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

Cheiron School
September 2015
SPring-8
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The short wavelength region of the electromagneticspectrum

n = 1 – δ + iβ

δ, β << 1
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Scanning x-ray fluorescence microprobe (µ-XRF)

J.H. Underwood and A.C. Thompson, NIM A266, 296 & 318 (1988).
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3Hard x-ray nanoprobe at SPring-8

Courtesy of S. Matsuyama andK. Yamauchi, Osaka University.
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4Sub-cellular elemental analysis using the hard x-ray
fluorescence nanoprobe at SPring-8

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

Courtesy of S. Matsuyama andK. Yamauchi, Osaka University.
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Hard x-ray microprobe at SPring-8
f1: 25298m	

mm	

Optical microscope	
PIN photodiode	mm	
f2: 15045m	

Undulator	

DCM	
TC1Slit	

Front end	

Courtesy of H. Mimura andK. Yamauchi, Osaka University.

Mirror manipulator	
Ion chamber	
Incident Slit	

SDD	Beam monitor	
Sample & Scanner	

Experimental hutch	

In-situ phase compensation

H. Mimura et al., Nature Physics, 6, 122 (2009)
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Breaking the 10 nm barrier in hard x-ray focusing
In-situ phase compensation

7 nm

Piezo-electric phasecompensator

Focusing mirror with phase error

Focusing mirror with phase error
H. Mimura et al., Nature Physics, 6, 122 (2009)
XRM2010, 16th, Aug, 2010	

Optical configuration for active phase compensation
Knife edge scanner

Fizeau interferometer

8APD@BL29XUL

X-ray

Deformable mirror

Focusing mirror(Mirror B)

Opticalinterferometer(ZYGO GPI)

CCD camera(for alignment)

Focusing mirrorFocalpoint

Ion chamber

Adaptive mirror

150mm

Zooming tube+ X-ray CCD

Zooming tube + CCD

APD placed at dark field

250mm

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

XRM2010, 16th, Aug, 2010	

Synchrotron-based art conservation at ESRFµ-XRF, µ-XRD, µ-XANES

18th Dynasty
Egyptian glassvase (1500 B.C.)
Courtesy ofMarine Cotte(ESRF, Grenoble,France)
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Examples of µ-XANES K-edge spectra
occurring in art materials
SulfurChlorine

Courtesy of Marine Cotte (ESRF, Grenoble, France)
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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)
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Synchrotron radiation x-ray tomographic microscopy
Swiss Light Source (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)
Courtesy of Marco Stampanoni, Swiss Light Source.

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

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

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Hard x-ray 3D x-ray tomography: microvascular
Swiss Light Source architecture of a mouse brain

SRXTM, 25 keV,
15 µm resolutionTomcat Beamline,
Swiss Light Source
M. Stampanoni,
T. Krucker et al.,Adv. Neur. Res. (2008)

Courtesy of Marco Stampanoni, Swiss Light Source.

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Two common soft x-ray microscopes

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Novel zone plates for dual functionality

Courtesy of Anne Sakdinawat, now at SLAC/Stanford
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High resolution zone plate microscopy• Well engineered• Sample indexing• Tiling for larger fieldof view
• Pre-focused• High sample throughput• Illumination important• Phase contrast

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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.)
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Differential interference contrast (DIC) imaging
at nanoscale magnetic edges
705 eV
Below Fe L3

711 eV
Above Fe L3

1 µm

XOR Zone plate
Courtesy of A. Sakdinawat, C. Chang and P. Fischer.

59 nm thick Gd25Fe75 layer
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λ = 1.52 nm (815 eV)Δr = 15 nm
N = 500
D = 30 µm
f = 300 µm
σ = 0.38
0.8 Δr = 12 nm

Cr/Si test pattern (Cr L3 @ 574 eV)
20(2000 X 2000,CheironSchool_Sept2015_Lec3.ppt
104 ph/pixel)

The water window for biological
x-ray microscopy

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Fast freeze cryo fixation strongly mitigatesradiation dose effects

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

Temperature (Celsius)

Fast Freeze
500ΔT = – 50°c
16 ms
Δt-50-100-150-100102030405060708090100Time (milliseconds)

W. Meyer-Ilse, G. Denbeaux, L. Johnson, A. Pearson (CXRO-LBNL)
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Organelle details imaged with cryogenic
preservation and high spatial resolution
Cryo x-ray microscopy of 3T3 fibroblast cells
ER

Filopodia

Nucleus

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)
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Bending magnet radiation used with a soft x-ray microscope
to form a high resolution image of a whole, hydrated mouseepithelial cellhw = 520 eV
32 µm x 32 µm
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)
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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)

λ = 2.4 nm
Courtesy of C. Larabell (UCSF & LBNL)
and M. LeGros (LBNL)
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Bio-nanotomography for 3D imaging of cells
Nanotomography of
Cryogenic Fixed Cells

λ = 2.4 nm (517 eV)
Δr = 35 nm
N = 320NA = 0.034
D = 45 µm
f = 650 µm
σ = 0.64
Resolution = 60 nm

Soft X-Ray Nanotomographyof a Yeast Cell

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

2 µm
DOF

10 µm

Courtesy of Anne Sakdinawat (LBNL & UCB.)
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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.
Δr = 45 nm
Color coding
identifies subcellularcomponents by their
x-ray absorption
coefficients
Courtesy of Carolyn Larabell, UCSF/LBNL.
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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)

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High resolution 3D image of a mouse cellby soft x-ray tomography
517 eV (2.4 nm)
Δr = 25 nm,1° intervals, ±60°29 nm nuclear
double membrane.

Courtesy ofGerd Schneider, BESSYII
and James McNally, NIH.
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Soft x-ray tomography of a single cell

517 eV (2.4 nm)
Δr = 25 nm,
1° intervals, ±60°29 nm nuclear
double membrane.

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

Courtesy of Gerd Schneider, BESSYII and James McNally, NIH.
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Nano-spectroscopy of sodium titanate
nanoribbons
In collaboration with:
C. Bittencourt,
U. Antwerp, Belgium

P. Guttmann et al., Nature Photonics(Dec. 2011).

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Environmental Consequencesof Portland cement
1.5 billion ton of cement

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

Courtesy of Professor Paulo Monteiro, CEE, UC Berkeley

Problem!

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

520 eV, 40 nm – spatial resolution
36Spectromicroscopy: high spatial and high spectral
resolution of surface and thin films

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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 2pOD
1.5

1.0

1 µm

0.55 µm

Protein (gray), Ca, K

0700

705710715

720725Different 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)
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A. Hitchcock, McMaster U.

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Hard x-ray zone plate microscopy• Shorter wavelengths, potentially better spatial
resolution and greater depth-of-field.
• Less absorption (β); phase shift (δ) 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.

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New	  ultra	  high	  aspect	  ra0o,	  high	  eﬃciency,	  hard	  x-­‐ray	  zone	  plates	  for	  high	  spa0al	  resolu0on	  at	  30-­‐50	  keV	  Δr = 100 nm
Δh = 6.6 µm

3.4 µCourtesy of Anne Sakdinawat, SLAC/Stanford; C. Chang and A. Sakdinawat, Nature Communications (27Jun2014).

X-ray focusing with orbital angular momentum control+1 OAMNo OAM-1 OAM

X-rays with various amounts of orbital angular momentum can beproduced using a spiral zone plate structure. These spiral zone platestructures can also be used in place of the objective zone plate in an x-ray
microscope for phase contrast imaging.
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Specialized x-ray diffractive optical elements enable
new science at synchrotron facilities

Spiral zone plate for
producing x-rays withcharge 50 orbital
angular momentum

Uniformly redundant
array for x-ray parallel
holography, alignment,
and other applications

Zone plate for x-ray
differential interference
contrast microscopy

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A lens is not necessarily required

Δrresol. = k1 λ / NA

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

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Coherent diffractive imaging (CDI)

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Lensless imaging of magnetic nanostructuresby x-ray spectro-holography

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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

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