Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Soft and Hard X-Ray Microscopy
David Attwood
University of California, Berkeley

Cheiron SchoolSeptember 2012
SPring-8
1

CheironSchool_Sept2012_Lec3.ppt

The short wavelength region of the electromagneticspectrum

n = 1 –  + i,  << 12

CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 20111

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Two common soft x-ray microscopes

3

CheironSchool_Sept2012_Lec3.ppt

Novel diffractive optics
Soft x-ray zone plate

DIC microscopy

Spiral zone plate for phase contrast

Cubic phase plate for DOF

Courtesy of Anne Sakdinawat, Chang Chang, Weilun Chao and Erik Anderson (LBNL & UCB.)
CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 201142

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

 = 1.52 nm (815 eV)
r = 15 nm
N = 500
D = 30 m
f = 300 m
 = 0.38
0.8 r = 12 nm

Cr/Si test pattern (Cr L3 @ 574 eV)(2000 X 2000,CheironSchool_Sept2012_Lec3.ppt
104 ph/pixel)

5Novel zone plates for specific functionality

Courtesy of Anne Sakdinawat, UC Berkeley
6

CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 20113

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

High resolution zone plate microscopy• Well engineered• Sample indexing• Tiling for larger fieldof view• Pre-focused• High sample throughput• Illumination important• Phase contrast

7

CheironSchool_Sept2012_Lec3.ppt

The water window for biological
x-ray microscopy

8

CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 2011

4

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Fast freeze cryo fixation strongly mitigatesradiation dose effects

Helium passes through LN, is cooled,
and directed onto sample windows

Temperature (Celsius)

Fast Freeze
500T = – 50°c
16 ms
t

-50
-100

-150
-10

0

10

20

30

40

50

60

70

80

90

100

Time (milliseconds)

W. Meyer-Ilse, G. Denbeaux, L. Johnson, A. Pearson (CXRO-LBNL)
9

CheironSchool_Sept2012_Lec3.ppt

Organelle details imaged with cryogenic
preservation and high spatial resolution
Cryo x-ray microscopy of 3T3 fibroblast cells
ERFilopodiaNucleus

Cell border
Nucleoli
C. Larabell, D. Yager, D. Hamamoto, M. Bissell, T. Shin (LBNL Life Sciences Division)
W. Meyer-Ilse, G. Denbeaux, L. Johnson, A. Pearson (CXRO-LBNL)
10

CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 2011

5

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Bending magnet radiation used with a soft x-ray microscope
to form a high resolution image of a whole, hydrated mouseepithelial cellhw = 520 eV32 m x 32 m
Ag enhanced Au labelingof the microtubule network,color coded blue.
Cell nucleus and nucleoli,
moderately absorbing,coded orange.
Less absorbing aqueous
regions coded black.
W. Meyer-Ilse et al.
J. Microsc. 201, 395 (2001)
Courtesy of C. Larabell and W. Meyer-Ilse (LBNL)
11

CheironSchool_Sept2012_Lec3.ppt

Bio-nanotomography for 3D imaging of cells
Nanotomography ofCryogenic Fixed Cells

Soft X-Ray Nanotomographyof a Yeast Cell

Courtesy of G. Schneider (BESSY)
Surf. Rev. Lett. 9, 177 (2002)

 = 2.4 nm
Courtesy of C. Larabell (UCSF & LBNL)
and M. LeGros (LBNL)
12

CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 2011

6

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Bio-nanotomography for 3D imaging of cells
Nanotomography ofCryogenic Fixed Cells

 = 2.4 nm (517 eV)
r = 35 nm
N = 320NA = 0.034
D = 45 m
f = 650 m
 = 0.64
Resolution = 60 nm

Soft X-Ray Nanotomographyof a Yeast Cell

 = 2.4 nm
Courtesy of C. Larabell (UCSF & LBNL)
and M. LeGros (LBNL)
13

CheironSchool_Sept2012_Lec3.ppt

Small DOF limits resolution for thick samples

Lateral Resolution =C. Larabell and M. LeGros,Molec. Bio. Cell 15, 957 (2004)

Depth of field == 28 nm 60 nm= 2 m

2 m
DOF

10 m

Courtesy of Anne Sakdinawat (LBNL & UCB.)
14

CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 20117

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Nanoscale 3-D biotomography
Mother daughter yeast cells just before separation
2-D slice from 3-D
Tomogram. Images
every 2°, 180° dataset, several minutes.
r = 45 nm

Color codingidentifies subcellularcomponents by their
x-ray absorption
coefficients
Courtesy of Carolyn Larabell, UCSF/LBNL.
15

CheironSchool_Sept2012_Lec3.ppt

Applications of soft x-ray microscopy
Biotomography at 60 nm resolution• Cryofixation• 2° angular intervals• Depth of focus limits resolution• New XM-2 dedicated to biologicalapplications, will become major
facility worldwide to draw biologists
to this evolving capability

Courtesy of C. Larabell (UCSF & LBNL)

16

CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 20118Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

High resolution (29 nm), 3D image of amouse cell by soft x-ray tomography
517 eV (2.4 nm)
r = 25 nm,1° intervals, ±60°29 nm nuclear
double membrane.

Courtesy ofGerd Schneider, BESSYII
and James McNally, NIH.
17

CheironSchool_Sept2012_Lec3.ppt

High resolution (29 nm), 3D imageof a mouse cell by soft x-ray tomography
2D slice from 3D data set
Details: 517 eV (2.4 nm)
r = 25 nm, 1° intervals, ±60°.
Note 29 nm nuclear membrane.
3D rendering

Courtesy of Gerd Schneider, BESSYII and James McNally, NIH.
18

CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 2011

9

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Magnetic x-ray microscopy using x-ray
magnetic circular dichroism (XMCD)

Magnetic X-Ray Microscopy•High spatial resolution in transmission•Bulk sensitive (thin films)•Complements surface sensitive PEEM
100 nmlines &spaces•Good elemental sensitivity•Good spin-orbit sensitivity•Allows applied magnetic field•Insensitive to capping layers•In-plane and out-of-plane measurements

1 mCourtesy of P. Fischer, (MPI, Stuttgart) and G. Denbeaux (CXRO/LBNL)
CheironSchool_Sept2012_Lec3.ppt

19

Magnetic domains imaged at different
photon energies
FeGd Multilayer

Contrast
reversal

1 m = 704 eV
below Fe L-edges

 = 707.5
eV
Fe L3-edge

 = 720.5
eV
Fe L2-edge

P. Fischer, T. Eimueller, M. Koehler (U. Wuerzberg)
S. Tsunashima (U. Nagoya) and N. Tagaki (Sanyo)
G. Denbeaux, L. Johnson, A. Pearson (CXRO-LBNL)
CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 20112010

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Magnetic recording of nanomagnetic patterns
to 15 nm spatial resolution
CoCrPt alloy
Co L3-edge at 778 eV(1.59 nm)

200 nmCourtesy of Peter Fischer (LBNL)
P. Fischer et al., Mat. Today 9, 26 (2006).

CheironSchool_Sept2012_Lec3.ppt

21

Time resolved studies of vortex dynamics inpatterned permalloy thin films
Pump and Probe setup requires:•Pump: Current pulse to “pump” sample•Probe: X-ray pulses (70ps) from ALS 2
Bunch mode•Perfect repeatability of dynamics

Sample:50 nm thick 2μmx4μm
permalloy (Ni80Fe20)
100nm thick gold waveguide(I along waveguide generates field to pumpsample)

BeforePump(t=0)

t=+1 ns

B.L. Mesler, P. Fischer,W. Chao, E. H. Anderson,
D.H. Kim J. Vac. Sci. Technol. B 25, 2598 (2007).

t=+1.6 ns

CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 2011

2211

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Differential interference contrast (DIC) imaging
at nanoscale magnetic edges
705 eVBelow Fe L3711 eV
Above Fe L3

1 m

XOR Zone plate

59 nm thick Gd25Fe75 layer

Courtesy of A. Sakdinawat, C. Chang and P. Fischer.
CheironSchool_Sept2012_Lec3.ppt

23

Environmental Consequencesof Portland cement
1.5 billion ton of cement

Problem!

Generates 1.5 billion
ton of CO2
Responsible for 7%
CO2 production inthe world

Courtesy of Professor Paulo Monteiro, CEE, UC Berkeley

Cheiron School, Japan, 29 September 201112

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Nanoscale x-ray imaging of cement processes:
early hydrates forming during the pre-induction
period1 μm

Early hydrates(Sheaf of wheat)

Grain

C3S hydrated for 34 min. insaturated lime and calcium
sulfate at w/c = 5, 1 s
exposure time, 516 eV, scalebar 1μm.
Courtesy of Professor Paulo Monteiro, CEE, UC Berkeley

Nanoscale x-ray imaging of cement processes

13 min34 min50 min10 min20 min1 hr 49 min

10 min34 min1 hr 8 min

Courtesy of Professor Paulo Monteiro, CEE, UC Berkeley

Cheiron School, Japan, 29 September 2011

520 eV, 40 nm – spatial resolution
2613

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Spectromicroscopy: high spatial and high spectral
resolution of surface and thin films

27

CheironSchool_Sept2012_Lec3.ppt

Biofilm from Saskatoon RiverRESULTS•Ni, Fe, Mn, Ca, K, O, C elemental map,
(there was no sign of Cr.)•Different oxidation states for Fe and Ni

Fe 2p

OD
1.5

1.0

1 μm

0.55 μm

Protein (gray), Ca, K

0700705

710715720

725

Different oxidation states (minerals) found for Fe & Ni

Tohru Araki, Adam Hitchcock (McMaster University)
Tolek Tyliszczak, LBNL
Sample from: John Lawrence, George Swerhone (NWRISaskatoon), Gary Leppard (NWRI-CCIW)
28

CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 201114

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

A. Hitchcock, McMaster U.

29

CheironSchool_Sept2012_Lec3.ppt

Patterned polymer photoresists
ALS-MES 11.0.2

M.K. Gilles, R. Planques, S.R. Leone
LBNL
Samples from B. Hinsberg, F. Huele
IBM Almaden

280 eV

290 eV

Exposure to UV light results in loss of carbonyl peak

Map chemical spectra taken of pure samples
onto a sample containing both componentsCourtesy of Mary Gilles, LBNL
CheironSchool_Sept2011_Lec3.ppt

Cheiron School, Japan, 29 September 2011

303015

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Hard x-ray zone plate microscopy• Shorter wavelengths, potentially better spatial
resolution and greater depth-of-field.
• Less absorption (); phase shift () dominates,
higher efficiency.
• Thicker structures required (e.g., zones), higher
aspect ratios pose nanofabrication challenges.
• Contrast of nanoscale samples minimal; will require
good statistics, uniform background, dose mitigation.

31

CheironSchool_Sept2012_Lec3.ppt

nanoXCT: Schematic and Challenges
X-ray Zone-plate Lens

Challenges for achieving nm scale resolution:• High resolution objective lens: limiting the ultimate resolution• High numerical aperture condenser lens:• Detector: high efficiency for lab. source and high speed for synchrotron sources• Precision mechanical system
Courtesy of Wenbing Yun and Michael Feser, Xradia

Cheiron School, Japan, 29 September 2011

32

16

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Xradia nanoXCT: Sub-25 nm Hard X-ray Image
Xradia Resolution Pattern• 50 nm bar width• 150 nm thick Au• 8keV x-ray energy• 3rd diffraction order

F. Duewer, M. Tang,
G. C. Yin, W. Yun,
M. Feser, et al.

Xradia nano-XCT
8-50S installed at
NSRRC, Taiwan

400 nm33

Elemental contrast by tuning energy across thecopper absorption edge ( Guan-Chian Yin et al)

8.4 keV

9.5 keV

9.5

8.4

Intensity difference between
E = 8.4 keV and 9.5 keV
34

Cheiron School, Japan, 29 September 201117Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Tomography of a Tungsten plug with “keyhole” at ~60 nm spatial resolution

Xradia and NSRRC

APL. 88, 241115 (2006)

Scanning x-ray fluorescence microprobe (-XRF)

J.H. Underwood and A.C. Thompson, NIM A266, 296 & 318 (1988).
CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 2011

3618

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

X-ray microprobe at SPring-8
Optical microscope
PIN photodiodemm
f1: 252m

m
f2: 15098m
45m

Undulator

DCM
TC1SlitSDD

Mirror manipulator
Ion chamber
Incident Slit

Beam monitor
Sample & Scanner

Experimental hutch

S. Matsuyama et al.,Rev. Sci. Instrum.
77, 103102 (2006)

Front end

Courtesy of K. Yamauchi andH. Mimura, Osaka University.
37

CheironSchool_Sept2012_Lec3.ppt

Sub-cellular elemental analysis using the hard x-ray
fluorescence microprobe at SPring-8

S. Matsuyama et al.,
X-Ray Spectrom.(2010).

Courtesy of K. Yamauchi andH. Mimura, Osaka University.
38

CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 201119

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

X-ray microprobe at SPring-8mm
f1: 252

Optical microscope
PIN photodiodemm
f2: 15098m
45m

Undulator

TC1Slit

SDD

Mirror manipulator

DCM

Ion chamber
Incident Slit

Front end

Courtesy of K. Yamauchi andH. Mimura, Osaka University.

Beam monitor
Sample & Scanner

Experimental hutch

In-situ phase compensation

H. Mimura et al., Nature Physics, 6, 122 (2009)39

CheironSchool_Sept2012_Lec3.ppt

Breaking the 10 nm barrier in hard x-ray focusing

40

In-situ phase compensation

7 nmPiezo-electric phasecompensator

Focusing mirror with phase error
rror

Focusing mirror with phase error
H. Mimura et al., Nature Physics, 6, 122 (2009)  

Cheiron School, Japan, 29 September 201120

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

*-$&)(!$".+-$)(!)+-$/ *#, )'* (,-$)(($!  " ,(( +$1 .$(- +! +)' - +41 +0 !)+'&&
 '$++)+
'$++)+).,$("'$++)+))$++)+ *-$&$(- +! +)' - +

' +!)+&$"(' (-).,$("'$++)+)&*)$(-)(#' ++))'$("-.
'$++)+
*-$/
 -$ '$++)+$ ''))'$("-. +0

*& -+%!$ & ''

H. Mimura et al., Nature Physics, 6, 122 (2009)  

Synchrotron-based art conservation at ESRF
-XRF, -XRD, -XANES

18th Dynasty
Egyptian glassvase (1500 B.C.)
Courtesy ofMarine Cotte(ESRF, Grenoble,France)
42

CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 201121

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Examples of -XANES K-edge spectra
occurring in art materials
SulfurChlorine

Courtesy of Marine Cotte (ESRF, Grenoble, France)
43

CheironSchool_Sept2012_Lec3.ppt

18th Dynasty Egyptian glass vase studied for anunderstanding of color and opaqueness in antiquity
1st production of glass objects Egypt (1500 B.C.),
opaque, colored, nanoscale calcium antimonate

Courtesy of Marine Cotte (ESRF, Grenoble, France)
44CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 201122

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Swiss Light Source

Synchrotron radiation x-ray tomographic microscopy(SRXTM)

Tomographic reconstruction of a 500 million
year old fossilized embryo from Southern China
P. Donoghue, S. Bengtson, M. Stampanoni
et al., Nature 442, (2006)

17.5 keV

Courtesy of Marco Stampanoni, Swiss Light Source.

Swiss Light Source

45

Tomographic reconstruction of a 500 million year
old fossilized embryo from Southern China
Markelia hunanensis
relative of modern roundworms
and arthropods

SRXTM, 17.5 keV,
15 m resolution
Tomcat Beamline,
Swiss Light Source
P. Donoghue, S. Bengtson, M.
Stampanoni et al., Nature 442, (2006)
Courtesy of Marco Stampanoni, Swiss Light Source.

Cheiron School, Japan, 29 September 2011

46

23

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

TOMCAT Microscope
Swiss Light Source

1 micron @ 10% MTF reached routinely
Courtesy of Marco Stampanoni, Swiss Light Source.

47

TOMCAT Microscope
Swiss Light Source

To CCD…

scintillator

objectivemirror

X-ray beam

rotation stage

centering device1 micron @ 10% MTF reached routinely
Courtesy of Marco Stampanoni, Swiss Light Source.

Cheiron School, Japan, 29 September 2011

48

24

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Swiss Light Source

Hard x-ray 3D x-ray tomography: microvasculararchitecture of a mouse brain

SRXTM, 25 keV,
15 m resolution
Tomcat Beamline,
Swiss Light Source

M. Stampanoni,
T. Krucker et al.,Adv. Neur. Res. (2008)

Courtesy of Marco Stampanoni, Swiss Light Source.

49

A lens is not necessarily requiredrresol. = k1  / NA

“Lensless” coherent diffraction imaging (CDI) is being
aggressively pursued.

50

CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 201125

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Coherent diffractive imaging (CDI)

51

CheironSchool_Sept2012_Lec3.ppt

Coherent diffractive imaging (CDI) examples
Femtosecond diffractive imaging
with a free electron laser

CDI with laboratory scalehigh harmonic generation (HHG)

Flash FEL,  = 32 nm (39 eV)
25 fsec, 1012 photons/pulse
62 nm resolution

HHG, n = 27, = 29 nm (43 eV )94 nm spatial resolutionChapman, et al. Nature Physics (2006)

Sandberg, et al. PNAS (2008)

Synchrotron based CDI of 100 nm Au spheres

Synchrotron CDI of Au particles
 = 0.083 nm (15 keV),
5 nm “resolution”
Schroer, et al. PRL (2008)

Cheiron School, Japan, 29 September 2011

Synchrotron based CDI of a freeze dried yeast cell
CDI
ALS/9.0.1
 = 1.66 nm(750 eV)

STXM
NSLS/X1 = 2.3 nm(540 eV)
42 nm
resolution

Tilted 3°

Tilted 4°

Shapiro, et al. PRL (2005)

30 nm fine features
52

CheironSchool_Sept2012_Lec3.ppt

26

Professor David Attwood, UC Berkeley
Soft and Hard X-Ray Microscopy

Lensless imaging of magnetic nanostructuresby x-ray spectro-holography

53

CheironSchool_Sept2012_Lec3.ppt

Lectures online at www.youtube.com

Amazon.com

UC Berkeley
www.coe.berkeley.edu/AST/sxreuv
www.coe.berkeley.edu/AST/srms
www.coe.berkeley.edu/AST/sxr2009
54

CheironSchool_Sept2012_Lec3.ppt

Cheiron School, Japan, 29 September 201127