FIB-Nanotomography in 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)
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Characterization of porous materials by Focused Ion Beam Nano-Tomography
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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
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)
Joy C. Andrews, Yijin Liu, and Piero PianettaStanford Synchrotron Radiation LightsourceStanford Linear Accelerator CenterYong S. ChuNational Synchrotron Light Source IIBrookhaven National Laboratory
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