Spectro-microscopies

Jean SusiniEuropean Synchrotron Radiation Facility,BP220, F-38043 Grenoble Cedex , France

School on X-Ray Imaging TechniquesESRF, Grenoble, 5-6 February, 2007

Spectro-microscopy ?

Imagingtaking a picture

Spectroscopyspread the light

Photometrymeasure how much light

Spectro-microscopies

E

X-Ray Fluorescence(XRF)

Fixed

ΔE

E

X-Ray Absorption (XAS)

ΔE

E1, E2, …

E

Infrared Absorption (FTIR)

ΔEEnergy

Sig

nal i

nten

sity

2 - Spectroscopy: ΔE

1 - Microscopy: < Δx, Δy < 1µm

The interactions photons-matter provide several contrast mechanisms

Photoionization,Compton scattering

Electron shift to excited states, valence-band photoemission

Molecular rotation, torsion

Molecular vibrational states

Symmetricstretching

Anti-symmetricstretching

Bending

Relative energy of electromagneticradiation

Dissociation

Vibration

Rotation

X-rays

ultraviolet

visible

infrared

Relevant interactions of X-ray photons with matter

An electron in the given shell (e.g. K) is ejected from the atom by an external primary excitation x-ray photon, creating a vacancy.

The excitation energy from the inner atom istransferred to one of the outer electrons causing itto be ejected from the atom.

“Auger” Electron

Higher energy core electron fills empty electron level, and ejects an x-ray photon of fixed energy.

X-ray fluorescence photon

X-ray Absorption

Synchrotron based micro-imaging techniques

X-ray spectroscopy

X-rayDiffraction & scatteringX-ray

fluorescence

InfraredFTIR-spectroscopy

• Composition• Quantification

Trace element mapping

• Short range structure• Electronic structure

Oxidation/speciation mapping• Molecular groups & structure

High S/N for spectroscopyFunctional group mapping

• Long range structureCrystal orientation mappingStress/strain/texture mapping

Phase contrast X-ray imaging

• 2D/3D MorphologyHigh resolutionDensity mapping

Broad emission spectrumWavelength/energy tunability

Low emittanceBrightnessCoherence

Synchrotron based micro-imaging techniquesX-ray

fluorescence

X-ray spectroscopy

Penetration• Bulk info

Long working distance• Space for sample

Long depth of field• 3D imaging

X-rayDiffraction & scattering

InfraredFTIR-spectroscopy

• Composition• Quantification

Trace element mapping

• Short range structure• Electronic structure

Oxidation/speciation mapping• Molecular groups & structure

High S/N for spectroscopyFunctional group mapping

• Long range structureCrystal orientation mappingStress/strain/texture mapping

Phase contrast X-ray imaging

• 2D/3D MorphologyHigh resolutionDensity mapping

Synchrotron based hard X-ray microprobe

Photodiode

Undulator

monochromator

X-ray lens

Aperture

Sampleraster scanned

Fluorescence detector

CCDDiffraction

CCDAlignment & imaging

• Spatial resolution : 0.05-2µm • Spectral resolution : ΔE/E ~ 10-2 - 10-4

• Averaged flux : 109 – 1013photons/s/µm2

LN2 dewarfor cryo

sample stage

Transmission detector stage

BEAM DIRECTION

Fluorescence detectors

Visible light microscope mechanics

Sample scanning stages

Overview of the ID22 end-station

• KB mirror system

• Sample stage

• Video microscope

• Si(Li) 1 element

• Si(Li) 13 elements

• Diffraction camera

• Imaging camera

X-ray Fluorescence: a brief reminder

Element specific

Co-localization

Quantification

X-rayKen

ergy

continuum

ML

Photo-electroncontinuum

ML

K

KαKβ

Eexc

Energy dispersivedetector

energy

Inte

nsity

X-ray Fluorescence: a brief reminder

Element specific

Co-localization

Quantification

X-rayKen

ergy

continuum

ML

Photo-electroncontinuum

ML

K

KαKβ

90°

Hor. Linearpolarization

monochromaticbeam

High flux

• Low scattering (background)• Low detection limit• X-ray absorption spectroscopyXRF geometry on synchrotron beamline

X-ray Fluorescence: a brief reminder

Element specific

Co-localization

Quantification

High sensitivity

Chemical info.

X-rayKen

ergy

continuum

ML

Photo-electroncontinuum

ML

K

KαKβ

90°

Hor. Linearpolarization

monochromaticbeam

High flux

• Low scattering (background)• Low detection limit• X-ray Absorption spectroscopy

X-ray Lines and Transitions

J. A. Bearden, Rev. Mod. Phys. 39, 78 (1967)

Photon energies, in electron volts, of principal K-, and L- shell emission lines

From http://xdb.lbl.gov/xdb.pdf

The fluorescence yield ωω is the probability that an x-ray photon will be

emitted as a result of ionization of a specific shell. For a given series of X-ray lines (e.g., the K series), ω is numerically equal to the ratio of K photons escaping from the atom to the ratio of original K-shell ionizations.

The fluorescence yield for K lines increases monotonically as a function of atomic number, and algebraic models accurately predict the empirical data.

This factor means that the sensitivity of the x-ray method decreases for the lighter elements. The decrease, however, is partly compensated by the higher photo-absorption cross sections in light elements.

Example:• zinc (Z=30) ωk = 0.45

• sodium (Z=11) ωk = 0.02

M.O. Krause, J. Phys. Chem. Ref. Data, 89,(1979)

Atomic number

Fluo

resc

ence

yie

ld ω

Phytoextraction in hyper accumulator plants

Green and low cost strategy for soil cleaning

Requires knowledge on the mechanisms of metal accumulation

M.P. Isaure et al., Biochimie, 2006

Anthropogenic activities

Cesium (Cs)Nuclear activities

Cadmium (Cd)Industrial (mining), agricultural

activities (fertilizers)

XRF mapping in Trichomes of Arabidopsis Thaliana

P

70 µm

S Cd K Ca

Eex: 5.8 keV, probe size: 0.4x0.2μm2, dwell time: 800 ms/pixel.

M.P. Isaure et al., Biochimie, in press

Cs accumulation in Arabidopsis Thaliana

10μm

20 µm

Eex: 5.8 keV, probe size: 0.4x0.2μm2, dwell time: 800 ms/pixel.

M.P. Isaure et al., Biochimie, 2006

Fluorescence tomography (3D-µXRF)

Pixel- by-pixel acquisition 2D-Slice or 3D-VolumeSinogram(s)

2D X-rayFocusing device

EDS

Sample

Beam

Diode #1

Diode #2

Fluorescence tomography (3D-µXRF)

The reconstruction problem is far more difficult compared to transmission tomography

self absorption corrections

µ(Ea, x) is a priori unknown

weak fluorescence signal for light elements

Pixel- by-pixel acquisition 2D-Slice or 3D-VolumeSinogram(s)

Fluorescence tomography (3D-µXRF)

Pixel- by-pixel acquisition 2D-Slice or 3D-VolumeSinogram(s)

Algorithmic solution:Optimal estimation of attenuation maps by combination of transmission, fluorescence and Compton tomographies

• B. Golosio et al., J. Appl. Phys. 94(1), 145 (2003)

Geometrical solution:Collimation of the detection angle to define a voxel: confocal geometry

• B. L. Vincze et al., Anal.Chem., 76(22) (2004)

Combining several signals

Photoelectric contribution to the absorption: σphoto~Z4

Transmission tomography• Provides the absorption coefficient

distribution µ(E0, x)

Compton tomography• provides information on the electronic density spatial

distribution

Compton scattering cross section σCompton~Z

Fluorescence tomography• information on the internal spatial

distribution of specific elements

QUANTIFICATION?Integration of the information(transmission+Compton+fluorescence)

B. Golosio et al., J. Appl. Phys. 94(1), 145 (2003)

• Foraminifera

Single-cell marine animals

Fossilized calcium carbonate shells

Proxies for past oceanic conditions (climate)

200 μm

Globorotalia inflata

X-ray fluorescence tomography

P. Bleuet et al., SPIE, 2006

X-ray radiograph

Transmission Ca Fe

Cu Zn Ni

Fluo-tomography: 3D volume rendering

Foraminifera (Globorotalia inflata)

Quantification on 2D slicesTransmission

50μm 0

50

cm-1

Compton

0

0.69

g/cm-3

50μm

0.009

0

g/cm-3

0.009

0

S0.008

0

g/cm-3

Cl0.522

0

g/cm-3

Ca

0.009

0

g/cm-3

Zn0.001

0

g/cm-3

Cu

X-ray Fluorescence Tomography – “confocal geometry”

Polycapillary

Coincidingfocii

FocussedX-ray beam

XRFdetector

Spatial resolution: 5-15µm

L. Vincze et al., Anal.Chem., 76(22) (2004)

Si(Li) detector

I0

Optical microscope

90°

Focusing lensSample

CCDAlignment

& imaging

ID18F

3D-Confocal XRF for tomography

diamond KK200

inclusion #2

Information on the geochemical environment and conditions in which the diamond was formed

Chemistry at several 100km depth

• Voxel-size: 7 × 7 × 7 µm3

• Full 3D analysis: 28830 spectra (voxel)• dwell time: 3 s/voxel

Y, Zr Sr

3D-rendering based on measured Sr, Y and Zr-Kα distributions

L. Vincze et al., Anal.Chem., 76(22) (2004)F.E. Brenker et al., EPSL, 236, (2005)

From 3D-confocal XRF to quantification (Vekemans et al. JAAS, 2004)

Element Phase 1 Phase 2Ca 29.6 % 18.6 %Sr 431 ppm 48 ppmY 9 ppm 61 ppmZr 35 ppm 233 ppm

Zr,Y rich phaseWalstromite structured CaSiO3

Sr-rich phaseLamite Ca2SiO4

depth Z

horizontal X

verti

cal Y

Sr, Th, Y/Zr composed image

unusual high Ca concentration

“existence of a Ca-rich diamond

reservoir at depth below 300 km”

Quantification using NIST SRM 613

CaMn

Fe

PbHf Th

SrZr

Y

Phase 1 (Sr)Phase 2 (Y,Zr)

Zr

YScatter peaks

Ca: 260 ppm, Sr: 1.2 ppm, Zr: 1.1 ppm

Detection limits (LT=100 s):

X-ray Absorption Near Edge Structure (XANES)

• Local site symmetry• Oxidation state • Orbital occupancy

1s

Continuumstates

Rydbergstates

σ

σ∗

ππ∗

X-rayEnergy

Nor

m. f

luo

yiel

d or

-alo

gI/I

0

XANES:electronic transitions to bound states,

nearly bond states or continuum

Chromium compounds

N

24 electrons

- 6 electronsCr VI (hexavalent)

- 3 electronsCr III (trivalent)

Chromium in cells

0

1(a.u.)

Potassium Cr (total)

Cr(VI)

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5.96 5.98 6 6.02 6.04 6.06 6.08 6.1

CrCl3PbCrO4

Tran

smitt

ed in

tens

ity (a

.u.)

Energy (keV)

R. Ortega et al. , Chem. Res. Toxicol., 5, (2005)

Micrograph

10 µm

Micro-XANES at the Sulfur K-edge in Pinna Nobilis

Mineral prisms

Organic matrix

0

0.2

0.4

0.6

0.8

1

2460 2470 2480 2490 2500

Energy (keV)

2.460 2.470 2.480 2.490 2.500

Energy (keV)

MethionineC-S-C

ChondroitineC-SO

4

CystineC-S-S-C

CysteineH-S-C

Y. Dauphin et al., Comparative Biochemistry and Physiology, Part A 132, (2002)

Chemical mapping of Sulphur species in Pinna Nobilis

Visible light microscope

Y. Dauphin et al., Journal of Marine Biology, 142, (2003) Y. Dauphin et al., J. Structural Biology, 132, (2003)

10µm

0.0

0.5 0.1

0.1a.u.

Fluorescence yield

• probe: 0.20x0.30 mm2

• dwell time: 2 sec/pixel

E=2473eV E=2482eV

SEM

Sulfur

Alternative strategy for fast and stable hard X-ray µ-XAS

Two-crystal fixed exit scanning monochromator+ large energy range for EXAFS+ high flux (XRF)+ mature technology- source of non-statistical noises(crystal motions, lack of beam stability, precision)

- relatively slow (10s-1s)

Wavelength (energy) dispersive polychromator+ fast (1s-1µs)+ stable- low flux- limited energy range and less flexible- fluorescence more difficult

ID24-ESRF: an energy dispersive spectrometer on undulator source

VFM1

HFM

VFM2

PLC

I0IF

VFM1

HFM

VFM2

PLC

I0IF

Slits: 50mm/s -> 1000eV/s

10-4< ΔE/E < 10-1

Tuning of flux vs ΔE/E

Scanning the slits = energy scan

1 pixel = 1 EXAFS or NEXAFS spectrum100x100pixels = 10000 spectra in 2 hrs acquisition

With an optimized optical scheme: 300x300nm2

5-15 keV5 x 5 µm2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

7100 7120 7140 7160 7180A

bsor

ptio

n (a

.u.)

Energy (eV)

S. Pascarelli et al., J. of Synchrotron Radiation, 2006

Redox and speciation mapping of metamorphic rocks

Sambagawa (Japan)

P, T conditions of formation of rocks

Thermodynamical models depend critically on oxidation state of Fe

Need for quantitative map of Fe redox at the µm level

S. Pascarelli et al., J. of Synchrotron Radiation, 2006

Redox and speciation mapping of metamorphic rocks

Sambagawa (Japan)

y (µ

m)

50

100

150

50 100 150 200 250 300 350

x (µm)

• Pixel size: 4x4µm2

• 3000spectra (1.5hrs)

0

1

2

7120 7140 7160 7180

Nom

aliz

ed a

bsor

banc

e

Energy (eV)

Chlorite Fe(II)Chlorite Fe(III)

Average

Single pixel

a)

From spectra to speciation mapping ?3 basic criteria:• Edge jump: Fe content• Edge position: Fe oxidation state• Absorbance at a defined energy: Fe speciation

Hei

ght

(µm

)

50

100

150

50 100 150 200 250 300 350Length (µm)

Length (µm)

Hei

ght

(µm

)

50

100

150 0.1

0.2

0.3

0.4

50 100 150 200 250 300 350Length (µm)

7121

7123

7125

7126

7122

7124

50 100 150 200 250 300 350Length (µm)

50

100

150Hei

ght

(µm

)

M. Muñoz et al., G-cubed, (2006)

Edge jumpFe content

Edge positionFe oxidation state

0.0

0.5

1.0

1.5

2.0

2.5

7100 7120 7140 7160 7180

Abs

orpt

ion

(a.u

.)

Energy (eV)

Chlorite Fe2+

Chlorite Fe3+

Phengite

Quartz

single pixel XANES

50 100 150 200 250 300 350Length (µm)

Hei

ght

(µm

)

50

100

150 0.750.800.850.900.95

Absorbance at a defined energyFe speciation

Quartz

Chlor 2+

Chlor 3+ Phengite

Blackening of Pompeian Cinnabar paintings

Red = cinnabar (HgS)

• M. Cotte, J. Susini, ESRF, Grenoble, France

• A. Moscato & C. Gratziu, Università di Pisa, Pisa,Italy

• A. Bertagnini, Istituto Nazionale di Geofisica e Vulcanologia, Pisa, Italy

• M. Pagano, Soprintendenza per i Beni Archeologici del Molise, Campobasso, Italy

Blackening of Pompeian Cinnabar paintings

Red = cinnabar (HgS)

• Excavation started in 1988 and was completed in 1992

• Rapid blackening since 1990

“Villa Sora”, in Torre del Greco near by Pompei

Blackening of Pompeian Cinnabar paintings

no alteration low alteration high alteration

• Cinnabar β−cinnabar?• Role of the lime substrate (CaCO3)?• Superficial or deep alteration?• Role of sulfur?• Other elements (e.g. cera punica) ?

X-ray fluorescence and elemental mapping

Energy (keV)

Cou

nts

SHg

AlMg

Na

SiCl

Compton

K

Rayleigh

1.2 1.6 2.0 4.03.63.22.82.4

104

103

102

101

Raw dataFit

(PyMca by A. Sole, ESRF)

Composition maps by X-ray Fluorescence

Hg Na

Si KAl

min

maxCl

S

S

ClHg Na

Si KAl

2mm

Semi-quantification?N

orm

aliz

ed fl

uore

scen

ce y

ield

(a.u

.)

Energy (keV)

2.46 2.47 2.48 2.49 2.50 2.51

Nor

mal

ized

fluo

resc

ence

yie

ld (a

.u.)

Energy (keV)

2.49 2.51 2.532.45 2.47

Sulfur K-edge XANES

XANES Sulfur K-edge

2.46 2.47 2.48 2.49 2.50

Energy (keV)

Nor

mal

ized

fluo

resc

ence

yie

ld (a

.u.)

HgS (cinnabar)

HgS (metacinnabar)

As2S3

Sb2S3

red Cd2S3

yellow Cd2S3

S8

Hg3S2Cl2

2.46 2.47 2.48 2.49 2.50 2.51 2.52N

orm

aliz

ed fl

uore

scen

ce y

ield

(a.u

.)

Energy (keV)

CaSO4.2H2O

CuSO4.H2O

PbSO4

FeSO4.nH2O

Na3Ca(Al3Si3O12)S

Al2(SO4)3

Sulfides Sulfates

Semi-quantification?N

orm

aliz

ed fl

uore

scen

ce y

ield

(a.u

.)

Energy (keV)

2.46 2.47 2.48 2.49 2.50 2.51

Nor

mal

ized

fluo

resc

ence

yie

ld (a

.u.)

2.45 2.47 2.49 2.51 2.53

Energy (keV)

Fit

Semi-quantification: sulfur compounds

gypsumcorderoite

sulfurcinnabar

Proportion (%)

0 20 40 60 80 100

(n=8)

(n=10)

(n=9)

(n=9)

(n=10)

(n=6)

Nor

mal

ized

fluo

resc

ence

yie

ld (a

.u.)

Energy (keV)

2.46 2.47 2.48 2.49 2.50 2.51

Semi-quantification: Chlorine compounds

2.81 2.82 2.83 2.84 2.85 2.86 2.87

Nor

mal

ized

fluo

resc

ence

yie

ld (a

.u.)

Energy (keV)

calomel

corderoite

terlinguaite

NaCl+KCl+CaCl2

Proportion (%)

0 20 40 60 80 100

(n=5)

(n=11)

(n=9)

(n=7)

(n=10)

Chemical mapping?

Nor

mal

ized

fluo

resc

ence

yie

ld (a

.u.)

Energy (keV)

2.49 2.51 2.532.45 2.47

[Sulfides] ∝I(2.471) - I(2.460)

I(2.510) - I(2.460)

[Sulfates] ∝I(2.482) - I(2.510)

I(2.510) - I(2.460)

2.471 2.482 2.5102.460

∝[re

duce

d su

lfur]

∝[o

xidi

zed

sulfu

r]

Chemical mapping: reduced vs oxidised Sulfur

min

max

Reduced sulfur Oxidised sulfurLight microscopy

SummaryAttributes of multi-keV X-Ray spectro-microscopies

X-ray Fluorescence

Micro-spectroscopy (XANES)

Larger focal lengths (> 20mm) Larger depth of focus (> 100µm)

• Space for sample environment• 3D imaging

Higher penetrationPhase contrast

• Chemical state specificity

• Microscopy on thick samples• Lower radiation damage (?)

• Trace element detection & mapping• Quantitative fluorescence analysis

Multi-technique approach• Micro-Fluorescence• Micro-diffraction• 3D imaging• Spectroscopies

In-situ experimentscontrolled sample environment

A common scientific case : trace element analysis in heterogeneous systems

Biology & MedicineEarth & Planetary Sciences

Environmental Sciences

Oxidation states

Quantification Co-localisationTrace elements

monitoringRedox reactions

2D/3D mappingXRF

XANES

Infrared spectroscopy

Asymmetric stretch2350 cm-1

Vertical bend666 cm-1

Symmetric stretch(not IR active)

Horizontal benddegenerate mode same

motion as above but rotated by 90o

CO2O = C = O

An IR active mode must involve a change in the dipole moment of the molecule = charge imbalance in the molecule

absorption =-log (It /Io)Io It

Infrared spectroscopy

Asymmetric stretch2350 cm-1

Vertical bend666 cm-1

Symmetric stretch(not IR active)

Horizontal benddegenerate mode same

motion as above but rotated by 90o

CO2O = C = O

Each functional group has an ensemble of motions ( vibrational) specificof the molecular group (fingerprint)

These motions ( or vibrational frequencies) are detected under« resonant » excitation in the energy domain 0.495 eV-0.062eV or 2.5 to 20µm or 4000-500 cm-1

There are databanks of spectra, which allow a rapid search and identification.

Infrared spectroscopy: some figures…

Vibration frequencies (and wave-numbers) are inversely proportional to atomic masses

• C-H stretch (3000 cm-1)• C-O stretch (1000-1300 cm-1)• C-Br stretch (600 cm-1)

Vibration frequencies (and wave-numbers) are proportional to bond strength

• C-C stretch (1000 cm-1)• C=C stretch (1600 cm-1)• C≡C stretch (2200 cm-1)

Near-IR: 12500 – 4000 cm-1

Mid-IR: 4000 – 400 cm-1

Far-IR: 400 – 10 cm-1

v

µ-w

0.8 to 2.5 µm

2.5 to 25 µm

25 to 100 µm

1 eV ~ 8100 cm-1

An example of spectrum (biological sample)

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Abs

orba

nce

1000 1500 2000 2500 3000 3500 4000

Wavenumbers (cm-1)

Amide IC=O

Amide IIC-N and N-H

CH2 and CH3stretching CH3 bending

Broad O-H and N-HStretching

PO2- stretching

C-O stretch

CO2

PO32- stretching of

phosphorylated proteins

Synchrotron infrared radiation: Two modes of emission

Bending magnet emission:

Edge emission:@ 10 µm

Synchrotron Infrared microscopy

Edge radiation

transfer line

spectrometermicroscope Beam

conditioning

Synchrotron source: brightness advantage

10000 1000 100 10 110 8

10 9

1010

1011

1012

1013

1014

1015

1016

1017

1018

1019

Practical limits

Universal Equation

2000K Black Body

Synchrotron Radiation

Phot

ons/

sec/

0.1%

bw/m

m2 /s

tr2

Wavelength (microns)BRIGHTNESS

Spatial Resolution

Signal-to-Noise

Data Collection

BROADBAND

Spectroscopy

Spectro-microscopy

Chemical mapping

A confocal microscope

Two confocal Schwarzschild objectives:

• focus the light onto the sample

• collect the light and relay it to the detector.

Diffraction-limited resolution of λ/2 ( λ: 2→12µm)

G.L. Carr, Rev. of Scientific Instruments, 2001, 72, 1613

Synchrotron vs Globar

0

5

10

15

20

0 10 20 30 40

Aperture (µm)P

eak

to p

eak

inte

nsity

(V)

Synchrotron

Globar

6×6 µm2 22×22 µm2

Synchrotron Globar

0102030405060708090

100

5 7 9 11 13Aperture (µm)

Iinte

nsity

ratio

ESRF-ID21

Synchrotron FTIR Microscopy

P. Dumas et al. Vibr. Spectr. 32 (2003).

500s

16s

0.0 1.0 (a.u.)

30 µm

cuticle

C. Merigoux et al.,Biochimica & Biophysica Acta, 1619, 53, (2003)

Ca - XRF0.2x0.2µm2

Infrared Spectra

Various calcium sites in human hair shaft

1000 1500 2000 2500 3000 3500 Wavenumbers (cm-1)

CH3-(CH2)n-XO

C NH

0.0 1.0 (a.u.)

30 µm

cuticle

C. Merigoux et al.,Biochimica & Biophysica Acta, 1619, 53, (2003)

Various calcium sites in human hair shaft

Two different « types » of lipids in cuticule and medulla

Ca - XRF0.2x0.2µm2

Protein distribution in cortex

Outlook

Multimodal analysisMultivariate data processing

Quantity Quality%

ppmppb structural

chemicalelemental

Scale

mm

µm

nm

3DIn-situ experiments

FluxDetection technology

Source and Optics