TEM by CVAS Bikanerboys

8/2/2019 TEM by CVAS Bikanerboys

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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


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INTRODUCTION

A TRANSMISSION ELECTRONMICROSCOPE, or TEM, has magnificationand resolution capabilities that are over a

thousand times beyond that offered by thelight microscope.

It is an instrument that is used to reveal theultrastructureof plant and animal cells as

well as viruses and may provide an imageof the very macromolecules that make upthese biological entities.

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INTRODUCTION

The TEM is a complex viewing systemequipped with a set of electromagneticlenses used to control the imagingelectrons in order to generate the

extremely fine structural details that areusually recorded on photographic film.

Since the illuminating electrons passthroughthe specimens, the information issaid to be a transmittedimage.

The modern TEM can achievemagnifications of one million times withresolutions of 0.1 nm.

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Basic Systems Making Up a

Transmission Electron Microscope

The transmission electron microscope (Figures6.20A, B and C) is made up of a number ofdifferent systems that are integrated to form onefunctional unit capable of orienting and imaging

extremely thin specimens. The illuminating systemconsists of the electron

gun and condenser lenses that give rise to andcontrol the amount of radiation striking thespecimen.

A specimen manipulation systemcomposed of thespecimen stage, specimen holders, and relatedhardware is necessary for orienting the thinspecimen outside and inside the microscope.

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Basic Systems Making Up a


The imaging systemincludes the objective,intermediate, and projector lenses that areinvolved in forming, focusing, and magnifyingthe image on the viewing screen as well as the

camerathat is used to record the image. A vacuum systemis necessary to remove

interfering air molecules from the column ofthe electron microscope. In the descriptions

that follow, the systems will be consideredfrom the top of the microscope to the bottom.

See Tables 6.3 and 6.4.

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TABLE 6.3 Major Column Components of the TEM*

Component Synonyms Function of Components

IlluminationSystem

Electron Gun Gun, Source Generates electrons and provides firstcoherent crossover of electron beam

Condenser Lens 1 C1, Spot Size Determines smallest illumination spot size onspecimen (see Spot Size in Table 6.4)

Condenser Lens 2 C2, Brightness Varies amount of illumination on specimenin combination with C1 (see Brightness inTable 6.4)

CondenserAperture

C2 Aperture Reduces spherical aberration, helps controlamount of illumination striking specimen


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SpecimenManipulationSystem

Specimen Exchanger Specimen Air Lock Chamber and mechanism for inserting specimenholder

Specimen Stage Stage Mechanism for moving specimen inside column ofmicroscope

Imaging System

Objective Lens Forms, magnifies, and focuses first image (seeFocus in Table 6.4)

Objective Aperture Controls contrast and spherical aberration

Intermediate Lens Diffraction Lens Normally used to help magnify image fromobjective lens and to focus diffraction pattern

Intermediate Aperture Diffraction

Aperture, FieldLimitingAperture

Selects area to be diffracted

Projector Lens 1 P1 Helps magnify image, possibly used in somediffraction work

Projector Lens 2 P2 Same as P1

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Observationand CameraSystems

Viewing

Chamber

Contains viewing screen for final image

BinocularMicroscope

Focusing Scope Magnifies image on viewing screen foraccurate focusing

Camera Contains film for recording

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Illuminating System

TABLE 6.4 Major Components on Control Panels of the TEM*

Component Synonyms Functions of Component

Filament Emission Effects emission of electrons upon heating

Bias Adjusts voltage differential between filamentand shield to regulate yield of electrons

High Voltage Reset HV, kV Reset Activates high voltage to gun

High Voltage Select HV, kV Select Selects amount of high voltage applied to gun

MagnificationControl

MAG Controls final magnification of image byactivating combinations of imaging lenses

Brightness C2 Controls current to second condenser lens

Gun Tilt Electronically tilts electron beam beneath gun

Gun Horizontal Electronically translates electron beambeneath gun

Spot Size C1 Controls final illumination spot size onspecimen

Objective Stigmator OBJ STIG Corrects astigmatism12


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Focus Wobbler Focus Aid Helps focus accurately at low magnifications

Exposure Meter Monitors illumination for accurate exposures

Vacuum Meter VAC Monitors vacuum levels in various parts ofscope

Focusing Control Focusfine, medium,coarse

Controls current to objective lens for accuratefocusing of image

Brightness Center Illumination Centration Translates entire illumination system ontoscreen center

Condenser Stigmator COND STIG Corrects astigmatism in condenser lenses

IntermediateStigmator

INT STIG Corrects astigmatism in intermediate lens

Bright/Dark Selects brightfield or darkfield operating mode

Main Main power switch to console

Main Evac EVAC Main switch to vacuum system

HV Wobbler HV Modulate Wobbles high voltage to locate voltage centerfor alignment

Objective Wobbler OBJ MODUL Wobbles current to objective lens foralignment

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Illuminating System

This system is situated at the top of themicroscope column and consists of theelectron gun (composed of the filament,

shield, and anode) and the condenserlenses.

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Figure 6.21(A) Diagram of an electron gun showingfilament, shield, and anode.The shield is connected directly to thehigh voltage, whereas the high voltageleading to the filament has a variableresistor (VR) to vary the amount of highvoltage.The output from the variable resistor is

then passed through two balancingresistors (BR) which are attached to thefilament.(B) Actual electron gun from TEMshowing filament (f), shield (s), andanode (a).

Compare to line drawing in 6.21(A).


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Figure 6.22Standard V-shaped tungsten filament (f) used in mostelectron microscopes. The filament is spotwelded tothe larger supporting arms, which pass through theceramic (c) insulator and plug into the electrical leads ofthe gun.


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Figure 6.21(A) Diagram of an electron gunShowing filament, shield, and

anode.The shield is connected directlyto the high voltage, whereas thehigh voltage leading to thefilament has a variable resistor(VR) to vary the amount of high

voltage.The output from the variableresistor is then passed throughtwo balancing resistors (BR)Which are attached to thefilament.

(B) Actual electron gun from TEMshowing filament (f), shield (s),and anode (a).Compare to line drawing in6.21(A).


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Figure 6.24 The self-biased electrongun.The shield (Wehnelt cylinder) is slightly

more negative than the filament tocontrol the release of electrons fromthe gun.

A variable bias resistor (seeFigure 6.21A) regulates the degree of

negativity of the filament.

The anode serves as a positiveattracting force and serves as anelectrostatic lens (in combination withthe shield) to help focus the electrons

into a crossover spot approximately 50m across.


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Viewing System andCamera The final image is projected onto a viewing screen

coated with a phosphorescent zinc-activatedcadmium sulfide powder attached to the screenwith a binder such as cellulose nitrate.

Most electron microscopes provide for aninclination of the viewing screen so that the imagemay be conveniently examined either with theunaided eye or with a stereomicroscopecalled thebinoculars.

With the stereomicroscope, although the imagemay appear to be rough due to the 100 m-sizedgrains of phosphorescent particles making up thescreen, it is necessary to view a magnified imagein order to focus accurately.

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Viewing System andCamera A shutter is provided to time the exposure so

that the proper negative density (as determinedby the previous calibration) may be obtained.

Most electron microscopes have timers thatvary from a fraction of a second to "hold"positions in which a timer may be used for verylong manual exposures.

Electron micrographs are exposed for 0.5 to 2seconds in order to record all density levels andto minimize image shift or drift(i.e., slowmovement of the image after exposure to thebeam).

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Viewing System andCamera Once the time has been selected, the illumination

level is adjusted with the C1 and C2 lens controlsuntil the exposure meter reaches the calibrationpoint.

The film is then advanced under the viewingscreen, and the screen is moved to permitelectrons to pass onto the film.

As one begins to raise the viewing screen, thebeam 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 thescreen is repositioned.

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High Contrast

High Resolution

Dark Field

Diffraction

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Major Operational Modes of the


During the alignment procedure, oneshould be aware that the conventionaltransmission electron microscope may beset up for operation in several differentoperational modes.

Depending on the design of themicroscope, this may involve relatively fewor many mutually exclusive adjustments.

In addition, certain specimen preparationtechniques may be utilized to furtherenhance these operational modes.

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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 contrastat the expense of high resolution.

As a result, this mode is generally usedat magnifications under 50,000 X.

The conditions that may be changed toenhance contrast are summarized below

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How to Obtain HighContrast 1. The focal length of the objective lens

is increased.

This necessitates using shorter specimenholder cartridges (Figure 6.30, left) in a top

entry stage to position the specimen higherin the objective lens.

In a side entry stage, adjustment of the z-axis 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, aworsening of chromatic aberration, and aloss 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 placedhigher in the objective lens, necessitating a longer focallength of the lens. (right) Standard top-entry specimengrid holder for high resolution work. The specimen gridis placed on the end of the grid holder shaft and held inplace with a sleeve that is slipped over the shaft (arrow).

These holders are placed in the specimen stage with thegrids in the downward position in the polepiece.


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How to Obtain HighContrast 2. Lower accelerating voltages are used. The

resulting lower energy electrons are more readilyaffected by differences in specimen density andthickness, and contrast will be thereby increased.

Unfortunately, this interaction with the specimengenerates a population of imaging electrons with awide range of energies, resulting in an increase inchromatic aberration.

Lower accelerating voltages are also more

damaging to the specimen, since the electrons areslowed down more and transfer more energy tothe specimen, resulting in excessive heating.

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How to Obtain High Contrast 2.Lower accelerating voltages are used

Lower energy electrons are moresusceptible to poor vacuum conditions,with the exacerbation of chromaticaberration.

Clean, high vacuums are needed tominimize electron energy losses, and themicroscope itself should be clean, sincethese electrons are more easily affected byastigmatism.

Lastly, it will be recalled that lower energyelectrons have longer wavelengths, so thatthe resolving power will be degraded.

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How to Obtain HighContrast 3. Smaller objective apertures should be

utilized.

These apertures will remove more of the

peripherally deflected electrons from thespecimen, so that the subtractive imagefrom the objective lens will be accentuatedin contrast (i.e., the signal-to-noise ratio isincreased).

Small apertures are more prone toastigmatism problems, making cleanvacuums and specimen anticontaminatorsessential.

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How to Obtain HighContrast 4. Photographic procedures may be employed.

Most images generated in the transmissionelectron microscope are enhanced for contrastusing photographic techniques.

During exposure of the electron micrograph, thesensitivity of the exposure meter may be adjustedto slightly overexpose the film.

Underdevelopment will then enhance the contrastrange in the final negative.

Details will necessarily be lost in the intermediatedensity ranges. Of course, during the printing ofthe negative, one may use higher contrastphotographic papers (see Chapter 8).

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How to Obtain HighContrast 5. The specimen may be prepared to enhance

contrast.

Standard fixation and staining techniques willincrease density by depositing the heavy metals

along various organelles. Certain embedding media (polyethelene glycol)

that may be dissolved or etched away will helpboost contrast, or one may utilize stained, frozensections without any embedding media.

The easiest approach is simply to cut thickersections; however, the resulting chromaticaberration and superimposition of structure willdegrade resolution.

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High Resolution

Most of the conditions used to achievehigh resolution in the electronmicroscope are the opposite

conditions discussed above for thehigh contrast mode.

Since contrast will be lacking in thesespecimens, efforts should be made to

boost contrast using appropriatespecimen preparation and darkroomtechniques, as described in the previoussection.

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How to Obtain High Resolution

1. The objective lens should be adjustedto give the shortest possible focallength and the proper specimen holders

used. In some systems, this is simply a matter of

pressing a single button, whereas, incertain microscopes several lens currents

must be changed concomitantly.

Perhaps it may even be necessary to inserta different polepiece in the objective lens.

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2. Adjustments to the gun, such as the use ofhigher accelerating voltages, will result in higherresolution for the reasons already mentioned inthe discussion on high contrast.

Chromatic aberration may be further lessened byusing field emission guns since the energy spreadof electrons generated from such guns isconsiderably narrower. (The energy spread fortungsten = 2 eV while field emission = 0.20.5 eV.)

In an electron microscope equipped with aconventional gun, a pointed tungsten filament willgenerate a more coherent, point source ofelectrons with better resolution capabilities.

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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, smallerapertures may be used but resolution will be diminished.

In addition, they must be kept clean since dirt will have a morepronounced effect on astigmatism.

Small condenser lens apertures will diminish sphericalaberration, 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 thermallydamage the specimen.

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4. Specimen preparation techniquesmay also enhance the resolution capability.

Extremely thin sections, for instance, willdiminish chromatic aberration.

Whenever possible, no supportingsubstrates should be used on the grid.

To achieve adequate support, this mayrequire 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 overthe holes.

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5. Miscellaneous conditionssuch as shorterviewing and exposure times will minimizecontamination, drift, and specimen damage, andhelp to preserve fine structural details.

Some of the newest microscopes have special

accessories for minimal electron dose observationof the specimen and may even utilize electronicimage intensifiers to enhance the brightness andcontrast of the image.

Anticontaminators over the diffusion pumps and

specimen area will diminish contamination andresolution loss.

High magnifications will be necessary, so carefuladjustment of the illuminating system is important.

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It may take nearly an hour for the eyes to totallyadapt to the low light levels, and this adaption willbe lost if one must leave the microscope room.

Alignment must be well done and stigmation mustbe checked periodicallyduring the viewing

session. The circuitry of the microscope should be

stabilized by allowing the lens currents and highvoltage 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 focalplane for optimum resolution.

In addition, they prevent accurate magnificationdetermination and are more prone to drift since thesupport films are often detached.

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Darkfield

In the normal operating mode of the transmissionelectron microscope, the unscattered rays of thebeam are combined with some of the deflectedelectrons 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, asshown in Figure 6.50, left, the unscatteredelectrons 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 moreaberrations and the image is of poor quality.

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Figure 6.50Schematic diagram showing two ways of setting upmicroscope for darkfield imaging: (left) displacementof 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 theillumination system so that the beam strikes thespecimen at an angle.

If the objective aperture is left normally centered, it willnow 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 eitherbrightfield or darkfield operation.

After alignment of the tilt for brightfield followed by a

darkfield alignment, one may rapidly shift from onemode to the other with the flip of a switch.

Both sets of controls also provide for separatestigmation controls to correct for any astigmatismintroduced by the tilting of the beam to large angles.

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Darkfield

The darkfield mode can be used toenhance contrast in certain types ofunstained specimens (thin frozen

sections) or in negatively stainedspecimens.

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 brightfieldmode. Specimen consists of inorganic salt crystals.


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Diffraction

In specimens that contain crystals of unknowncomposition, the diffraction technique may be usedto measure the spacing of the atomic crystallinelattice and determine the composition of thecrystal, since different crystals have uniquespacings of their lattices.

Thediffraction phenomenonis based on thereflection or diffraction of the electron beam tocertain angles by a crystalline lattice.

Instead of focusing a conventional image of thecrystal on the viewing screen using the objectivelens, one uses the intermediate or diffraction lensto focus on the back focal plane to see theselected area diffraction (SAD) on the screen.

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Diffraction

Since the crystalline lattice diffractselectrons to form bright spots on theviewing screen (similar to the mirrored

rotating sphere sometimes used inballrooms to reflect a light source onto thewalls), 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 peripheralspots represent rays diffracted at variousangles. 45


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Diffraction

The distance of these spots from the bright centralspot is inversely proportional to the spacing of thecrystalline lattice.

A crystal with small lattice spacings will diffract the

electrons to greater angles to give spots that arespaced far from the central spot.

This is unfortunate for biologists, since organiccrystals, such as protein, with large latticespacings will diffract the beam so little that the

spots will be crowded around the central brightspot and engulfed by its brilliance.

With organic crystals, the specialized technique ofhigh dispersion electron diffraction must be used.

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Diffraction Practices

After the crystal is located (using thestandard bright-field imaging mode), thecrystal is centered on the viewing screenand the objective aperture removed.

After placing the TEM in the selected areadiffraction (SAD) mode, an SAD aperture ofthe appropriate size is inserted to selectthe 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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Refocus the image using the objective lens focuscontrols to bring the image into the same plane asthe intermediate aperture. (If contrast isinadequate at this point, temporarily reinsert theobjective 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 thesecond condenser (C2) lens is spread to preventburning of the viewing screen.

For photography, adjust the size of the diffractionpattern using the camera length control, readjustthe C2 lens so that the pattern is very dim, andfocus the central bright spot as small as possibleusing the intermediate lens.

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In order to cut down on the glare from the brightcentral spot, a physical beam stopper is inserted tocover it.

Exposures are usually made for 30 to 60 secondsin the manual mode since the illumination levels

will be very low. Single crystals will generate separate spots while

polycrystalline specimens will produce so manyspots around the central point that they will blendto form a series of concentric rings (Figure 6.52).

Some biological applications of diffraction may beto confirm that a crystal present in human lungtissue is a form of asbestos, or to identify anunknown crystal in a plant or bacterial cell. Seealso Chapter 15 and the reference sources at the

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Figure 6.52Diffraction pattern obtained from polycrystallinespecimen showing characteristic ring pattern.


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Electrons, Waves, and Resolution

Physicists have demonstrated that, besides beingdiscrete particles having a negative charge and a massof 9.1X10-23 kg, electrons also have wave properties.

In fact, the wavelength () of an electron is expressed bythe 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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After appropriate substitutions associatingkinetic energy to mass, velocity, andaccelerating voltage, the equation may beexpressed: =1.23/(V)1/2

Where: V = accelerating voltage Therefore, if one were operating a

transmission electron microscope at anaccelerating voltage of 60 kV, thewavelength of the electron would be 0.005nm, and the resolving power of thesystemafter substitution of these valuesinto Equation 6.2should beapproximately 0.003 nm.

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Determine resolving power

Radius of Airy Disc: the radius of theAiry disc as measured to the first darkring is express by Equation 6.1:

r= 0.612 /n (sin )

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In fact, the actual resolution of a modernhigh resolution transmission electronmicroscope is closer to 0.1 nm.

The reason we are not able to achieve thenearly 100-fold better resolution of 0.003nm is due to the extremely narrow apertureangles (about 1,000 times smaller than thatof the light microscope) needed by the

electron microscope lenses to overcome amajor resolution limiting phenomenoncalled spherical aberration.

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In addition, the diffraction phenomenonas well as chromatic aberration andastigmatism (to be discussed later) all

degrade the resolution capabilities of theTEM.

To appreciate these problems, it is

necessary to understand how lensesfunction.

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Magnification

Besides forming images with high resolution,the lenses of the electron microscope are ableto further magnify these images.

Magnification refers to the degree of

enlargement of the diameter of a final imagecompared to the original.

In practice, magnification equals a distancemeasured between two points on an imagedivided by the distance measured betweenthese same two points on the original object, or

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Magnification

Consequently, if the image distancebetween two points measures 25.5 mmwhile the distance between these same

two points on the object measures 5mm, then the magnification is

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Magnification

As will be discussed later, there are atleast three magnifying lenses in anelectron 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 systemas shown in Equation 6.6.

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Magnification

Equation 6.6: Calculation of TotalMagnification, MT, of the TEM

where: MT = total magnification or mag

MO = mag of objective lens

MI = mag of intermediate lensMP = mag of projector lens(es)

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Magnification

For example, if the transmission electronmicroscope is operating in the highmagnification 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 thevalues would be: 50 0.5 50 =1.250.

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Magnification

Intermediate magnifications may be produced byvarying the current to the various lenses.

Sometimes, it is desirable to view as much of thespecimen as possible in order to evaluate quickly thequality 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 magnificationmode, which can be accomplished by shutting off theobjective 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, theobjective lens is used the least.

Normally it is maintained at around 50 or 100 while the

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Magnification

Although it is theoretically possible toincrease the magnification indefinitely, thequality of the image magnified is

dependent on the resolving power of thelenses in the system.

Consequently, the term usefulmagnificationis used to define the

maximum magnification that should beused for a particular optical system. It isdefined 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 wouldbe 1,000 because the resolving power of the humaneye is about 0.2 mm, while the resolving power of thelight 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 ofapproximately 1,000,000, or a thousand times greaterthan the light microscope.

In practice, due to the diminished illumination at suchhigh magnifications, microscopists would probably take

the micrograph at a magnification of 250,000

andphotographically enlarge the negative to the neededmagnification.

However, only rarely do biologists need such highmagnifications.

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Magnification

Depending on the model of TEM, the magnificationchanges may occur as a series of discrete steps oras an infinitely variable or ''zoom" magnificationseries.

Modern electron microscopes have digital displaysthat give the approximate total magnification whenone varies the magnification control.

Older microscopes usually have an analog gaugethat may either read the current to one lens, or

they may have a magnification knob with a seriesof click stops that may be correlated to a particularmagnification.

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Magnification

All microscopes (including lightmicroscopes) must be calibrated in order todetermine more accurately the total

magnification, since a number of variablesmay cause the magnification to vary by asmuch as 20 to 30% over a short period oftime.

Even modern instruments with directreading digital displays are guaranteed tobe accurate to only 5 to 10% of thestated values.

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Thank you!

TEM by CVAS Bikanerboys

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