Biophysical and physicochemical methods for analyzing plants in vivo and in situ (III): y g p ( )

X-ray spectroscopy for localising&quantifying metalslocalising&quantifying metals

and for investigating metal ligands

Hendrik Küpper, Advanced Course on Bioinorganic Chemistry & Biophysics of Plants, summer semester 2013

X-ray spectroscopyGeneral comments on sample preparation techniques

a) chemical fixation and resin embedding

Advantages: over many years best established procedure in many laboratories

Disadvantages: Metals will inevitably be re-distributed ARTEFACTS

b) freeze substitution or freeze drying

Advantages: less element re-distributiuon than in (a)

Disadvantages: still at least intracellular (vacuole wall) re-distribution artefacts inevitable

c) frozen-hydrated tissues

Advantages: hardly any element-redistribution METHOD OF CHOICE!

Disadvantages: Required rapid-freeze techniques and cryostage ( e pensi e)( expensive)

e) non-frozen fresh tissues

Advantages: NO preparation necessary, “in vivo” situation

Disdvantages: Strong beam damage MORE artefacts than in (c)!

(1) X-ray emission spectroscopy(a) Energy Dispersive X-ray Analysis (EDXA)

Use of an electron microscope as an X-ray spectrometer( ) gy y y ( )

Signals generated in the scanning electron microscope (SEM)

incident electron beam

backscattered electrons

characteristic x-ray photons

backscattered electrons

secondary electronsBremsstrahlung

y

visible light(cathode luminescence)

specimen

absorbedabsorbedelectrons

transmitted and inelastically scattered electrons

elastically scatteredelectrons

Principle of Energy Dispersive X-rayDispersive X ray Analysis (EDXA)

P i i l f P ti lPrinciple of Particle Induced X-ray

Emission (PIXE)Emission (PIXE)

The origin of the different lines in an EDXA spectrum

Analysis of EDXA spectra

peaks of characteristic x-ray photons

spectral windowou

nts

continuous backgroundof bremsstrahlung

spectral windowC

o of bremsstrahlung

Energy / keV

Analysis: a) recording of complete spectrum, subtraction of background --> quantification of peak areas by comparison to internal standardstandardb) recording of counts in spectral window --> dot maps, line scans

Detection limits of EDXA

Methods of plant analysis using EDXASampling of single-cells saps with micropipettesSampling of single cells saps with micropipettes

micropipette filled with silicon oil, connected to i fill d i f t lli diffair-filled syringe for controlling pressure difference

turgor pressure of punctured cell fills pipette Sample preparation:turgor pressure of punctured cell fills pipette with 5-20 picolitres (10-12 l) of cell sap

Sample preparation:1) transfer to storage grid,addition of internal standard (e.g. RbF)standard (e.g. RbF) and matrix (e.g. mannitol)

2) transfer to analysis grid,drying with isopentaney g p

Analysis:1) recording of EDXA spectra in SEM2) data processing

20typical dried sample

20 µmanalysis grid

Küpper H, Zhao F, McGrath SP (1999) Plant Physiol 119, 305-11

Methods of plant analysis using EDXAQuantification of elements in single-cells sapsQuantification of elements in single cells saps

1) net peak area is normalised by internal standard (an element not Evaluation of the method

Ad t(

naturally present in the sample, e.g. Rb)

2) ratio obtained from 1) is quantified

Advantages:- potentially very accurate- enables measurement of

small concentrationsusing calibration curve

nts

small concentrations

Disadvantages:only few types of cells are accessible

10000Cadmium

RbK

cou

n - only few types of cells are accessible to sampling with micropipettes

- risk of preparation artefacts- no distinction between cytoplasm and

1000

per

100

0 no distinction between cytoplasm and vacuole, measurement of cell walls impossible

- very difficult to obtain information

1 10 100100co

unts

concentration / mM

very difficult to obtain information about heterogeneity of element distribution inside the analysed tissueconcentration / mM y

Küpper H, Zhao F, McGrath SP (1999) Plant Physiol 119, 305-11

Methods of plant analysis using EDXAFreeze fracturingFreeze-fracturing

Excise sample from plant, mount in/on stub or vice.p p ,The EDXA spectrum of the vice must not interfere with that of the sample!

Freeze the sample in melting nitrogen slush, transfer to cooled (-170°C) preparation chamber

Fracture sample with fast-moving blade (to cut rather than break the cells)

Produce conductive sample surface by evaporating carbon wireProduce conductive sample surface by evaporating carbon wire

T f t l d ( 150°C) l t i SEM lTransfer to cooled (-150°C) sample stage in SEM, analyse

Küpper H, Zhao F, McGrath SP (1999) Plant Physiol 119, 305-11

Methods of plant analysis using EDXAAnalysis of bulk-frozen samples

Effect of shadingshading inside a sample leads to absorption ofleads to absorption of low-energy x-rays

Dot map of O Kα line (0 6)Dot -map of O Kα line (0.6) Normal x-ray spectrumX-ray spectrum in shadow of trichome

Effect of acceleration voltagehigh acceleration voltage leads to deeper

sec. e-scatt. e-

inc. e-X-rays high acceleration voltage leads to deeper

penetration into the sample!y

fluorescence

ionisation volume

Methods of plant analysis using EDXAQualitative and semi quantitative analysis of bulk frozen samplesQualitative and semi-quantitative analysis of bulk-frozen samples

Line scansS f h Z K l h li

Dot mapsScan of the Zn K alpha line(0.6x half width) along the straight line. Amplitude represents the counts/s inside the selected

pScan of the Zn K alpha line(0.6x half width) over the whole image. Each dot represents one x-ray count inside the selectedcounts/s inside the selected

spectral window.ray count inside the selected spectral window.

Küpper H, Lombi E, Zhao FJ, McGrath SP (2000) Planta 212, 75-84

EDXA imaging application example:Ni silicate accumulation in cell walls of Berkheya coddii

Dot maps (K α lines) of the upper side of a Berkheya coddii leaf. Quantitative relation between Si and Ni in metal accumulation spots: 3.5 (± 1) Si / Ni (P = 0.0055)

Dot maps (K α lines) showing the development of metal accumulation spots. U t l fUp: senescent leaf, down: young-mature leaf;blue: Ca, red: Mn, yellow: Ni

Küpper H (2001, doctoral thesis). UFO Atelier für Gestaltung und Verlag, Allensbach (ISBN 3-935511-07-8)4

EDXAQuantitative analysis ofQuantitative analysis of

bulk-frozen samples

Counts in spectra (A)can be normalised to either thecan be normalised to either the background (B) or an internal standard. The oxygen Kα line has proven to be a reliablehas proven to be a reliable internal standard in bulk-frozen samples, in particular in aqueous compartments like q pvacuoles (C).

Küpper H, Lombi E, Zhao FJ, McGrath SP (2000) Planta 212, 75-84

EDXA quantification application example:Al accumulation in epidermal cell walls of tea (Camellia sinensis)Al accumulation in epidermal cell walls of tea (Camellia sinensis)

7

Young leaves

4

5

6

1

2

3

mM

Old leaves300350400A

l

100150200250

050

100

upper epidermis palisade spongy lower epidermisCut OCW V ICW V CW V CW ICW V OCW

Electronoptic picture of an old C. upper epidermis palisade spongy lower epidermismesophyll

p psinensis leaf (upper epidermis) and dot map of the Al K α line

Carr HP, Lombi E, Küpper H, McGrath SP, Wong MH* (2003) Agronomie 23, 705-710

Methods of plant analysis using EDXAAnalysis of bulk-frozen samples

Evaluation of the freeze-fracturing methodAdvantages:- All types of cells and tissues can be analysed y y- In situ-analysis with very little risk of preparation artefacts- Easy analysis of the heterogeneity of element distribution, by use of dot-maps even in

an imaging way

Disadvantages:- Limited sensitivity (min. 1mM) and accuracy (shading)

El t i d d ti ith l t t t t b li bl tifi d- Elements in dead tissues with low water content cannot be reliably quantified

(1) X-ray emission spectroscopy(b) Proton induced X-ray emission (PIXE) imaging( ) y ( ) g g

K Ca Ni

From:Siegele R, Kachenko AG, Bhatia NP, Wang YD, Ionescu M, Singh B, Baker AJM, Cohen DD,From:Siegele R, Kachenko AG, Bhatia NP, Wang YD, Ionescu M, Singh B, Baker AJM, Cohen DD, 2008, X-ray spectrometry 37, 133-6

Imaging of potassium, calcium and nickel in a leaf of Hybanthus floribundus

more sensitive than EDX, but no observation of frozen-hydrated samples (samples have to be freeze-dried) increased risk of artefacts

(1) X-ray emission spectroscopy(c) X-ray fluorescence imaging (XRF)

From: Kim SA,

Punshon T, Lanzirotti ALanzirotti A,

Li L, Alonso JM, Ecker JR, Kaplan J,

Guerinot ML, 2006,

Science 314, 1295-8

Imaging of Fe, Mn, and Zn in seeds of Arabidopsis thaliana

MUCH more sensitive than EDX and PIXE, but in contrast to EDX it requires a synchrotron instead of an SEM for excitation limitation of beamtime

(1) X-ray emission spectroscopy(c) µ-XRF: Sample preparation and measurement

From work of Mishra S, Wellenreuther G, Küpper Hon As-stress in Ceratophyllum demersum

measured at the DESY (Hamburg)

cryostream

( g)

camerad t t

detector

cameradetector

(2) X-ray absorption and fluorescence spectroscopyWhere it is done...Where it is done...

How Synchrotron radiation is generated

DESY

Bending Magnet

Wiggler

Undulator

Free Electron LaserFree Electron Laser

X-ray absorption (I)

X-ray absorption (II)

XAS techniques

What can we learn from XAS?

Example of what can we learn from XANES (I)

Example of what can we learn from XANES (II)

Principle of Extended X-ray Absorption Fine Structure (EXAFS)(EXAFS)

Principle of single vs. multiple scattering contributions in EXAFSEXAFS

Effects of single vs. multiple scattering contributions in EXAFSEXAFS

Preparation of plant material for XAS (EXAFS and XANES)

Excise sample from plant

Freeze the sample in melting nitrogen slushFreeze the sample in melting nitrogen slush

grind sample in mortar cooled by dry ice

fill the still frozen-hydrated powder into an EXAFS cuvette, seal with Kapton tapeThe EXAFS spectrum of the cuvette must not interfere with that of the sample!

T f t l d (20 K) l h ld f b li lTransfer to cooled (20 K) sample holder of beamline, analyse

Küpper H, Mijovilovich A, Meyer-Klaucke W, Kroneck PMH (2004) Plant Physiology 134 (2), 748-757

Analysis of EXAFS data (I)

Analysis of EXAFS data (II)

1.01.21.4

ores

cenc

e1.01.21.4

measured data fit with datasets of model compounds

resc

ence

Cd-ligands: model compounds Cd in hyperaccumulator leaves

0.20.40.60.8

0

0 8

1.0

1.2 XASis

ed X

-ray

fluo

0 20.40.60.81.0

1.0

1.2

XAS

sed

X-r

ay fl

uo

26700 26800 26900 27000 271000.0

6

26750 268000.8

Nor

mal

Excitation energy [eV] 26700 26800 26900 27000 27100

0.00.2

6

26750 26800

Nor

mal

i

Excitation energy [eV]

measured datafi i h h i l d l

4-2024

AFS

[Chi

*k3 ]

0

2

4fit with theoretical model

FS [C

hi*k

3 ]

2 4 6 8 10 12

-6-4

30k / Å-1

EXAFSEXA

2 4 6 8 10 12

-4

-2

24k / Å-1

EXAFSEXA

F

15202530

EXAFS Fourier transform

m A

mpl

itude

1216

20

24 measured data fit with theoretical model fit with datasets of

model compounds

Am

plitu

de

0 1 2 3 4 5 605

10

Di t [Å]

Tran

sfor

m

0 1 2 3 4 5 60

4

8

EXAFS Fourier transform

Tran

sfor

m A

oxygen (aqueous) oxygen (citrate) histidine sulphur (glutathione)

Distance [Å] 0 1 2 3 4 5 6 Distance [Å]

Küpper H, Mijovilovich A, Meyer-Klaucke W, Kroneck PMH (2004) Plant Physiology 134 (2), 748-757

Application example: Speciation of cadmium and zinc hyperaccumulated by Thlaspi caerulescens (Ganges ecotype)yp y p ( g yp )

1.0Z

0.6

0.8 young leaves mature leaves mature stems

Zn

0 0

0.2

0.4 histidine contribution

of

*k3

0.0

0.8

1.0

lran

sfor

m

Cd

increase in sulphur contribution

0.4

0.6 young leaves senescent leaves mature stems

Four

ier T

1 2 3 4 50.0

0.2

Küpper H, Mijovilovich A, Meyer-Klaucke W, Kroneck PMH (2004) Plant Physiology 134, 748-757

Distance [Å]

Application example: Speciation of cadmium and zinc hyperaccumulated by Thlaspi caerulescens (Ganges ecotype)

80

g to

Cd

80

g to

Cd

60

nds

bind

ing

60

nds

bind

in

20

40

of a

ll lig

an

20

40

of a

ll lig

an

young mature senescent dead0

Perc

ent o

Developmental stage of leavesstems petioles leaves

0

Per

cent

o

Tissue sulphur ligands N/O ligands

Küpper H, Mijovilovich A, Meyer-Klaucke W, Kroneck PMH (2004) Plant Physiology 134, 748-757

Microscopic X-ray absorption spectroscopyConfocal µ-XANES: Sample mounting and measurement

E

SX

M

P

From work of Mishra S, Wellenreuther G, Küpper H on As-stress in Ceratophyllum demersum, measured at the DESY (Hamburg)

µm

All slides of my lectures can be downloaded

from my workgroup homepage www uni konstanz de Department of BiologyWorkgroups Küpper labwww.uni-konstanz.de Department of Biology Workgroups Küpper lab,

or directlyhttp://www.uni-konstanz.de/FuF/Bio/kuepper/Homepage/AG_Kuepper_Homepage.html

and

on the ILIAS websiteon the ILIAS website