FIB-Nanotomography from Materials Science to Life Science · 2013-06-17 · Crossbeam Microscopy Metz 2013 CIME Centre Interdisciplinaire de Microscopie Electronique (EPFL-CIME) FIB-Nanotomography

Crossbeam Microscopy Metz 2013

CIME

Centre Interdisciplinaire de Microscopie Electronique(EPFL-CIME)

Centre Interdisciplinaire de Microscopie Electronique(EPFL-CIME)

FIB-Nanotomographyfrom Materials Science to Life Science

Marco Cantoni, Graham KnottEcole Polytechnique Fédérale Lausanne


central facility for electron microscopyo 6 TEMs:

CM-12, TECNAI Spirit, TF-20, OSIRISCM300, JEM2200FS, soon TITAN3

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 Electronique

Director: 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, freezesubstitution…)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 CantoniSince 2007: BIO‐EM: Graham Knott


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


outline

1. Introduction to FIB Nano-Tomography (FIB-NT)

2. low kV imaging in a SEM/FIB, the right selection of your detector, resolution in z direction

3. Applications in Materials Science- easier segmentation with multiple detectors- porous samples

4. Applications in Life ScienceAtlas 3D


FIB-Nanotomography

Journal of Microscopy, Vol. 216, Pt 1 October 2004, pp. 84–95


“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 “Image” ~1min / slice (~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


2) low kV imaging in a SEM/FIB, the right selection of your detector

“direct” tomography: cutting and imaging

What you (detector) see is what you get !


0.5 mmNb3Sn multifilament superconducting cable

Nb3Sn superconductor multifilament cable:14’000 Nb3Sn filaments (diameter ~5um) in Cu matrix

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


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

10keV‐0keV 1.6keV‐0keV 1.6keV‐1.4keV

Scatter range in Nb3Sn:

"monte CArlo SImulation of electroN trajectory in sOlids".http://www.gel.usherbrooke.ca/casino/index.html


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 contrast

2048x1536x1700, (10x10x10nm voxel), 28hours


Tribology: wear trace on steelTribo-corrosion

J. Perret, S.Mischler IMX-LMCHGrain orientation contrast of small grains(grain size < 100nm)

SE (Evervard-Thornley)

BSE

Orientation contrastidentification of grain texture

10um

10um

2048x1536x1200 volume: 20x15x12um10x10x10nm voxel



growth of ZnO films, photovoltaics

10x10x10nm voxel size, 2048x1536x2200 pixel/slices

C. Balif, S. Nicolay, D. Alexander

Orientation contrastidentification of grain texture





Solid Oxide Fuel Cell cathodeP. Tanasini, LENI

Porous, non-conducting:Resin infiltration, gold coating


The

righ

t co

ndit

ions

1.87kV, EsB detector


Image:2048x153610nm pixel size

2200 images36hours


Segmentation and analysis


Comparison with Transmission X-ray Microscopy (TXM)capillary condenser

sample objective ZP

optically‐coupled CCD at image plane

tomography

rotation 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 PieroPianettaStanford 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


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‐InLens

10x10x10nm voxel size, 2048x1536x2000 pixel/slices2 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)



FIB/SEM Nanotomography, volume reconstructionTypical voxel sizes

Cement, (10nm)3 voxel Solar cell: ZnO, (10nm)3 voxel Nb3Sn, (10nm)3 voxel

IC, (10nm)3 voxel clay, (10nm)3 voxel


FIB-NT compared with other 3D-techniques

New possibilities in 3D-microscopy:Combination with quantitative analytical SEM techniques: EBSD, EDX

• Voxel size ~5‐10nm• Dwell time ~10µsec.• 1 slice, image / min.• HT: 1‐2kV• Escape depth of signal (BSE) ≤ 5nm

3D‐EDX of NiTi/Stainless steelPierre Burdet, Marco Cantoni


FIB-NT in Life-Science


How do cells attach to a surface..?

SEM: critical point drying, metal coating

FIB Nanotomography of biocompatible materials

K. Dittmar, A. Tourvielle, H. Hofmann EPFL-IMX-LTPM.Cantoni, Graham Knott 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

versus

FIB 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



Segmentation based on grey levels

Medical steel Ceramic coating: TiO2


Cell outer membrane and more…



more than 1 cell:brain research a big Challenge in Life Science

~1’000’000’000 neurons~1’000’000’000’000 connections

Simulation of brain functionwith 10’000 neurons


Serial thin sectioning

55 nm sections floated on water

Tissue blocks are sectionedHIGH RESOLUTION NEEDEDTEM thin sections prepared by

Ultramicrotomy

Graham Knott, UNIL/EPFL-CIME


brain tissue, resin embeddedPrepared as for ultramicrotome

cutting (serial section TEM)


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

Energy loss: 0.2kV

10nm


2048 x 1536 x 1600 Volume: 10 x 8 x 8 um voxel: 5x5x5nm

2 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

Big volumes


smaller voxel: 3nm x 3nm x 3nm

X – Y plane (image) Y -Z plane, (virtual)



Reconstruction:Christel Genoud

2weeks of work


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-ssTEM)

• Image contrast and resolution TEM quality

• Stable and reliable automated acquisition (less artifacts than ultra-microtomy)

FIB Nanotomographyin life science


In vivo image of layer 5 pyramidal dendrites

In fixed section After laser branding

Mouse, GFPm line, green fluorescent protein (GFP)2‐photon imaging done by Anthony Holtmaat, Geneva


Trimmed block ready for serial sectioning

Final block for cutting serial sections


Correlated 2 photon and FIBSEM microscopy



FIB-NT compared with other 3D-techniques

• isotropic voxel size ~3/4/5-10nm• dwell time ~5-10µsec., 1 slice, 1

image / min.• typ. volumes: (5….30µm)3

• HT: 1-2kV• Escape depth of signal (BSE) ≤ 5nm• No cutting artefacts• Cuts virtually everything (resin,

metal, ceramics)

Malaria parasite in red blood cells8x8x8 nm voxel


Have fun !with your Auriga 40

Thank you

Documents

FIB-Nanotomography from Materials Science to Life Science · 2013-06-17 · Crossbeam Microscopy Metz 2013 CIME Centre Interdisciplinaire de Microscopie Electronique (EPFL-CIME) FIB-Nanotomography