REPORT ON TRANSMISSION ELECTRON MICROSCOPE Course ME-228 Materials and Structural Property Correlations Course Instructor Prof M S !

o"#i Su"$itted "y

Prati% Nir&ali '()*+*, Niles&%u$ar C&a-&an '()*2., C&etan Po/ar '()*+0, Ma&es& 1ada$ '()028,

Trans$ission electron $icroscopy

A TEM image of the polio virus. The polio virus is 30 nm in size. Trans$ission electron $icroscopy (TEM) is a microscopy technique where y a eam of electrons is transmitte! through an ultra"thin specimen# interacting with the specimen as it passes through. An image is forme! from the interaction of the electrons transmitte! through the specimen$ the image is magnifie! an! focuse! onto an imaging !evice# such as a fluorescent screen# on a layer of photographic film# or to e !etecte! y a sensor such as a %%& camera. TEMs are capa le of imaging at a significantly higher resolution than light microscopes# owing to the small !e 'roglie wavelength of electrons. This ena les the instrument(s user to e)amine fine !etail*even as small as a single column of atoms# which is tens of thousan!s times smaller than the smallest resolva le o +ect in a light microscope. TEM forms a ma+or analysis metho! in a range of scientific fiel!s# in oth physical an! iological sciences. TEMs fin! application in cancer research# virology# materials science as well as pollution an! semicon!uctor research. At smaller magnifications TEM image contrast is !ue to a sorption of electrons in the material# !ue to the thic,ness an! composition of the material. At higher magnifications comple) wave interactions mo!ulate the intensity of the image# requiring e)pert analysis of o serve! images. Alternate mo!es of use allow for the TEM to o serve mo!ulations in chemical i!entity# crystal orientation# electronic structure an! sample in!uce! electron phase shift as well as the regular a sorption ase! imaging.
The first TEM was built by Max Knoll and Ernst Ruska in 1931, with this group developing the first TEM with resolving power greater than that of light in 1933 and the first o!!er ial TEM in 1939"

3istory4
Initial de-elop$ent

The first practical TEM# -riginally installe! at .. / 0ar en"1er,e an! now on !isplay at the &eutsches Museum in Munich# /ermany

Ernst A e originally propose! that the a ility to resolve !etail in an o +ect was limite! y the wavelength of the light use! in imaging# thus limiting the useful o taina le magnification from an optical microscope to a few micrometers. &evelopments into ultraviolet (23) microscopes# le! y 4oehler# allowe! for an increase in resolving power of a out a factor of two. 5owever this require! more e)pensive quartz optical components# !ue to the a sorption of 23 y glass. At this point it was elieve! that o taining an image with su "micrometer information was simply impossi le !ue to this wavelength constraint. .t ha! earlier een recognize! y 6l7c,er in 89:9 that the !eflection of ;catho!e rays; (electrons) was possi le y the use of magnetic fiel!s. This effect ha! een utilise! to uil! primitive catho!e ray oscilloscopes (%<-s) as early as 89=> y 0er!inan! 'raun# inten!e! as a measurement !evice. .n!ee! in 89=8 it was recognize! y <iec,e that the catho!e rays coul! e focuse! y these magnetic fiel!s# allowing for simple lens !esigns. ?ater this theory

was e)ten!e! y 5ans 'usch in his wor, pu lishe! in 8=@A# who showe! that the lens ma,er(s equation# coul! un!er appropriate assumptions# e applica le to electrons. .n 8=@9# at the Technological 2niversity of 'erlin A!olf Matthias# 6rofessor of 5igh voltage Technology an! Electrical .nstallations# appointe! Ma) 4noll to lea! a team of researchers to a!vance the %<- !esign. The team consiste! of several 6h& stu!ents inclu!ing Ernst <us,a an! 'o!o von 'orries. This team of researchers concerne! themselves with lens !esign an! %<- column placement# which they attempte! to o tain the parameters that coul! e optimise! to allow for construction of etter %<-s# as well as the !evelopment of electron optical components which coul! e use! to generate low magnification (nearly 8B8) images. .n 8=38 the group successfully generate! magnifie! images of mesh gri!s place! over the ano!e aperture. The !evice use! two magnetic lenses to achieve higher magnifications# argua ly the first electron microscope. .n that same year# <einhol! <u!en erg# the scientific !irector of the Ciemens company# ha! patente! an electrostatic lens electron microscope.

I$pro-in5 resolution
At this time the wave nature of electrons# which were consi!ere! charge! matter particles# ha! not een fully realise! until the pu lication of the &e 'roglie hypothesis in 8=@>. The group was unaware of this pu lication until 8=3@# where it was quic,ly realize! that the &e 'roglie wavelength of electrons was many or!ers of magnitu!e smaller than that for light# theoretically allowing for imaging at atomic scales. .n April 8=3@# <us,a suggeste! the construction of a new electron microscope for !irect imaging of specimens inserte! into the microscope# rather than simple mesh gri!s or images of apertures. 1ith this !evice successful !iffraction an! normal imaging of aluminium sheet was achieve!# however e)cee!ing the magnification achieva le with light microscopy ha! still not een successfully !emonstrate!. This goal was achieve! in Ceptem er 8=33# using images of cotton fi ers# which were quic,ly acquire! efore eing !amage! y the electron eam. At this time# interest in the electron microscope ha! increase!# with other groups# such as Al ert 6re us an! Dames 5illier at the 2niversity of Toronto who constructe! the first TEM in north America in 8=39# continually a!vancing TEM !esign. <esearch continue! on the electron microscope at Ciemens in 8=3A# the aim of the research was the !evelopment improvement of TEM imaging properties# particularly with regar! to iological specimens. At this time electron microscopes were eing fa ricate! for specific groups# such as the ;EM8; !evice use! at the 24 Eational 6hysical ?a oratory. .n 8=3= the first commercial electron microscope# picture!# was installe! in the 6hysics !epartment of .. / 0ar en"1er,e. 0urther wor, on the electron microscope was hampere! y the !estruction of a new la oratory constructe! at Ciemens y an air"rai!# as well as the !eath of two of the researchers# 5einz M7ller an! 0rie!ric, 4rause !uring 1orl! 1ar ...

6urt&er researc&
After 1orl! 1ar ..# <us,a resume! wor, at Ciemens# where he continue! to !evelop the electron microscope# pro!ucing the first microscope with 800, magnification. The fun!amental structure of this microscope !esign# with multi"stage eam preparation optics# is still use! in mo!ern microscopes. The worl!wi!e electron microscopy community a!vance! with electron microscopes eing manufacture! in Manchester 24# the 2CA (<%A)# /ermany (Ciemens) an! Dapan . The first international conference in electron microscopy was in &elft

in 8=F@# with more than one hun!re! atten!ees. ?ater conferences inclu!e! the ;0irst; international conference in 6aris# 8=:0 an! then in ?on!on in 8=:F. 1ith the !evelopment of TEM# the associate! technique of scanning transmission electron microscopy (CTEM) was re"investigate! an! !i! not ecome !evelope! until the 8=>0s# with Al ert %rewe at the 2niversity of %hicago !eveloping the fiel! emission gunan! a!!ing a high quality o +ective lens to create the mo!ern CTEM. 2sing this !esign# %rewe !emonstrate! the a ility to image atoms using annular !ar,"fiel! imaging. %rewe an! cowor,ers at the 2niversity of %hicago !evelope! the col! fiel! electron emission source an! uilt a CTEM a le to visualize single heavy atoms on thin car on su strates.

!ac%5round
Electrons
Theoretically# the ma)imum resolution# d# that one can o tain with a light microscope has een limite! y the wavelength of the photons that are eing use! to pro e the sample# G an! the numerical aperture of the system# NA.

Early twentieth century scientists theorise! ways of getting aroun! the limitations of the relatively large wavelength of visi le light (wavelengths of F00H>00 nanometers) y using electrons. ?i,e all matter# electrons have oth wave an! particle properties (as theorize! y ?ouis"3ictor !e 'roglie)# an! their wave"li,e properties mean that a eam of electrons can e ma!e to ehave li,e a eam of electromagnetic ra!iation. The wavelength of electrons is foun! y equating the !e 'roglie equation to the ,inetic energy of an electron. An a!!itional correction must e ma!e to account for relativistic effects# as in a TEM an electron(s velocity approaches the spee! of light# c.

where# h is 6lanc,(s constant# m0 is the rest mass of an electron an! E is the energy of the accelerate! electron. Electrons are usually generate! in an electron microscope y a process ,nown as thermionic emission from a filament# usually tungsten# in the same manner as a light ul # or alternatively y fiel! electron emission. The electrons are then accelerate! y an electric potential (measure! in volts) an! focuse! y electrostatic an! electromagnetic lenses onto the sample. The transmitte! eam contains information a out electron !ensity# phase an! perio!icity$ this eam is use! to form an image.

Source for$ation

?ayout of optical components in a asic TEM

Cingle crystal ?a'A filament

5airpin style tungsten filament 0rom the top !own# the TEM consists of an emission source# which may e a tungsten filament# or a lanthanum he)a ori!e (?a'A) source. 0or tungsten# this will e of the form of either a hairpin"style filament# or a small spi,e"shape! filament. ?a' A sources utilize small single crystals. 'y connecting this gun to a high voltage source (typically I800"300 ,3) the gun will# given sufficient current# egin to emit electrons either y thermionic or fiel! electron emission into the vacuum. This e)traction is usually ai!e! y the use of a 1ehnelt cylin!er. -nce e)tracte!# the upper lenses of the TEM allow for the formation of the electron pro e to the !esire! size an! location for later interaction with the sample. Manipulation of the electron eam is performe! using two physical effects. The interaction of electrons with a magnetic fiel! will cause electrons to move accor!ing to the right han! rule# thus allowing for electromagnets to manipulate the electron eam. The use of magnetic fiel!s allows for the formation of a magnetic lens of varia le focusing power# the lens shape originating !ue to the !istri ution of magnetic flu). A!!itionally# electrostatic fiel!s can cause the electrons to e !eflecte! through a constant angle. %oupling of two !eflections in opposing !irections with a small interme!iate gap allows for the formation of a shift in the eam path# this eing use! in TEM for eam shifting# su sequently this is e)tremely important to CTEM. 0rom these two effects# as well as the use of an electron imaging system# sufficient control over the eam path is possi le for TEM operation. The optical configuration of a TEM can e rapi!ly change!# unli,e that for an optical microscope# as lenses in the eam path can e ena le!# have their strength change!# or e !isa le! entirely simply via rapi! electrical switching# the spee! of which is limite! y effects such as the magnetic hysteresis of the lenses.

Optics
The lenses of a TEM allow for eam convergence# with the angle of convergence as a varia le parameter# giving the TEM the a ility to change magnification simply y mo!ifying the amount of current that flows through the coil# qua!rupole or he)apole lenses. The qua!rupole lens is an arrangement of electromagnetic coils at the vertices of the square# ena ling the generation of a lensing magnetic fiel!s# the he)apole configuration simply enhances the lens symmetry y using si)# rather than four coils.

Typically a TEM consists of three stages of lensing. The stages are the con!ensor lenses# the o +ective lenses# an! the pro+ector lenses. The con!ensor lenses are responsi le for primary eam formation# whilst the o +ective lenses focus the eam !own onto the sample itself. The pro+ector lenses are use! to e)pan! the eam onto the phosphor screen or other imaging !evice# such as film. The magnification of the TEM is !ue to the ratio of the !istances etween the specimen an! the o +ective lens( image plane. A!!itional qua! or he)apole lenses allow for the correction of asymmetrical eam !istortions# ,nown as astigmatism. .t is note! that TEM optical configurations !iffer significantly with implementation# with manufacturers using custom lens configurations# such as in spherical a erration correcte! instruments#J8>K or TEMs utilising energy filtering to correct electron chromatic a erration.

7isplay
.maging systems in a TEM consist of a phosphor screen# which may e ma!e of fine (80" 800 Lm) particulate zinc sulphi!e# for !irect o servation y the operator. -ptionally# an image recor!ing system such as film ase! or !ope! MA/ screen couple! %%&s. Typically these !evices can e remove! or inserte! into the eam path y the operator as require!.

Co$ponents

The electron source of the TEM is at the top# where the lensing system (F#> an! 9) focuses the eam on the specimen an! then pro+ects it onto the viewing screen (80). The eam control is on the right (83 an! 8F) A TEM is compose! of several components# which inclu!e a vacuum system in which the electrons travel# an electron emission source for generation of the electron stream# a series of electromagnetic lenses# as well as electrostatic plates. The latter two allow the operator to gui!e an! manipulate the eam as require!. Also require! is a !evice to allow the insertion into# motion within# an! removal of specimens from the eam path. .maging !evices are su sequently use! to create an image from the electrons that e)it the system.

8acuu$ syste$
To increase the mean free path of the electron gas interaction# a stan!ar! TEM is evacuate! to low pressures# typically on the or!er of 80NF 6a. The nee! for this is twofol!B first the allowance for the voltage !ifference etween the catho!e an! the groun! without generating an arc# an! secon!ly to re!uce the collision frequency of electrons with gas atoms to negligi le levels*this effect is characterise! y the mean free path. TEM components such as specimen hol!ers an! film cartri!ges must e routinely inserte! or replace! requiring a system with the a ility to re"evacuate on a regular asis. As such# TEMs are equippe! with multiple pumping systems an! airloc,s an! are not permanently vacuum seale!. The vacuum system for evacuating a TEM to an operating pressure level consists of several stages. .nitially a low or roughing vacuum is achieve! with either a rotary vane pump or !iaphragm pumps ringing the TEM to a sufficiently low pressure to allow the operation of a tur omolecular or !iffusion pump which rings the TEM to its high vacuum level necessary for operations. To allow for the low vacuum pump to not require continuous operation# while continually operating the tur omolecular pumps# the vacuum si!e of a low"pressure pump may e connecte! to cham ers which accommo!ate the e)haust gases from the tur omolecular pump. Cections of the TEM may e isolate! y the use of gate valves# to allow for !ifferent vacuum levels in specific areas# such as a higher vacuum of 80 NF to 80N> 6a or higher in the electron gun in high resolution or fiel! emission TEMs. 5igh"voltage TEMs require ultra high vacuums on the range of 80 N> to 80N= 6a to prevent generation of an electrical arc# particularly at the TEM catho!e. As such for higher voltage TEMs a thir! vacuum system may operate# with the gun isolate! from the main cham er either y use of gate valves or y the use of a !ifferential pumping aperture. The !ifferential pumping aperture is a small hole that prevents !iffusion of gas molecules into the higher vacuum gun area faster than they can e pumpe! out. 0or these very low pressures either an ion pump or a getter material is use!. 6oor vacuum in a TEM can cause several pro lems# from !eposition of gas insi!e the TEM onto the specimen as it is eing viewe! through a process ,nown as electron eam in!uce! !eposition# or in more severe cases !amage to the catho!e from an electrical !ischarge. 3acuum pro lems !ue to specimen su limation are limite! y the use of a col! trap to a!sor su limate! gases in the vicinity of the specimen.

Speci$en sta5e4

TEM sample support mesh ;gri!;# with ultramicrotomy sections TEM specimen stage !esigns inclu!e airloc,s to allow for insertion of the specimen hol!er into the vacuum with minimal increase in pressure in other areas of the microscope. The specimen hol!ers are a!apte! to hol! a stan!ar! size of gri! upon which the sample is place! or a stan!ar! size of self"supporting specimen. Ctan!ar! TEM gri! sizes is a 3.0: mm !iameter ring# with a thic,ness an! mesh size ranging from a few to 800 Lm. The sample is place! onto the inner meshe! area having !iameter of appro)imately @.: mm. 2sual gri! materials are copper# moly !enum# gol! or platinum. This gri! is place! into the sample hol!er which is paire! with the specimen stage. A wi!e variety of !esigns of stages an! hol!ers e)ist# !epen!ing upon the type of e)periment eing performe!. .n a!!ition to 3.0: mm gri!s# @.3 mm gri!s are sometimes# if rarely# use!. These gri!s were particularly use! in the mineral sciences where a large !egree of tilt can e require! an! where specimen material may e e)tremely rare. Electron transparent specimens have a thic,ness aroun! 800 nm# ut this value !epen!s on the accelerating voltage. -nce inserte! into a TEM# the sample often has to e manipulate! to present the region of interest to the eam# such as in single grain !iffraction# in a specific orientation. To accommo!ate this# the TEM stage inclu!es mechanisms for the translation of the sample in the OM plane of the sample# for P height a!+ustment of the sample hol!er# an! usually for at least one rotation !egree of free!om for the sample. Thus a TEM stage may provi!e four !egrees of free!om for the motion of the specimen. Most mo!ern TEMs provi!e the a ility for two orthogonal rotation angles of movement with specialize! hol!er !esigns calle! !ou le"tilt sample hol!ers. -f note however is that some stage !esigns# such as top"entry or vertical insertion stages once common for high resolution TEM stu!ies# may simply only have O"M translation availa le. The !esign criteria of TEM stages are comple)# owing to the simultaneous requirements of mechanical an! electron"optical constraints an! have thus generate! many unique implementations. A TEM stage is require! to have the a ility to hol! a specimen an! e manipulate! to ring the region of interest into the path of the electron eam. As the TEM can operate over a wi!e range of magnifications# the stage must simultaneously e highly resistant to mechanical !rift# with !rift requirements as low as a few nmQminute while eing a le to move several umQminute# with repositioning accuracy on the or!er of nanometers. Earlier !esigns of TEM accomplishe! this with a comple) set of mechanical !owngearing !evices# allowing the operator to finely control the motion of the stage y several rotating ro!s. Mo!ern !evices may use electrical stage !esigns# using screw gearing in concert with stepper motors# provi!ing the operator with a computer" ase! stage input# such as a +oystic, or trac, all.

Two main !esigns for stages in a TEM e)ist# the si!e"entry an! top entry version. J8=K Each !esign must accommo!ate the matching hol!er to allow for specimen insertion without either !amaging !elicate TEM optics or allowing gas into TEM systems un!er vacuum.

A !iagram of a single a)is tilt sample hol!er for insertion into a TEM goniometer. Titling of the hol!er is achieve! y rotation of the entire goniometer The most common is the si!e entry hol!er# where the specimen is place! near the tip of a long metal ( rass or stainless steel) ro!# with the specimen place! flat in a small ore. Along the ro! are several polymer vacuum rings to allow for the formation of a vacuum seal of sufficient quality# when inserte! into the stage. The stage is thus !esigne! to accommo!ate the ro!# placing the sample either in etween or near the o +ective lens# !epen!ent upon the o +ective !esign. 1hen inserte! into the stage# the si!e entry hol!er has its tip containe! within the TEM vacuum# an! the ase is presente! to atmosphere# the airloc, forme! y the vacuum rings. .nsertion proce!ures for si!e entry TEM hol!ers typically involve the rotation of the sample to trigger micro switches that initiate evacuation of the airloc, efore the sample is inserte! into the TEM column. The secon! !esign is the top"entry hol!er consists of a cartri!ge that is several cm long with a ore !rille! !own the cartri!ge a)is. The specimen is loa!e! into the ore# possi ly utilising a small screw ring to hol! the sample in place. This cartri!ge is inserte! into an airloc, with the ore perpen!icular to the TEM optic a)is. 1hen seale!# the airloc, is manipulate! to push the cartri!ge such that the cartri!ge falls into place# where the ore hole ecomes aligne! with the eam a)is# such that the eam travels !own the cartri!ge ore an! into the specimen. Cuch !esigns are typically una le to e tilte! without loc,ing the eam path or interfering with the o +ective lens.

Electron 5un4

%ross sectional !iagram of an electron gun assem ly# illustrating electron e)traction The electron gun is forme! from several componentsB the filament# a iasing circuit# a 1ehnelt cap# an! an e)traction ano!e. 'y connecting the filament to the negative component power supply# electrons can e ;pumpe!; from the electron gun to the ano!e plate# an! TEM column# thus completing the circuit. The gun is !esigne! to create a eam of electrons e)iting from the assem ly at some given angle# ,nown as the gun !ivergence semiangle# R. 'y constructing the 1ehnelt cylin!er such that it has a higher negative charge than the filament itself# electrons that e)it the filament in a !iverging manner are# un!er proper operation# force! into a converging pattern the minimum size of which is the gun crossover !iameter. The thermionic emission current !ensity# J# can e relate! to the wor, function of the emitting material an! is a 'oltzmann !istri ution given elow# where A is a constant# S is the wor, function an! T is the temperature of the material.

This equation shows that in or!er to achieve sufficient current !ensity it is necessary to heat the emitter# ta,ing care not to cause !amage y application of e)cessive heat# for this reason materials with either a high melting point# such as tungsten# or those with a low wor, function (?a'A) are require! for the gun filament. 0urthermore oth lanthanum he)a ori!e an! tungsten thermionic sources must e heate! in or!er to achieve thermionic emission# this can e achieve! y the use of a small resistive strip. To prevent thermal shoc,# there is often a !elay enforce! in the application of current to the tip# to prevent thermal gra!ients from !amaging the filament# the !elay is usually a few secon!s for ?a'A# an! significantly lower for tungsten .

Electron lens4

&iagram of a TEM split polepiece !esign lens Electron lenses are !esigne! to act in a manner emulating that of an optical lens# y focusing parallel rays at some constant focal length. ?enses may operate electrostatically or magnetically. The ma+ority of electron lenses for TEM utilise electromagnetic coils to generate a conve) lens. 0or these lenses the fiel! pro!uce! for the lens must e ra!ially symmetric# as !eviation from the ra!ial symmetry of the magnetic lens causes a errations such as astigmatism# an! worsens spherical an! chromatic a erration. Electron lenses are manufacture! from iron# iron"co alt or nic,el co alt alloys# such as permalloy. These are selecte! for their magnetic properties# such as magnetic saturation# hysteresis an! permea ility. The components inclu!e the yo,e# the magnetic coil# the poles# the polepiece# an! the e)ternal control circuitry. The polepiece must e manufacture! in a very symmetrical manner# as this provi!es the oun!ary con!itions for the magnetic fiel! that forms the lens. .mperfections in the manufacture of the polepiece can in!uce severe !istortions in the magnetic fiel! symmetry# which in!uce !istortions that will ultimately limit the lenses( a ility to repro!uce the o +ect plane. The e)act !imensions of the gap# pole piece internal !iameter an! taper# as well as the overall !esign of the lens is often performe! y finite element analysis of the magnetic fiel!# whilst consi!ering the thermal an! electrical constraints of the !esign. The coils which pro!uce the magnetic fiel! are locate! within the lens yo,e. The coils can contain a varia le current# ut typically utilise high voltages# an! therefore require significant insulation in or!er to prevent short"circuiting the lens components. Thermal !istri utors are place! to ensure the e)traction of the heat generate! y the energy lost to resistance of the coil win!ings. The win!ings may e water coole!# using a chille! water supply in or!er to facilitate the removal of the high thermal !uty.

Apertures4

Apertures are annular metallic plates# through which electrons that are further than a fi)e! !istance from the optic a)is may e e)clu!e!. These consist of a small metallic !isc that is sufficiently thic, to prevent electrons from passing through the !isc# whilst permitting a)ial electrons. This permission of central electrons in a TEM causes two effects simultaneouslyB firstly# apertures !ecrease the eam intensity as electrons are filtere! from the eam# which may e !esire! in the case of eam sensitive samples. Cecon!ly# this filtering removes electrons that are scattere! to high angles# which may e !ue to unwante! processes such as spherical or chromatic a erration# or !ue to !iffraction from interaction within the sample. Apertures are either a fi)e! aperture within the column# such as at the con!ensor lens# or are a mova le aperture# which can e inserte! or with!rawn from the eam path# or move! in the plane perpen!icular to the eam path. Aperture assem lies are mechanical !evices which allow for the selection of !ifferent aperture sizes# which may e use! y the operator to tra!e off intensity an! the filtering effect of the aperture. Aperture assem lies are often equippe! with micrometres to move the aperture# require! !uring optical cali ration.

I$a5in5 $et&ods
.maging metho!s in TEM utilize the information containe! in the electron waves e)iting from the sample to form an image. The pro+ector lenses allow for the correct positioning of this electron wave !istri ution onto the viewing system. The o serve! intensity of the image# .# assuming sufficiently high quality of imaging !evice# can e appro)imate! as proportional to the time"average amplitu!e of the electron wavefunctions# where the wave which form the e)it eam is !enote! y T.

&ifferent imaging metho!s therefore attempt to mo!ify the electron waves e)iting the sample in a form that is useful to o tain information with regar!s to the sample# or eam itself. 0rom the previous equation# it can e !e!uce! that the o serve! image !epen!s not only on the amplitu!e of eam# ut also on the phase of the electrons# although phase effects may often e ignore! at lower magnifications. 5igher resolution imaging requires thinner samples an! higher energies of inci!ent electrons. Therefore the sample can no longer e consi!ere! to e a sor ing electrons# via a 'eer(s law effect# rather the sample can e mo!elle! as an o +ect that !oes not change the amplitu!e of the incoming electron wavefunction. <ather the sample mo!ifies the phase of the incoming wave$ this mo!el is ,nown as a pure phase o +ect# for sufficiently thin specimens phase effects !ominate the image# complicating analysis of the o serve! intensities. 0or e)ample# to improve the contrast in the image the TEM may e operate! at a slight !efocus to enhance contrast# owing to convolution y the contrast transfer function of the TEM# which woul! normally !ecrease contrast if the sample was not a wea, phase o +ect.

Contrast for$ation4

%ontrast formation in the TEM !epen!s greatly on the mo!e of operation. %omple) imaging techniques# which utilise the unique a ility to change lens strength or to !eactivate a lens# allow for many operating mo!es. These mo!es may e use! to !iscern information that is of particular interest to the investigator.

!ri5&t field4 The most common mo!e of operation for a TEM is the right fiel! imaging mo!e. .n this mo!e the contrast formation# when consi!ere! classically# is forme! !irectly y occlusion an! a sorption of electrons in the sample. Thic,er regions of the sample# or regions with a higher atomic num er will appear !ar,# whilst regions with no sample in the eam path will appear right H hence the term ; right fiel!;. The image is in effect assume! to e a simple two !imensional pro+ection of the sample !own the optic a)is# an! to a first appro)imation may e mo!elle! via 'eer(s law# more comple) analyses require the mo!elling of the sample to inclu!e phase information. 7iffraction contrast4

Transmission electron micrograph of !islocations# which are faults in the structure of the crystal lattice at the atomic scale Camples can e)hi it !iffraction contrast# where y the electron eam un!ergoes 'ragg scattering# which in the case of a crystalline sample# !isperses electrons into !iscrete locations in the ac, focal plane. 'y the placement of apertures in the ac, focal plane# i.e. the o +ective aperture# the !esire! 'ragg reflections can e selecte! (or e)clu!e!)# thus only parts of the sample that are causing the electrons to scatter to the selecte! reflections will en! up pro+ecte! onto the imaging apparatus. .f the reflections that are selecte! !o not inclu!e the unscattere! eam (which will appear up at the focal point of the lens)# then the image will appear !ar, wherever no sample scattering to the selecte! pea, is present# as such a region without a specimen will appear !ar,. This is ,nown as a !ar,"fiel! image. Mo!ern TEMs are often equippe! with specimen hol!ers that allow the user to tilt the specimen to a range of angles in or!er to o tain specific !iffraction con!itions# an! apertures place! a ove the specimen allow the user to select electrons that woul! otherwise e !iffracte! in a particular !irection from entering the specimen.

Applications for this metho! inclu!e the i!entification of lattice !efects in crystals. 'y carefully selecting the orientation of the sample# it is possi le not +ust to !etermine the position of !efects ut also to !etermine the type of !efect present. .f the sample is oriente! so that one particular plane is only slightly tilte! away from the strongest !iffracting angle (,nown as the 'ragg Angle)# any !istortion of the crystal plane that locally tilts the plane to the 'ragg angle will pro!uce particularly strong contrast variations. 5owever# !efects that pro!uce only !isplacement of atoms that !o not tilt the crystal to the 'ragg angle (i. e. !isplacements parallel to the crystal plane) will not pro!uce strong contrast. Electron ener5y loss4 2tilizing the a!vance! technique of EE?C# for TEMs appropriately equippe! electrons can e re+ecte! ase! upon their voltage (which# !ue to constant charge is their energy)# using magnetic sector ase! !evices ,nown as EE?C spectrometers. These !evices allow for the selection of particular energy values# which can e associate! with the way the electron has interacte! with the sample. 0or e)ample !ifferent elements in a sample result in !ifferent electron energies in the eam after the sample. This normally results in chromatic a erration H however this effect can# for e)ample# e use! to generate an image which provi!es information on elemental composition# ase! upon the atomic transition !uring electron" electron interaction. EE?C spectrometers can often e operate! in oth spectroscopic an! imaging mo!es# allowing for isolation or re+ection of elastically scattere! eams. As for many images inelastic scattering will inclu!e information that may not e of interest to the investigator thus re!ucing o serva le signals of interest# EE?C imaging can e use! to enhance contrast in o serve! images# inclu!ing oth right fiel! an! !iffraction# y re+ecting unwante! components. P&ase contrast4 %rystal structure can also e investigate! y 5igh <esolution Transmission Electron Microscopy (5<TEM)# also ,nown as phase contrast. 1hen utilizing a 0iel! emission source# of uniform thic,ness# the images are forme! !ue to !ifferences in phase of electron waves# which is cause! y specimen interaction. .mage formation is given y the comple) mo!ulus of the incoming electron eams. As such# the image is not only !epen!ent on the num er of electrons hitting the screen# ma,ing !irect interpretation of phase contrast images more comple). 5owever this effect can e use! to an a!vantage# as it can e manipulate! to provi!e more information a out the sample# such as in comple) phase retrieval techniques.

7iffraction4

%rystalline !iffraction pattern from a twinne! grain of 0%% Austenitic steel As previously state!# y a!+usting the magnetic lenses such that the ac, focal plane of the lens rather than the imaging plane is place! on the imaging apparatus a !iffraction pattern can e generate!. 0or thin crystalline samples# this pro!uces an image that consists of a pattern of !ots in the case of a single crystal# or a series of rings in the case of a polycrystalline or amorphous soli! material. 0or the single crystal case the !iffraction pattern is !epen!ent upon the orientation of the specimen an! the structure of the sample illuminate! y the electron eam. This image provi!es the investigator with information a out the space group symmetries in the crystal an! the crystal(s orientation to the eam path. This is typically !one without utilising any information ut the position at which the !iffraction spots appear an! the o serve! image symmetries. &iffraction patterns can have a large !ynamic range# an! for crystalline samples# may have intensities greater than those recor!a le y %%&. As such# TEMs may still e equippe! with film cartri!ges for the purpose of o taining these images# as the film is a single use !etector.

%onvergent 'eam 4i,uchi lines from Cilicon# near the J800K zone a)is Analysis of !iffraction patterns eyon! point"position can e comple)# as the image is sensitive to a num er of factors such as specimen thic,ness an! orientation# o +ective lens !efocus# spherical an! chromatic a erration. Although quantitative interpretation of the contrast shown in lattice images is possi le# it is inherently complicate! an! can require e)tensive computer simulation an! analysis# such as electron multislice analysis. More comple) ehaviour in the !iffraction plane is also possi le# with phenomena such as 4i,uchi lines arising from multiple !iffraction within the crystalline lattice. .n convergent eam electron !iffraction (%'E&) where a non"parallel# i.e. converging# electron wavefront is pro!uce! y concentrating the electron eam into a fine pro e at the sample surface# the interaction of the convergent eam can provi!e information eyon! structural !ata such as sample thic,ness.

T&ree di$ensional i$a5in5

A three !imensional TEM image of a parapo)avirus As TEM specimen hol!ers typically allow for the rotation of a sample y a !esire! angle# multiple views of the same specimen can e o taine! y rotating the angle of the sample along an a)is perpen!icular to the eam. 'y ta,ing multiple images of a single TEM sample at !iffering angles# typically in 8U increments# a set of images ,nown as a ;tilt series; can e collecte!. This metho!ology was propose! in the 8=>0s y 1alter 5oppe. 2n!er purely a sorption contrast con!itions# this set of images can e use! to construct a three"!imensional representation of the sample. The reconstruction is accomplishe! y a two"step process# first images are aligne! to account for errors in the positioning of a sample$ such errors can occur !ue to vi ration or mechanical !rift. Alignment metho!s use image registration algorithms# such as autocorrelation metho!s to correct these errors. Cecon!ly# using a technique ,nown as filtere! ac, pro+ection# the aligne! image slices can e transforme! from a set of two"!imensional images# Ij(x,y)# to a single three"!imensional image# I'j(x,y,z). This three !imensional image is of particular interest when morphological information is require!# further stu!y can e un!erta,en using computer algorithms# such as isosurfaces an! !ata slicing to analyse the !ata. As TEM samples cannot typically e viewe! at a full 890U rotation# the o serve! images typically suffer from a ;missing we!ge; of !ata# which when using 0ourier ase! ac, pro+ection metho!s !ecreases the range of resolva le frequencies in the three !imensional reconstruction. Mechanical techniques# such as multi"a)is tilting# as well as numerical techniques e)ist to limit the impact of this missing !ata on the o serve! specimen morphology. 3ariants on this metho!# referre! to as single particle analysis# use images of multiple i!entical o +ects at !ifferent orientations to pro!uce the image !ata require! for three !imensional reconstruction. Assuming that o +ects !o not have significant preferre! orientations# this metho! !oes not suffer from the missing !ata we!ge# however it assumes that the !ifferent o +ects image! can e treate! as if the !ata was generate! from a single o +ect.

Sa$ple preparation
Cample preparation in TEM can e a comple) proce!ure. TEM specimens are require! to e at most hun!re!s of nanometers thic,# as unli,e neutron or O"<ay ra!iation the electron eam

interacts rea!ily with the sample# an effect that increases roughly with atomic num er square! (z@). 5igh quality samples will have a thic,ness that is compara le to the mean free path of the electrons that travel through the samples# which may e only a few tens of nanometers. 6reparation of TEM specimens is specific to the material un!er analysis an! the !esire! information to o tain from the specimen. As such# many generic techniques have een use! for the preparation of the require! thin sections. Materials that have !imensions small enough to e electron transparent# such as pow!ers or nanotu es# can e quic,ly prepare! y the !eposition of a !ilute sample containing the specimen onto support gri!s or films. .n the iological sciences in or!er to withstan! the instrument vacuum an! facilitate han!ling# iological specimens can e fi)ate! using either a negative staining material such as uranyl acetate or y plastic em e!!ing. Alternately samples may e hel! at liqui! nitrogen temperatures after em e!!ing in vitreous ice. .n material science an! metallurgy the specimens ten! to e naturally resistant to vacuum# ut still must e prepare! as a thin foil# or etche! so some portion of the specimen is thin enough for the eam to penetrate. %onstraints on the thic,ness of the material may e limite! y the scattering cross"section of the atoms from which the material is comprise!.

Tissue sectionin54
'y passing samples over a glass or !iamon! e!ge# small# thin sections can e rea!ily o taine! using a semi"automate! metho!. This metho! is use! to o tain thin# minimally !eforme! samples that allow for the o servation of tissue samples. A!!itionally inorganic samples have een stu!ie!# such as aluminium# although this usage is limite! owing to the heavy !amage in!uce! in the less soft samples. To prevent charge uil!"up at the sample surface# tissue samples nee! to e coate! with a thin layer of con!ucting material# such as car on# where the coating thic,ness is several nanometers. This may e achieve! via an electric arc !eposition process using a sputter coating !evice.

Sa$ple stainin54

A section of a cell of Bacillus subtilis# ta,en with a Tecnai T"8@ TEM. The scale ar is @00 nm. &etails in light microscope samples can e enhance! y stains that a sor light$ similarly TEM samples of iological tissues can utilize high atomic num er stains to enhance contrast. The stain a sor s electrons or scatters part of the electron eam which otherwise is pro+ecte! onto the imaging system. %ompoun!s of heavy metals such as osmium# lea!# or uranium may e use! prior to TEM o servation to selectively !eposit electron !ense atoms in or on the

sample in !esire! cellular or protein regions# requiring an un!erstan!ing of how heavy metals in! to iological tissues.

Mec&anical $illin54
Mechanical polishing may e use! to prepare samples. 6olishing nee!s to e !one to a high quality# to ensure constant sample thic,ness across the region of interest. A !iamon!# or cu ic oron nitri!e polishing compoun! may e use! in the final stages of polishing to remove any scratches that may cause contrast fluctuations !ue to varying sample thic,ness. Even after careful mechanical milling# a!!itional fine metho!s such as ion etching may e require! to perform final stage thinning.

C&e$ical etc&in54
%ertain samples may e prepare! y chemical etching# particularly metallic specimens. These samples are thinne! using a chemical etchant# such as an aci!# to prepare the sample for TEM o servation. &evices to control the thinning process may allow the operator to control either the voltage or current passing through the specimen# an! may inclu!e systems to !etect when the sample has een thinne! to a sufficient level of optical transparency.

Ion etc&in54

CEM image of a thin TEM sample mille! y 0.'. The thin mem rane shown here is suita le for TEM e)amination$ however# at I300"nm thic,# it woul! not e suita le for high" resolution TEM without further milling. .on etching is a sputtering process that can remove very fine quantities of material. This is use! to perform a finishing polish of specimens polishe! y other means. .on etching uses an inert gas passe! through an electric fiel! to generate a plasma stream that is !irecte! to the sample surface. Acceleration energies for gases such as argon are typically a few ,ilovolts. The sample may e rotate! to promote even polishing of the sample surface. The sputtering rate of such metho!s is on the or!er of tens of micrometers per hour# limiting the metho! to only e)tremely fine polishing. More recently focusse! ion eam metho!s have een use! to prepare samples. 0.' is a relatively new technique to prepare thin samples for TEM e)amination from larger specimens. 'ecause 0.' can e use! to micro"machine samples very precisely# it is possi le to mill very thin mem ranes from a specific area of interest in a sample# such as a semicon!uctor or metal. 2nli,e inert gas ion sputtering# 0.' ma,es use of significantly more

energetic gallium ions an! may alter the composition or structure of the material through gallium implantation.

Modifications
The capa ilities of the TEM can e further e)ten!e! y a!!itional stages an! !etectors# sometimes incorporate! on the same microscope. An electron cryomicroscope (%ryoTEM) is a TEM with a specimen hol!er capa le of maintaining the specimen at liqui! nitrogen or liqui! helium temperatures. This allows imaging specimens prepare! in vitreous ice# the preferre! preparation technique for imaging in!ivi!ual molecules or macromolecular assem lies. A TEM can e mo!ifie! into a scanning transmission electron microscope (CTEM) y the a!!ition of a system that rasters the eam across the sample to form the image# com ine! with suita le !etectors. Ccanning coils are use! to !eflect the eam# such as y an electrostatic shift of the eam# where the eam is then collecte! using a current !etector such as a fara!ay cup# which acts as a !irect electron counter. 'y correlating the electron count to the position of the scanning eam (,nown as the ;pro e;)# the transmitte! component of the eam may e measure!. The non"transmitte! components may e o taine! either y eam tilting or y the use of annular !ar, fiel! !etectors. .n"situ e)periments may also e con!ucte! with e)periments such as in"situ reactions or material !eformation testing. Mo!ern research TEMs may inclu!e a erration correctors# to re!uce the amount of !istortion in the image. .nci!ent eam Monochromators may also e use! which re!uce the energy sprea! of the inci!ent electron eam to less than 0.8: e3. Ma+or TEM ma,ers inclu!e DE-?# 5itachi 5igh"technologies# 0E. %ompany (from merging with 6hilips Electron -ptics)# %arl Peiss an! E.-E. Lo/ -olta5e electron $icroscope 'L8EM, The low voltage electron microscope (?3EM) is a com ination of CEM# TEM an! CTEM in one instrument# which operate! at relatively low electron accelerating voltage of : ,3. ?ow voltage increases image contrast which is especially important for iological specimens. This increase in contrast significantly re!uces# or even eliminates the nee! to stain. Cectione! samples generally nee! to e thinner than they woul! e for conventional TEM (@0"A: nm). <esolutions of a few nm are possi le in TEM# CEM an! CTEM mo!es.

Li$itations
There are a num er of !raw ac,s to the TEM technique. Many materials require e)tensive sample preparation to pro!uce a sample thin enough to e electron transparent# which ma,es TEM analysis a relatively time consuming process with a low throughput of samples. The

structure of the sample may also e change! !uring the preparation process. Also the fiel! of view is relatively small# raising the possi ility that the region analyse! may not e characteristic of the whole sample. There is potential that the sample may e !amage! y the electron eam# particularly in the case of iological materials.

Resolution li$its
The limit of resolution o taina le in a TEM may e !escri e! in several ways# an! is typically referre! to as the information limit of the microscope. -ne commonly use! value is a cut"off value of the contrast transfer function# a function that is usually quote! in the frequency !omain to !efine the repro!uction of spatial frequencies of o +ects in the o +ect plane y the microscope optics. A cut"off frequency# max# for the transfer function may e appro)imate! with the following equation# where %s is the spherical a erration coefficient an! G is the electron wavelengthB

0or a @00 ,3 microscope# with partly correcte! spherical a errations (;to the thir! or!er;) an! a %s value of 8 Vm# a theoretical cut"off value might e 8Q max W F@ pm. The same microscope without a corrector woul! have %s W 0.: mm an! thus a @00"pm cut"off 6ractically# the spherical a errations are suppresse! in the est# ;a erration"correcte!; microscopes. Their resolution is however limite! y electron source geometry an! rightness an! chromatic a errations in the o +ective lens system. .ntriguingly# the frequency !omain representation of the contrast transfer function may often have an oscillatory nature# which can e tune! y a!+usting the focal value of the o +ective lens. This oscillatory nature implies that some spatial frequencies are faithfully image! y the microscope# whilst others are suppresse!. 'y com ining multiple images with !ifferent spatial frequencies# the use of techniques such as focal series reconstruction can e use! to improve the resolution of the TEM in a limite! manner. The contrast transfer function can# to some e)tent# e e)perimentally appro)imate! through techniques such as 0ourier transforming images of amorphous material# such as amorphous car on. More recently# a!vances in a erration corrector !esign have een a le to re!uce spherical a errations.an! to achieve resolution elow 0.: XngstrYms (:0 pm) at magnifications a ove :0 million times. .mprove! resolution allows for the imaging of lighter atoms that scatter electrons less efficiently# such as lithium atoms in lithium attery materials. The a ility to !etermine the position of atoms within materials has ma!e the 5<TEM an in!ispensa le tool for nanotechnology research an! !evelopment in many fiel!s# inclu!ing heterogeneous catalysis an! the !evelopment of semicon!uctor !evices for electronics an! photonics.