A RF plasma oxygen ion source on NanoSIMS for subcellular trace element detection

(1)

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, France

2Bordeaux Imaging Center, Pôle d'imagerie électronique, Bordeaux, France

3CAMECA, 29 Quai des Grésillons, Gennevilliers, France

4Martin-Luther-Universität Halle-Wittenberg

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

The 7^th International NanoSIMS user meeting

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

Leipzig, Germany, 22-24^th of August 2017

(2)

Equipex MARSS project

FTMS NanoSIMS

TOFSIMS HR MC-ICPMS

(3)

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

(4)

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

(5)

A new RF plasma O

^-

primary ion source on NanoSIMS

Collaboration with

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

(6)

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^-1at 8 kV

(7)

5E+02 5E+03 5E+04 5E+05 5E+06

10 15 20 25 30 35

Ion beamcurrent(pA)

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 beam using a Wien filter located after the source

O^- ions represent approximately 88% of the distribution

(8)

37 nm

0 2000 4000 6000 8000

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

Intensity (cps)

Distance (µm)

Si oxide grain sample over Al substrate Image size: 3 x 3 µm

Probe intensity: 0.15 pA

27

Al

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)

(9)

0 2000 4000 6000 8000

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

Intensity (cps)

Distance (µm)

27Al⁺ _{37 nm}

0 20000 40000 60000

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

Intensity (cps)

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

Intensity (cps)

Distance (µm)

100 nm

27Al⁺

100%

84%

16%

Comparison with Duoplasmatron and Cs primary ion sources

RF plasma source, 0.15pA

Duoplasmatron Source, 0.1pA

Cs⁺ source, 0.17pA

(10)

0,01 0,1 1 10 100

10 100 1000

Probe current (pA)

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 sample16 times higher with RF plasma source Achievable lateral resolution improved by a factor of 3

40 nm 3 x (RF)

120 nm (Duo)

(11)

0,00 0,01 0,10 1,00 10,00 100,00

Normalized counts (cps/nm² )

O- Duoplasmatron

O- RF plasma source

Fe

Al 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)

(12)

Bioimaging with NanoSIMS

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

in plant (algae) cells

(13)

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 F

V N Nu C T P S

TEM analysis

(70 nm thin section) resolution down to 1 nm

1 µm

S

(14)

Cs

⁺

source

256pix 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 source

256pix FOV 8µm 1,4pA 10ms/pix

23Na

[0-331]

40Ca ³¹P ⁵⁶Fe ⁶³Cu

[min-max]

(15)

O

^-

Duoplasmatron source

40Ca

[0-144]

[0-95] [0-38]

23Na ³¹P

[0-6]

56Fe

[0-5]

63Cu

[min-max]

23Na

63Cu [0-7]

[0-118]

[0-1771] [0-68]

[0-229]

40Ca

23Na ³¹P ⁵⁶Fe ⁶³Cu

(16)

NanoSIMS analysis of Chlamydomonas reinhardtii cells

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

Duoplasmatron 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

(17)

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

(18)

Na ^Max

Min

P

Ca

Fe

Cu

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

Subcellular element imaging by NanoSIMS (RF plasma O

^-

ion source)

1 µm

S

(19)

Scheme of a Acidocalcisome

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

(20)

Correlative imaging TEM - NanoSIMS

(21)

resin block

70 nm

300 nm

40

Ca

⁺ ²⁴

Mg

⁺

56

Fe

⁺ ⁶⁶

Zn

⁺

2 µm 2 µm

Max

Min

1 µm cw

vg cv vg

th

vg vg cv

th th

ac

ac cv

ac cv cv

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.

(22)

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

(23)

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

(24)

A RF plasma oxygen ion source on NanoSIMS for subcellular trace element detection