Make  a  table  (e.g.  in  Excel)  with  a  row  for  the  observation  tools

§Optical  Microscopy§ SEM§ TEM  § AFM§ STM  

where  you  describe  briefly  the  following  properties:  

§ Basic  operating  principle  

§ Approximate  sensor  resolution  (10-­xm)  

§ Applications  (i.e.  specimen  type)  

§ Prominent  limitations  

Problem  1

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§Optical  Microscopy

§Operating  principle:  Visible  light  (electromagnetic  radiation  in  the  visible  spectrum)  and  a  system  of  lenses  is  used  to  magnify  images  of  small  objects.

§ Resolution:  Around  200  nm  

§ Applications:  Everyday  lab  use,  observe  samples  directly  without  altering  them,  determine  protein  concentrations  in  living  cells  using  UV-­fluorescence  imaging,  image  samples  where  no  resolution  lower  than  200  nm  is  needed

§ Limitations:  Resolution  limited  by  diffraction.  The  resolution  limit  is  a  function  of  the  wavelength  of  light.  Theoretical  optical  resolution:  d  =  0.61  λ  /  N.A.  Transparent  samples  or  different  samples  with  similar  transparencies  are  difficult  to  image.

Problem  1

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§ Scanning  Electron  Microscopy  (SEM)

§Operating  principle:  An  electron  beam  is  focused  onto  a  conductive  sample  using  electromagnetic  coils.  The  beam  is  scanned  over  the  sample  and  the  interaction  products  (often  SE)  are  detected.  These  signals  are  used  to  create  an  image  where  each  pixel  corresponds  to  the  signal  strength  on  the  corresponding  position  on  the  sample.

§ Resolution:  Few  nanometers

§ Applications:  Image  microstructures,  surface  topography,  material  compositions

§ Limitations:  The  samples  must  be  solid.  It  is  not  possible  to  observe  wet  samples  and  living  cells.  Often  a  high  vacuum  and  a  conducting  coating  is  needed,  which  both  can  alter  the  sample.  Due  to  electron  scattering,  the  electrons  from  the  electron  beam  interacts  with  a  larger  area  than  just  on  the  focused  spot.  This  limits  the  resolution.  

Problem  1

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§ Transmission  Electron  Microscopy  (TEM)

§Operating  principle:  A  beam  of  electrons  interacts  with  and  eventually  passes  through  an  ultrathin  sample  and  is  magnified  by  using  electromagnetic  lenses.

§ Resolution:  Few  nanometers (better  than  SEM)  

§ Applications:  Observing  thin  samples  (e.g.  thin  slices  of  brain  tissue),  Crystallography  (atomic  arrangement)  

§ Limitations:  Long  sample  preparation  time,  a  thin  sample  is  needed,  sample  preparation  can  alter  the  sample,  resolution  limited  by  electron  diffraction,  higher  acceleration  voltage  (smaller  wavelength)  increase  the  resolution,  but  simultaneously  increase  the  damage  introduced  in  the  sample.  High  vacuum  is  needed.

Problem  1

4

§ Scanning  TunnelingMicroscopy  (STM)

§Operating  principle:  Quantum  mechanical  tunneling of  electrons  from  the  probe  through  the  sample.  

§ Resolution:  Sub  nanometer

§ Applications:  Obtaining  atomic  level  details  of  samples,  manipulation  of  atoms

§ Limitations:  Electrically  conductive  sample  needed.  Slow  scanning  rate  and  small  scanning  area.  Sharp  edges  and  overhangs  cannot  be  imaged.  The  size  of  the  probe  tip  sets  a  resolution  limit  (smallest  tip  =  one  atom),  needs  to  operate  under  ultra-­high  vacuum.

Problem  1

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§ Atomic  Force  Microscopy  (AFM)

§Operating  principle:  A  laser  diode  is  used  to  detect  the  deflection  of  a  cantilever  probe  moving  in  very  close  proximity  to  the  surface

§ Resolution:  Sub  nanometer

§ Applications:  Observing  and  manipulating  nanometer-­sized  objects,  nanolithography,  measuring  magnetic  surface  pattern  (MFM),  measuring  biological  samples  in  a  wet  environment

§ Limitations:  Slow  scanning  rate  and  small  scanning  area.  Sharp  edges  and  overhangs  cannot  be  imaged.  The  sizeof  the  probe  tip  sets  a  resolution  limit  (smallest  tip  =  one  atom).  The  tip  can  crush  into  the  sample  when  largetopological  heights  differences  are  present.  The  tip  can  damage  the  sample.  Friction  and  adhesion  can  influence  the  measurement.  

Problem  1

6

Given  an  oil  immersion  objective  with  63x  magnification,  Numerical  Aperture  (N.A.)  =  1.4,  a  standard  10x  eyepiece,  and  a  UV  laser  of  405  nm

a. What  resolving  power  does  system  have,  i.e.  what  is  the  minimal  distance  between  two  points  you  can  theoretically  distinguish?  

b. What  resolving  power  does  system  have,  i.e.  what  is  the  minimal  distance  between  two  points  you  can  theoretically  distinguish?  

c. What  does  depth  of  field  mean  and  how  does  this  relate  to  N.A.?  

Problem  2

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a. What  resolving  power  does  system  have,  i.e.  what  is  the  minimal  distance  between  two  points  you  can  theoretically  distinguish?  

§ Theoretical  resolution:  d  =  0.61  λ /  N.A.  =  176.5  nm

b. How  large  would  an  object  of  800  nm  length  appear?  

§M=M1M2 with  M1,  M2 =  magnification  of  objective  and  ocular

§ 0.8  μm  x  63  x  10  =  504  μm  

c. What  does  depth  of  field  mean  and  how  does  this  relate  to  N.A.?  

§ DOF  is  the  range  of  distance  along  the  optical  axis  in  which  the  specimen  can  move  without  losing  the  sharpness  of  the  image.  This  depends  on  the  resolution  of  the  microscope

§ The  higher  the  N.A.  the  shallower  this  distance

Problem  2

8

Comparison  of  a  lense with  smaller  N.A.  to  one  with  a  larger  N.A.

How  do  images  taken  with  optical  and  transmission  electron  microscopy  differ?  What  influences  whether  you  see  an  area  as  being  bright  or  dark?  

§ The  basic  principles  of  both  microscopes  are  very  similar  but  in  the  TEM  electrons  are  used  instead  of  light

§ In  optical  microscopy  you  therefore  see  things  like  you  see  them  with  your  eyes,  i.e.  reflection  and  refraction  appear  as  in  real  life  (but  with  a  lower  resolution  than  TEM)

§ In  electron  microscopy,  the  interaction  between  the  electrons  and  the  sample  is  observed.  The  resulting  images  can  look  similar  but  have  a  higher  resolution  and  can  be  influenced  by  material  properties  such  as  conductivity  and  electromagnetic  effects  etc.

§ Bright  Field:  The  areas  which  absorb  light  (e.g.  electrons)  appear  dark  whereas  the  areas  with  a  low  interaction  between  sample  and  light  (e.g.  electrons)  appear  bright.

§ Dark  Field:  The  light  (e.g.  electron  beam)  which  barely  interacts  with  the  sample  does  not  hit  the  detector  (dark  area).  Only  the  light  (e.g.  electron  beam)  which  is  scattered  at  the  sample  can  reach  the  detector.  These  areas  appear  bright.  

Problem  3

9

AFM  and  STM:  

a. What  properties  are  needed  for  AFM  cantilevers  in  the  different  modes?  

b. What  is  the  main  advantage  of  using  tapping  mode  compared  to  using  contact  mode?  

c. What  is  a  key  advantage  of  Scanning  Probe  Microscopes  over  Electron  or  Optical  Microscopes?  

Problem  4

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a. What  properties  are  needed  for  AFM  cantilevers  in  the  different  modes?  

§ Three  operation  modes:  Contact,  tapping  and  non-­contact

§ In  the  contact  mode,  the  static  deflection  of  the  cantilever  is  used  to  calculate  the  force.  Because  the  measurement  of  a  static  signal  is  prone  to  noise  and  drift,  low  stiffness  cantilevers  are  used  to  boost  the  deflection  signal.  Silicon  nitride  probes  are  often  used.  

§ For  the  tapping  mode  and  non-­contact  mode  the  stiffness  needs  to  be  higher to  get  higher  resonance  frequencies.  Operating  at  higher  frequencies  results  in  lower  noise  levels.  Silicon  probes  are  typically  used.  

Problem  4

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b. What  is  the  main  advantage  of  using  tapping  mode  compared  to  using  contact  mode?  

§ In  the  tapping  mode,  the  image  is  obtained  by  measuring  the  forces  generated  by  the  intermittent  contact  of  the  tip  with  the  sample

§ This  lessens  the  damage  to  the  sample  compared  to  the  contact  mode.  Hence,  it  is  possible  to  image  more  delicate  structures  using  tapping  mode

Problem  4

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c. What  is  a  key  advantage  of  Scanning  Probe  Microscopes  over  Electron  or  Optical  Microscopes?  

§ The  resolution  is  not  limited  by  diffraction.  

§ Scanning  Probe  Microscopes  use  a  physical  probe  to  scan  the  specimen  to  obtain  information  of  samples.  Only  the  size  of  the  probe  limits  the  resolution.

Problem  4

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You  have  an  SEM  and  an  AFM.  Which  instrument  do  you  use  in  the  following  applications  and  why  (explain)?

a. To  investigate  the  vertical  surfaces  of  your  etch  trenches.  The  Trench  is  3  micron  in  width  and  15  micron  in  depth  

b. To  investigate  the  surface  of  nanotubes  lying  horizontally  on  a  substrate.  The  nanotubes  are  20  nm  in  diameter  and  100  nm  in  length  

Problem  5

14

a. To  investigate  the  vertical  surfaces  of  your  etch  trenches.  The  Trench  is  3  micron  in  width  and  15  micron  in  depth  

§ You  use  an  SEM  and  tilt  the  stage  so  that  you  can  see  the  vertical  surfaces.  The  AFM  cantilever  tip  cannot  access  the  vertical  surfaces  of  etch  trenches.  

Problem  5

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b. To  investigate  the  surface  of  nanotubes  lying  horizontally  on  a  substrate.  The  nanotubes  are  20  nm  in  diameter  and  100  nm  in  length  

§ Investigating  the  surface  of  nanotubes  needs  atomic  resolution.  

§ AFM  should  be  chosen.  

Problem  5

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