

This is in contrastto the light

microscope, where larger apertureangles result in rather narrow depthsof field, making it necessary to focusthrough the various levels to view theentire depth in the specimen.

Depth of field(Dfi) occurs in the objectplane, 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

and depth of focus.

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Viewing System and Camera

The final image is projected onto a viewing screencoated with a phosphorescent zinc-activated cadmiumsulfide powderattached to the screen with a binder suchas cellulose nitrate.

Most electron microscopes provide for an inclination ofthe viewing screen so that the image may beconveniently examined either with the unaided eye orwith a stereomicroscopecalled the binoculars.

With the stereomicroscope, although the image may

appear to be rough due to the 100 m-sizedgrains ofphosphorescent particles making up the screen, it isnecessary to view a magnified image in order to focusaccurately.

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Major Operational Modes ofthe Transmission Electron

Microscope

High Contrast

High Resolution

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High Contrast

A constant problem with biologicalspecimens is their low contrast.

In the high contrast mode, the instrument

is adjusted to give contrast at theexpense of high resolution.

As a result, this mode is generally used at

magnifications under 50,000X. 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 isincreased.

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 enhancecontrast.

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High Resolution

Most of the conditions used to achievehigh resolutionin the electron microscopeare the opposite conditionsdiscussed

above for the high contrast mode. Since contrastwill be lacking in these

specimens, efforts should be made toboost contrast using appropriate specimenpreparation and darkroom techniques.

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Magnification

There are at least three magnifying lenses in an electronmicroscope: the objective, intermediate,and projectorlenses.

The final magnification is calculated as the product of

the individual magnifying powers of all of the lenses inthe system.

Equation. Calculation of Total Magnification, MT, of the TEM

where: MT= total magnification or mag

MO= mag of objectivelens

MI= mag of intermediatelens

MP= mag of projector lens(es)

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Comparison of Light Microscope to TEM &SEM

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TEM-SMR (2)