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