A RF plasma oxygen ion source on NanoSIMS for

subcellular trace element detection

Dirk Schaumlöffel1, Julien Malherbe1, Étienne Gontier2

François Hillion3, François Horréard3, Dirk Dobritzsch4

1Université de Pau et des Pays de l’Adour / CNRS

Institut des Sciences Analytiques et de Physico-Chimie

pour l'Environnement et les Matériaux, UMR 5254 IPREM/LCABIE, Pau, France2Bordeaux Imaging Center, Pôle d'imagerie électronique, Bordeaux, France3CAMECA, 29 Quai des Grésillons, Gennevilliers, France4Martin-Luther-Universität Halle-Wittenberg

Institut für Biochemie und Biotechnologie, Abteilung Pflanzenbiochemie, Halle (Saale), Germany

The 7th International NanoSIMS user meeting

“NanoSIMS & correlative microscopy: exploring physical and biochemical boundaries”

Leipzig, Germany, 22-24th of August 2017

Equipex MARSS project

FTMS NanoSIMS

TOFSIMSHR MC-ICPMS

NanoSIMS delivery and installation in Pau (March – June 2017)

NanoSIMS 50L scheme

Normal, co-axial

objective/extraction lens

6 moveable trolleys (EM/FC)

O- Duoplasmatron

primary ion source

Moveable Cs+ primary ion source

Magnetic Sector Mass analyzer

with 7 mass parallel detection

1 fixed detector (EM/FC)

Sample

CCD camera

SED

TIC

O- RF plasma

primary ion source

A new RF plasma O- primary ion source on NanoSIMS

Collaboration with

Hyperion™ source manufactured by Oregon Physics (Hillsboro, OR)

RF

gas inlet

RF coil

magnetic filter

extraction and skimmer block

+-

dielectric plasma tube

+-

Upper plasma

region

e-, O+, O-

Lower plasma

region

e-, O+, O-

O- beam

Schematic view of the RF plasma O- primary ion source

Source diameter : 70 - 80 µm (manufacturer specification 35-50 µm )

Brightness: ~ 100 mA×cm-2×sr-1 at 8 kV

5E+02

5E+03

5E+04

5E+05

5E+06

10 15 20 25 30 35

Ion

beam

cu

rren

t(p

A)

Wien filter plate voltage (V)

O3-

O2-

O-

18O-

OH-

5.104

5.103

5.102

5.105

5.106

Oxygen ion distribution of the primary beamusing a Wien filter located after the source

O- ions represent approximately 88% of the distribution

37 nm

0

2000

4000

6000

8000

0,4 0,5 0,6 0,7 0,8 0,9 1

Inte

nsity (

cp

s)

Distance (µm)

Si oxide grain sample over Al substrate

Image size: 3 x 3 µm

Probe intensity: 0.15 pA

27Al

Line scan (left image) showing intensity

variation from 16 to 84 %:

determination of probe size (resolution)

Determination of the size of the O- primary ion beam(probe size)

0

2000

4000

6000

8000

0,4 0,5 0,6 0,7 0,8 0,9 1

Inte

nsity (

cp

s)

Distance (µm)

27Al+ 37 nm

0

20000

40000

60000

0,8 0,9 1 1,1 1,2 1,3 1,4

Inte

nsity (

cp

s)

Distance (µm)

47 nm

28Si-

100%

84%

16%

100%

84%

16%

0

1000

2000

3000

4000

0,5 0,6 0,7 0,8 0,9 1 1,1

Inte

nsity (

cp

s)

Distance (µm)

100 nm

27Al+

100%

84%

16%

Comparison with Duoplasmatron and Cs primary ion sources

RF plasma source,0.15pA

DuoplasmatronSource, 0.1pA

Cs+ source, 0.17pA

0,01

0,1

1

10

100

10 100 1000

Pro

be c

urr

en

t (p

A)

Probe size (nm)

Série1

Série2

Série3

Cs+ source

O- duoplasmatron

O- RF plasma

1

0.1

0.01

10

100

Comparison of the sample current density

for the Cs+ , O- duoplasmatron and O- RF sources

16 x

0.13 pA (Duo)

2.0 pA (Cs)

2.1 pA (RF)

Current density at the sample 16 times higher with RF plasma source

Achievable lateral resolution improved by a factor of 3

3 x40 nm(RF)

120 nm (Duo)

0,00

0,01

0,10

1,00

10,00

100,00

No

rmalized

co

un

ts (

cp

s/n

m²

)

O- Duoplasmatron

O- RF plasma source

FeAl Cu Zn Pb

1

0.1

0.01

10

100

0.001

O- Duoplasmatron

(14-32 pA, beam size 476-613 nm)

O- RF plasma

(23-94 pA, beam size 180-300 nm)

Comparison of normalized counts for selected elements

using O- duoplasmatron and O- RF sources

Count rate normalized to acquisition time, probe size, and isotope abundance

30x30 µm

256x256 pixel

reference materials

Increased secondary ion yield increased apparent element sensitivity (by factor 5 to 45)

Bioimaging with NanoSIMS

Use of the RF plasma oxygen primary ion source for the localizationof major (Na, Ca, P) and trace (Fe, Cu,) elements

in plant (algae) cells

Application to a model organism

Model system: Chlamydomonas reinhardtii

• single celled green micro algae• commonly found in soil and fresh water• exists in different strains• model organism to study cell response to metal stress

10

µm

: Flagella: Vacuoles: Nucleus: Nucleolus: Chloroplast: Thylakoid: Pyrenoid: Starch

FVNNuCTPS

TEM analysis(70 nm thin section)resolution down to 1 nm

1 µm

S

Cs+ source256pix 8µm 1 pA 10ms/pix

[800-1500]

12C14N

[75-500] [0-14]

31P

[14-60]

[min-max]

12C 31P 32S

23Na[0-721] [0-4] [0-4][0-4]

O- RF plasma source256pix FOV 8µm 1,4pA 10ms/pix

23Na

[0-331]

40Ca 56Fe 63Cu31P

[min-max]

O- Duoplasmatron source256pix FOV 8µm 1,5pA 8ms/pix

40Ca

[0-144][0-95] [0-38]

23Na 31P

[0-6]

56Fe

[0-5]

63Cu

[min-max]

256pix FOV 8µm 1,5pA 15ms/pix

23Na

63Cu

[0-7][0-118][0-1771] [0-68][0-229]

40Ca23Na 31P 56Fe 63Cu

NanoSIMS analysis of Chlamydomonas reinhardtii cells

Comparison conventional Duoplasmatron O- ion source and new RF plasma O- ion source

New RF plasma O- ion sourceDuoplasmatron O- ion source

20 x 20 µm

11 min (Duo)

5.5 min (RF)

256x256 pixel

1 plane

23Na

40Ca

relative intensity: Max

Min

acidocalcisomes

pyrenoid with

starch plates

300 nm

thin sections

Lateral resolution in biological cell imaging (C. reinhardtii)

Line scans on Ca containing vacuoles/acidocalcisomes

O- Duoplasmatron

[min-max]

O- RF plasma

1.5pA ; FOV 8µm ; 256pix ; 8ms/pix

86pA ; FOV 20µm ; 256pix ; 5ms/pix 1.4pA ; FOV 8µm ; 256pix ; 10ms/pix

2.5pA ; FOV 20µm ; 256pix ; 10ms/pix

NaMax

Min

P

Ca

Fe

Cu

Single cell imaging: 12 x 12 µm, 22 min, 512x512 pixel, 5 ms/pixel 2 planes

Pyr

eno

idw

ith

sta

rch

pla

tes

Gra

nu

les

?A

cid

oca

lcis

om

es

Subcellular element imaging by NanoSIMS (RF plasma O- ion source)

1 µm

S

Scheme of a Acidocalcisome

R. Docampo, W. de Souza, K. Miranda, P. Rohloff, S. N. J. Moreno, Nature Reviews 2005, 3, 251-261

Correlative imaging TEM - NanoSIMS

resin block

70 nm

300 nm

40Ca+ 24Mg+

56Fe+ 66Zn+

2 µm 2 µm

2 µm2 µm

Max

Min

1 µm

cw

vg

cv

vg

th

th

vg

cvvg

th

th

ac

ac

cv

ac

cvcv

cw

vg vg

th

th

TEM

NanoSIMS

ac

ac

cv

vg

vg

RF plasma O- source: 10 × 10 µm FOV; 256 × 256 pix; dwell time 25 ms/pix; 27 min.

Conclusions: advantages of the new RF plasma O- source

• Higher beam density = better sensitivity for metals (Ca, Fe, Cu, Mn….)

• Higher lateral resolution than conventional oxygen sources = sharper

images enabling the observation of smaller details

• Less maintenance = less instrument downtime

• Stability: < 1.6 % variation of primary current over 14h

• High resolution images of trace elements in biological cell opens new

application fields

Acknowledgements

• French ANR-EQUIPEX program (Equipment of Excellence)

Project: ANR-11-EQPX-0027 – Mass Spectrometry Center MARSS

• CAMECA

• French Ministry of Research (PhD fellowship)

• Campus France – DAAD

University of Pau/CNRS-IPREM

Florent Penen PhD student

Marie-Pierre Isaure Lecturer

Anne-Laure Bulteau CNRS researcher (now ENS Lyon)

University Potsdam (Germany)

Tanja Schwerdtle (neurotoxicology)Tanja Schwerdtle (neurotoxicology)Tanja Schwerdtle (neurotoxicology)

Julia Bornhorst (neurotoxicology

Thank you for

your attention !