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Electron microscopy
and image analysis I
Catherine Vénien-Bryan!
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12+34%3'"556&'
Electron Microscope
Optical Microscope
Eye
Microscope - A device with a lens or series of lenses
that enlarge (magnify) the appearance of an object.
Does not apply to SEM.
Image - Perception of an object using your eyes (vision).
One can sense an object without vision (touch, etc..).
Requires visible light.
Resolution
is limited to approx.
1/2 the wavelength of illuminating source
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Two-dimensional crystals Resolution References [high resolution (better than 4Å)]
Bacteriorhodopsine p3 3.5Å, 3.0Å Henderson et al., 1990, Kimura et al., 1997
Plant LHC-II 3.4 Å Kühlbrandt et al., 1994 Tubulin dimer 3.7 Å Nogales et al., 1998 Aquaporin 3.8 Å, 4.0 Å, 1.8 Å Murata et al., 2000; Mitra et al.,
2002; Gonen et al., 2005 Two-dimensional crystals [low resolution (better than 9Å)]
Porin PhoE 6.0 Å Jap et al. 1991 Plant photosystem II RC 8.0 Å Rhee et al. 1998 Yeast H+-ATPase 8.0 Å Auer et al. 1998 Gap junction channel 7.5 Å Unger et al. 1999 Gluthatione transferase 6.0 Å Schmidt-Krey et al. 2000 NhaA Na/H antiporter 7.0 Å Williams 2000 Glycerol channel GlpF 6.9 Å Stahlberg et al. 2000 OxlT, oxalic acid transporter 6.5 Å Hirai et al. 2002 SecYEG complex 8.0 Å Breyton et al. 2002 EmrE multidrug transporter 7.0 Å Ubarretxena-Belandia et al. 2003 Rhodopsin frog 7.5 Å Unger et al. 1997 Metarhodopsine I 5.5 Å Ruprecht et al. 2004 Helical structure Acetylcholine receptor 4.0 Å Miyazawa et al., 2003 Bacterial flagellum 4.0 Å Yonekura et al., 2003 Microtubule 8.0 Å Li et al., 2002 Calcium ATPase 8.0 Å Zhang et al., 1998 Tobacco mosaic virus 10.0 Å Jeng et al., 1989
Structure MW Resolution References
Single particles E.coli 70S ribosome 2.5 MDa 11.5Å, 9Å Gabashvili et al., 2000
Valle et al., 2003 Bacteriophage SPP1 connectors
1.0 MDa 10Å Orlova et al., 2003
50S ribosomal subunit 1.6MDa 7.5Å Matadeen et al., 2001 GroEL 0.8MDa 8.7Å, 11.5Å Ranson et al., 2001
Ludtke et al., 2001 Glutamate synthase 1.2 MDa 9.5Å Cottevieille et al., 2008
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=JZ!F=5#0%&+0!J%&#+$'%!Z<#9D5#H!*+!0/'!$<#9D5#!6/*9/!E5(<-&0'+!0/'!&E>-*0<('+!&#(!>/&+'+!5$!0/'!'-'90%5#!(*`%&9D5#!>&f'%#!$5%E'(!*#!0/'!,&9T!$59&-!>-&#'!5$!0/'!5,8'9D7'!-'#+I!"0!9&#!,'!%'>%'+'#0'(!&+j!
!
PhCF = sin["
2(Cs#3u4 $ 2%Z#u2)]
u= spatial frequency A= Amplitude contrast (in cryo 0.07 negligeable) " = wavelength electrons Cs = lens sphericity aberration %z = defocus value for the image
?/&+'!95#0%&+0!0%&#+$'%!$<#9D5#!
?/&+'!$&905%+j!@;>/'%*9&-!&,'%%&D5#!! !@X'$59<+!7&-<'!!
CE>-*0<('!95#0%&+0!0%&#+$'%!$<#9D5#!
CE>-*0<('!$&905%+j!@F5,8'9D7'H!&>'%0<%'+!@;>&D&-!95/'%'#9'!'#7'-5>'!F#5#@>&%&--'-L!95#7'%.'#0!,'&EH!@J'E>5%&-!95/'%'#9'!'#7'-5>>'!F#5#!E5#59/%5E&D9!,'&EL!*#+0&,*-*D'+!5$!0/'!.<#!&#(!-'#+'+I!
!
AmCF = cos["
2(Cs#3u4 $ 2%Zu2)]
`5%"-+&"'"-+%&Q$-'Q,%L85%'('
2!W!cos&#
Amplitude contrast 10%
2!(1-W)!sin&#
Phase contrast 90%
=JZ!050&-!
W = 10 %, Cs = 1 mm, ""= 0,025 Å %z = -3,5 "m
Do not forget the envelope
A
B
C
D
Spatial frequencies
Spatial frequencies
Spatial frequencies
fréquences spatiales
cont
rast
e co
ntra
st
cont
rast
co
ntra
st
diffractogramme
CTFx envelope
envelope
CTF
Envelope function from the CTF :
Determine the maximum of the transmitted spatial frequencies (limit of the information i.e. the highest resolution attainable with the microscope)
E(u) = Es(u)Ec(u)Ed(u)Ev(u)ED(u) Es(u): instability of the source Ec(u): Chromatic aberration Ed(u): Specimen drift
Ev(u): spécimen vibration ED(u): detector
Low-pass filter Image
Pixel size: 5.5 Å
Low-pass filter High pass-filter
Image
Pixel size 5.5 Å
object CTF defocus 1 Image altered by CTF
Image altered by CTF and contrast inverted
X =
CTF
Resolution limit
11 Å
object CTF defocus 2 Image altered by CTF
Image altered by CTF and contrast inverted
X =
Resolution limit
CTF
17 Å
object CTF defocus 3 Image altered by CTF
X =
CTF
25 Å
Image altered by CTF and contrast inverted
Resolution limit
object CTF defocus 4 Image altered by CTF
X =
CTF
Resolution limit
30 Å
Image altered by CTF and contrast inverted
FTC FTC2 + '
CTF defocus value-500 nm CTF defocus value-1000 nm
Wiener Filter Wiener Filter
CTF x filter CTFxfilter
defocus 1 defocus 2
After CTF correction
CTF
Wiener Filter Wiener filter
CTF1
CTF12 + '
("N(CTFn x filtern)
CTF2
CTF22 + '
"#!=JZ!95%%'9D5#L!6'!&f'E>0!05!%'0%*'7'!0/'!<#(*+05%0'(!5,8'90!$%5E!0/'!*E&.'I!J/*+!&f'E>0!*+!95E>%5E*+'(!,3!0/'!>%'+'#9'!5$!#5*+'{!*I'IL!%'957'%3!5$!0/'!5,8'90!*+!#'7'%!*('&-I!!;*#9'!0/'!=JZ!&-6&3+!/&+!K'%5!9%5++*#.+L!>&%0!5$!0/'!*#$5%E&D5#!&,5<0!0/'!5,8'90!*+!-5+0I!!J/*+!*+!6/3!6'!E&T'!<+'!5$!(#,#3*1%&4*)#(%.5'*&$#6%*'%6&7#3#$'%6#8.90(%(#:$)(;%".-&$)%'"*'%'"#%
3#(01+$)%<=>(%?$@AB%C.&$'1D%@*E#3%*--3.-3&*'#%F#&)"+$)B%9.,#3%'"#%F".1#%>.03%(-*9#%F&'".0'%
)*-G%
!#%*1(.%*--1D%*%H1'#3I%!&#$#3%H1'#3%&$%.36#3%'.%3#'3&#,#%(.4#%.8%'"#%&$8.34*+.$G%
`5%"-+&"'S-+%&Q$-"'d,%L85%'
A6$L"-5%'24L-5&L5=4$&'
S-+%&24&&45%'A6$L"-5%'T4L-5&L5=.''?SAT@'
UL+%%4%3'A6$L"-5%'T4L-5&L5=.'?UAT@'
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UAT'+==64L+85%&'
Y&9T!+9&f'%*#.!&#(!+'95#(&%3!'-'90%5#+!!&%'!95--'90'(!,3!('0'905%+!0/'#!!,3!('0'905%+!!&#(!95#7'%0'(!*#05!&!+*.#&-!!
A6$L"-5%'A%$-3.'V5&&'U=$L"-5&L5=.''
@J/'!5,8'90!*+!'G>5+'(!05!&!,'&E!5$!'-'90%5#+!6*0/!&!T#56#!!L!#&%%56!%&#.'!5$!T*#'D9+!'#'%.3!@;5E'!'-'90%5#+!6*--!<#('%.5!*#'-&+D9!+9&f'%*#.!F0/'3!-5++!'#'%.3!&#(!0/'*%!0%&8'905%3!*+!E5(*_'(H!@J/'!&E5<#0!5$!'#'%.3!-5++!9&#!,'!E'&+<%'(!7*&!&#!'-'90%5#!+>'90%5E'0'%!&#(!*#0'%>%'0'(!!*#!0'%E+!5$!6/&0!9&<+'(!0/'!'#'%.3!-5++I!!@""#'-&+D9!*#0'%&9D5#+!*#9-<('!>/5#5#!'G9*0&D5#+L!*#0'%!&#(!*#0%&!,&#(!0%&#+*D5#+L!!@">-&+E5#!'G9*0&D5#+L!*##'%!+/'--!*5#*K&D5#+L!&#(!|'%'#T57!%&(*&D5#I!!J/'!*##'%@+/'--!*5#*K&D5#+!&%'!>&%D9<-&%-3!<+'$<-!$5%!('0'9D#.!0/'!'-'E'#0&-!95E>5#'#0+!!5$!&!E&0'%*&-I!!V&>>*#.!5$!0/'!'-'E'#0+!5$!0/'!E&0'%*&-!!
A6$L"-5%'A%$-3.'V5&&'U=$L"-5&L5=.''
7$Q$-$%L$&'
p*./0!&#(!A-'90%5#!V*9%5+95>3!AIVI!;-&30'%L!4I;I!;-&30'%!C9&('E*9!?%'++!
=5%%'-&D7'!V*9%5+95>3!*#!Y*5-5.3!j!"#+0%<E'#0&D5#!&#(!V'0/5(+L!VICI!4&3&0I!C9&('E*9!>%'++!
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