characterization of porous materials by focused ion beam nano-tomography
DESCRIPTION
By Marco Cantoni (EPFL)TRANSCRIPT
FIB-Nanotomographyin Materials and Life Science
Marco Cantoni,Graham Knott, Pierre Burdet
Ecole Polytechnique Fédérale Lausanne
CIME
Centre Interdisciplinaire de Microscopie Electronique(EPFL-CIME)
Centre Interdisciplinaire de Microscopie Electronique(EPFL-CIME)
central facility for electron microscopyo 5 TEMs:
TECNAIs: Spirit, TF-20, OSIRISCM300, JEM2200FS
o 3 SEMs (2 FEI XLF-30,1 Zeiss MERLIN)
o 1 FIB (ZEISS NVision40)
o Yearly ≈240 operators from 60 different labs of 4 faculties. 13’000-15’000 "beam hours“
o open to everybodyMainly as a “Do it yourself” we train you... you do yourself your observations
o For « small » needs, we do the investigation for you, feasibility studies
CIME: Centre Interdisciplinaire de Microscopie ElectroniqueDirector: Prof. Cécile Hébert
Science and Technology of Engineering
Materials Sciencealloys, ceramics (+powder),polymers, cement/concretbiomaterials…
Microengineeringmicromachininglithographybio-med. eng.
Life Sciences
Conventional TEM (fixation, staining, high-pressure freezing, freeze substitution…)Cryo TEM under development
Basic Sciences
PhysicsMetals, alloys, ceramics,Semiconductors, nanoparticles, fullerenes, thin films…
ChemistryCatalystselectro-active coatings…
Architecture, Civil and Environmental Eng.
CorrosionWoodWaste transforming bacteria
Facility Manager:
EM for Phys./Chem./Mat. S. : Marco Cantoni
Graham Knott: BIO-EM (since 2007)
Since August 2008: Zeiss NVision 40e-beam: ZEISS Gemini, 1-30kV, 1nm @ 30kV, 2.5nm @1 kV
Ion-beam: 1-30kV, 4nm @ 30kV
EDS X-MAX (SDD) 80mm2 detector
Kleindiek micromanipulator (TEM prep)
2-3 Ga Sources / year (~5000 beam hours)
FIB Applications @ CIME
• Materials Science:– TEM Lamellae preparation– cross-sectioning, SE/BSE imaging, EDX– 3D reconstruction– 3D EDX (in collaboration with ZEISS and OXFORD
INSTRUMENTS)– 3D reconstruction of biocompatible materials
• Life Science:– Serial Sectioning of cells and brain tissue:
SUPER-STACKS
WYSIWYG: What You (detector) See Is What You Get
3D FIB/SEM: volume reconstruction
outline• low kV imaging in a SEM/FIB, the right
selection of your detector• Applications in Materials Science, porous
samples• Life Science, biological samples…?• Automatic Segmentation• (3D EDX)
0.5 mm
Nb3Sn multifilament superconducting cableNb3Sn superconductor multifilament cable:
14’000 Nb3Sn filaments (diameter ~5um) in Cu matrix
3D FIB/SEM: volume reconstruction
Solid State BSE detectoracceleration voltage:20kV, 15kV
Mechanical polishing <-> Ar ion beam polished
EDX maps
Sn
Cu
Nb
in-chamber ET-detector, SE
in-column “InLens”, SE-detector
in-column, “energy-selective” EsB, BSE-detector
Low kV:acceleration voltage: 1.8 kVNo solid state BSE detector
0.5 mmNb3Sn multifilament superconducting cable
Nb3Sn superconductor multifilament cable:14’000 Nb3Sn filaments (diameter ~5um) in
Cu matrix
1.8kV EsB detector: Materials & orientation contrast
3D FIB/SEM: volume reconstruction
Materials & grain contrast2048x1536x1700, (10x10x10nm voxel), 28hours
10keV100nm300nm
1.6keV(low loss, EsB grid at 1.4kV)
2-3nm(20nm)
1.6keV10nm20nm
HTBSE esc. depthpenetration
What is the spatial resolution of BSE electrons ?
Energy selective BS
27nm300nm 27nm
10keV-0keV 1.6keV-0keV 1.6keV-1.4keV
Scatter range in Nb3Sn:
“Leitmotiv”Isometric voxel size
x = y = z
• Slice thickness (z) = image pixel size (x,y)Z dimension ~ X or Y, typical: 10nm, possible 5nm (3nm)
• Image dimensions / data size (8-bit grey level tiff):– 1024 x 786: 800 slices -> 640 Mb– 2048 x 1572: 1600 slices -> 5 Gb– 3096 x 2358: 3000 slices -> 21 Gb
• Acquisition time ~1min / slice(40-60 slices / hour)-> high S/N ratio, beam current (1-1.5nA), detector efficiency
• Dwell times/pixel 5- 15µsec. (detector signal -> 256 grey levels)
• High throughput: minimise overhead, no tilting, rotating, drift correction
• Z- Resolution: low kV !!!
3D FIB/SEM: volume reconstruction
Pb-free solder SnAgCu:“one detector is not enough”
ETD (SE classic)
InLens: SE low energy
EsB: Energy selective Backscattered
M. Maleki, EPFL-LMAF
10x10x10nm voxel size, 2048x1536x2000
2 images (2x3Mb) / slice …! (DUAL Channel !)
1.6keV, EsB & InLens-SE detector
12Gb data
EsB InLens SE10µm
Phase 1. Dark in EsB image, White in SE-InLens10x10x10nm voxel size, 2048x1536x2000 pixel/slices
2 images (3Mb) / slice …… 12Gb data
10x10x10nm voxel size, 2048x1536x2000 pixel/slices2 images (3Mb) / slice …… 12Gb data
Phase 2: White in SE-InLens - Dark in EsB image
Solid Oxide Fuel Cell cathodeP. Tanasini, LENI
The
righ
t co
ndit
ions
1.87kV, EsB detector
Image:2048x153610nm pixel size
2200 images36hours
Rendering of dense volume
Segmentation and analysis
Comparison with Transmission X-ray Microscopy (TXM)capillary condenser
sample objective ZP
optically‐coupled CCD at image plane
tomographyrotation axis
pin hole
beam stop
LC‐SLC LS‐ZP LZP‐CCD
GeorgeJ.Nelson,WilliamM.Harris,JeffreyJ.Lombardo,JohnR.Izzo,Jr.,andWilsonK.S.Chiu*
Joy C. Andrews, Yijin Liu, and Piero PianettaStanford Synchrotron Radiation LightsourceStanford Linear Accelerator CenterYong S. ChuNational Synchrotron Light Source IIBrookhaven National Laboratory
TXM
FIB
LSM
YSZ
Pore
FIB data down-sampled to 25nm voxel size
GeorgeJ.Nelson,WilliamM.Harris,JeffreyJ.Lombardo,JohnR.Izzo,Jr.,
andWilsonK.S.Chiu*Department of Mechanical Engineering,
University of Connecticut
How do cells attach to a surface..?
SEM: critical point drying, metal coating
FIB Nanotomography of biocompatible materialsK. Dittmar, A. Tourvielle, H. Hofmann EPFL-IMX-LTP, M.Cantoni EPFL-CIME
Biocompatibility of implants (ceramic coatings)Drug delivery from implants
FIB Cross-section of a fixed, epoxy-embedded and stained sample
Does this cell like the coating…?
FIB milling of“hollow” structure
versusFIB milling of
massive “homogenous block”
Image stack: 1024x786 pixel: (10nm image pixel size)
2kV, 60um Aperture, high current, EsB detector (grid 1.5kV)
600 slices, 20nm thickness, milling current 700pA
Rendering of iso-surfaces
Medical steel Ceramic coating: TiO2
Rendering
Cell outer membrane and more…
Volume: 10x8x8 um, 10x10x10nm voxel
Biological samples….brain tissue, resin embedded
Which detector…?In-chamber SE (Everhard-Thornley)
in-Lens SEin-Lens BSE (energy selective)
TEM , 100kVthin (50nm) section
SEM (FIB) , 1.4kV“surface”, (<5nm escape depth)
Brain tissue: synapsevesicles (~50nm), mitochondria
2048 x 1536 x 1600 Volume: 10 x 8 x 8 um voxel: 5x5x5nm2 days of fully automated acqusition, 5 ~GB of Data
Milling current 700pA,20sec. milling , 1.2min.imaging / slice
• Voxel: 7.5x7.5x7.5nm
• Image 3096x2304
• 3300 slices (48hours)
• 23x17x24 um
• 9700um3
• ~7000 synapses
• 23Gb data
Bigger volumes
Automated segmentation of neuronal structuresIlastik v0.5 - Fred Hamprecht, University of Heidelberg
Synapse recognition - Anna Kreshuk
Automated segmentation of neuronal structuresIlastik v0.5 - Fred Hamprecht, University of Heidelberg
• Specimen preparation (fixation, staining, dehydration, resin infiltration same as for BIO-TEM)
• Image contrast and resolution TEM quality
• Stable and reliable automated acquisition (less artifacts than ultra-microtomy)
FIB Nanotomographyin life science
• Specimen preparation (fixation, staining, dehydration, resin infiltration same as for BIO-TEM)
• Image contrast and resolution TEM quality
• Stable and reliable automated acquisition (less artifacts than ultra-microtomy)
FIB Nanotomographyin life science
FIB-NT compared with other 3D-techniques
New possibilities in 3D-microscopy:Combination with quantitative analytical SEM techniques: EBSD, EDX
10x10x10 nm voxel, ZnO film
• isotropic voxel size ~5-10nm• Dwell time ~5-10µsec.• 1 slice, image / min.• HT: 1-2kV• Escape depth of signal (BSE) ≤ 5nm
8x8x8 nm voxel, malaria parasite
New detectors speed up the acquisition !dreaming of 1M counts/sec.
50-100k counts/sec. are more realistic at the moment
The “SDD age”
2008 (“SDD age”), FIB @ CIME, use the full potential of the machine
3D-EDX, Pierre Burdet: Ph.D. Thesis
Goal: FIB Nano-Tomography based on EDX-elemental mapsnew generation of EDX detectors (SDD)Develop algorithms do “deconvolute” the interaction volume of characteristic X-ray
Ion beam
Sample: Al/Zn, Jonathan Friedli, STI-LSMX
o Stack of 269 EDX mapso High tension : 5kVo Voxel size : 20 x 20 x 40 nmo Pixel per map : 256 x 192 (x 269)o Time per slice : 4+1 minuteso Time of acquisition : 24 hours
�200 �100 0 100 200position�nm�
200
400
600
800Intensity
Zn L�
Al K�
evaluation of delocalisation: Model system
– Simulated linescan across the interface normal to y • Signal is shifted towards Al because of the incident angle• Positions of threshold (10 %, 50 % and 90 %) are used to compare with other geometries
Zn Al100 nm
90 %
10 %
50 %
– Potential• NiTi – stainless steel welding
– Biomedical application• Complex microstructure
– Intermetallic phases• Fracture location
– In weld close to NiTi
SS
SSNiTi ?
NiTi
Laser
300µm
Welding process100 m
SSNiTi
Longitudinal cut through welded wires
Jonas Vannod, EPFL-CIME /LSMX
N. L. Abramycheva, V. Mosko, Univ. Ser. 2: Khimiya 40 (1999) 139-143
• Segmentation based on ternary diagram
• Green 4: Between Ni3Ti and Fe2Ti• Red 5: Fe2Ti• Blue 6: -(FeNi)
SE image with high Fe phases
4
6
5
Ternary diagram
z
z
y
2 m
x
• Small microstructure– EDX phases used as mask– Threshold on SE contrast
6a
z
z
y
2 m
2
3
6b
Ternary diagram
x
Phases visualization
123
6
5
4
Ternary diagram
2
3
4
6
5
1
z y
x
2 m
Thank you for your attention