Transmission SIMS: A novel approach to achieving
higher secondary ion yields of intact biomolecules
Department of Micro Engineering, Kyoto University
K. Nakajima, K. Nagano, T. Marumo, K. Yamamoto, K. Kimura
Japan Atomic Energy Agency
K. Narumi, Y. Saitoh
National Institute of Advanced Industrial Science and Technology
K. Hirata
K. Nakajima et al, APL 104 (2014) 114103.
1
SIMS: secondary ion mass spectrometry
Excellent lateral and depth resolution
Sensitivity is extremely high
Often used for analysis and
imaging of biomolecules
D. Jeramie et al, Nature Reviews Microbiology 9, 683.
http://www.teknoscienze.com/
Transmission SIMS
Sensitivity for large biomolecules is
rather poor because biomolecules are
easily destroyed by ion impact
in combination with high energy cluster ions, secondary ion yield of
intact biomolecule is enhanced and fragment ions are suppressed
2
OUTLINE
1. Introduction
Analysis and imaging of biomaterials using SIMS
2. Transmission SIMS using 6 MeV Cu4+
In the forward direction, yield of intact amino acid ion is enhanced but
fragment ions are more enhanced
3. Transmission SIMS using 5 MeV C60+
Yield of intact amino acid ion is enhanced by one order of magnitude and
fragment ions are suppressed in the forward direction
The mechanism of the enhancement and suppression is discussed in
terms of deposited-energy distribution at the surface
4. Development of new molecular imaging technique
Transmission SIMS + PEEM
5. Summary
3
Sensitivity of conventional SIMS is poor for biomolecules
Y. Nakata et al, Nucl. Instr. Meth. in Phys. Res. B 256 (2007) 489.
10 keV Ar+ → Arginine film
Many fragment ions
Arginine
m = 174.2
1 × 10-5 ions/incident ion
Yield of intact molecular ion is very low
Fragment ions are dominant
To improve the sensitivity of biomolecules
1. Use of high energy ions → PDMS, MeV-SIMS
2. Use of cluster ions → Cluster SIMS
4
Plasma Desorption Mass Spectrometry (PDMS)
Fission fragments → arginine
D.E. Torgerson et al, Biochem. Biophys. Res. Commun. 60 (1974) 616.
MeV-SIMS: becomes popular for the analysis of biomaterials
6 MeV Cu4+ → animal cells
H. Yamada et al, Surf. Interface Anal. 43 (2011) 363.
5
Cluster SIMS: C60 ion
J.S. Fletcher et al, Anal. Chem. 78 (2006) 1827.
C60+ → Irganox 1010
～ 10-3 ions/incident ion
Large yields compared to conventional SIMS
Yield increases with impact energy
→ further enhancement with MeV C60+ ?
6
Another possibility to enhance the yield in SIMS
In the conventional SIMS, 2ndary ions emitted in the backward
direction are measured
primary ion
Backward
emission
Thin film sample
2ndary ions
Forward
emission
2ndary ions
Primary ions can directly transfer their momentum to the
target atoms in the forward direction → Enhancement of
secondary ion yield is expected in the forward direction
MeV ions, cluster ions, transmission geometry
→ Transmission SIMS using MeV C60 ion
7
Setup: TOF-SIMS with 5 MeV C60 ion
MCP
start
TDC
Mass spectrum
Phenylalanine
m = 165.2
a-SiN film (20nm)
5 MeV C60+
6 MeV Cu4+
Phenylalanine film: deposited on
self-supporting a-SiN film by
vacuum evaporation
Measurement of forward emission
6 MeV Cu4+ impact is also measured
as a reference
SSBD
20000
COUNTS
stop
10000
0
0
50
100
m/z
150
6 MeV Cu4+
Forward emission
Backward emission
5 MeV C60+
Forward emission
Backward emission
8
200
What happens when 5 MeVC60 → a-SiN
5 MeV C60
30 keV Ga
(Conventional SIMS)
6 MeV Cu
(MeV-SIMS)
Stotal
25.9 keV/nm
1.71 keV/nm
3.52 keV/nm
Selec
21.9 keV/nm
0.16 keV/nm
3.33 keV/nm
Snucl
4.0 keV/nm
1.55 keV/nm
0.19 keV/nm
TEM image
Tilt image: 25°
a-SiN irradiated with
with 5 MeV
C60 C60Film
Irradiation
5 MeV
ionthickness
should100have
nm
large effect on the target Track length ~44 nm
C60
Projected range ~ 190 nm
10nm
10nm
Cylindrical ion track is produced
Density is reduced inside the ion track? → HAADF-STEM
9
HAADF-STEM: semi-quantitative analysis is possible
HAADF-STEM image
Normalized Density
5 MeV C+60
a-SiN(100nm)
1
Radial density distribution
0.8
0
10nm
2
4
6
Distance from center (nm)
8
10
a-SiN irradiated with 5 MeV C60
Missing volume ~400 nm3 → 3000 atoms!
This suggests very large sputtering yield
10
Sputtering yield measurement using high-resolution RBS
20000
15000
pristine
irradiated
C60
a-SiN
random channeling
5000
0
5
N
3
10
250
5
O
ENERGY (keV)
1
Si
300
102
DEPTH (nm)
10
20
30
experimental
pristine irradiated
SRIM
simulation
Si
40
N
O
C
SRIM cannot explain
such large yields
0.5
SRIM calculation
5
10-2
0
CONCENTRATION
10000
SPUTTERING YIELD (atoms/ion)
COUNTS (arb. units)
104
Sputtering
yield
is as large as several thousand
400 keV He+
a-SiN/Si(001)
2
5
10-1
2
5
100
ENERGY OF C60 (MeV)
0
0
10
2
101
5
20
16
DEPTH (10
-2
cm )
30
Ballistic collision cascade is negligible → Thermal spike plays an important role.
Sputtering yield is estimated using unified thermal spike (u-TS) model
11
Unified thermal spike (u-TS) model
M. Toulemonde et al, Phys. Rev. B 83, 054106 (2011).
The u-TS model describes temperature evolution using two thermal diffusion equations.
Both Se and Sn are included
Thermal evaporation mechanism
P. Sigmund, C. Claussen, J. Appl. Phys. 52 (1981) 990.
𝚽𝚽𝒊𝒊 𝑻𝑻𝒂𝒂 𝒓𝒓, 𝒕𝒕
SPUTTERING YIELD (atoms/ion)
104
5
C60
= 𝑵𝑵𝒊𝒊
𝒌𝒌𝑩𝑩 𝑻𝑻𝒂𝒂 𝒓𝒓, 𝒕𝒕
−𝑼𝑼𝒊𝒊
𝒆𝒆𝒆𝒆𝒆𝒆
𝟐𝟐𝝅𝝅𝑴𝑴𝒊𝒊
𝒌𝒌𝑩𝑩 𝑻𝑻𝒂𝒂 𝒓𝒓, 𝒕𝒕
experimental
u-TS model (U=0.94 eV)
SRIM
5
10000
102
5 MeV C60
r = 2 nm
8000
a-SiN
3 nm
6000
4 nm
5 nm
4000
6 nm
7 nm
8 nm
9 nm
2000
0
10-14
a-SiN
103
Evolution of atomic temperature
TEMPERATURE (K)
𝝏𝝏𝑻𝑻𝒆𝒆 𝟏𝟏 𝝏𝝏
𝝏𝝏𝑻𝑻𝒆𝒆
=
𝒓𝒓𝑲𝑲𝒆𝒆
− 𝒈𝒈 𝑻𝑻𝒆𝒆 − 𝑻𝑻𝒂𝒂 + 𝑨𝑨 𝒓𝒓, 𝒗𝒗, 𝒕𝒕
𝝏𝝏𝒕𝒕
𝒓𝒓 𝝏𝝏𝒓𝒓
𝝏𝝏𝒓𝒓
𝝏𝝏𝑻𝑻𝒂𝒂 𝟏𝟏 𝝏𝝏
𝝏𝝏𝑻𝑻𝒂𝒂
𝑪𝑪𝒂𝒂
=
𝒓𝒓𝑲𝑲𝒂𝒂
+ 𝒈𝒈 𝑻𝑻𝒆𝒆 − 𝑻𝑻𝒂𝒂 + 𝑩𝑩 𝒓𝒓, 𝒕𝒕
𝝏𝝏𝒕𝒕
𝒓𝒓 𝝏𝝏𝒓𝒓
𝝏𝝏𝒓𝒓
𝑪𝑪𝒆𝒆
10-13
10-12
TIME (s)
10-11
10-10
u-TS model reproduces observed
sputtering yield
5
10-2
2
5
10-1
2
5
100
ENERGY OF C60 (MeV)
2
5
101
12
u-TS model also reproduces observed track radius
produced by swift heavy ions as well as C60 ions
Ion tracks produced by swift heavy ions
shell radius
core radius
TRACK RADIUS (nm)
6
Lines: u-TS calculation
4
Shell radius
420 MeV Au
200 MeV Au
100 MeV Xe
200 MeV Kr
0
0
5
10
15
Core radius
20
25
ENERGY LOSS (keV/nm)
u-TS model is a reliable model to
describe and understand the ionsolid interactions, including MeV
C60 ions
30
10
TRACK RADIUS (nm)
2
Ion tracks produced by C60 ions
C60
8
a-SiN
core radius
shell radius
Shell radius
35
6
Lines: u-TS calculation
4
Core radius
2
0
0
1
2
3
4
ENERGY of C60 (MeV)
5
13
Summary: effect of MeV C60 ion impact on materials
A very intense thermal spikes are produced
TEMPERATURE (K)
105
5 MeV C60
6 MeV Cu
a-SiN
a-SiN
T > 10,000 K at r < 3 nm
T > 1,000 K at r < 13 nm
104
103
102 -1
10
Temperature distribution
around impact position
5
100
5
101
DISTANCE FROM CENTER (nm)
As a result of such a very intense thermal spike,
1) Cylindrical ion tracks are produced:
diameter a few nm length several tens nm
2) Extremely high sputtering yield is observed:
14
several thousand atoms/incident ion → large 2ndary ion yield?
SIMS measurement with 6 MeV Cu4+ ion
MCP
start
TDC
Mass spectrum
Phenylalanine
m = 165.2
a-SiN film (20nm)
5 MeV C60+
6 MeV Cu4+
Phenylalanine film: deposited on
self-supporting a-SiN film by
vacuum evaporation
Measurement of forward emission
6 MeV Cu4+ impact is also measured
as a reference
SSBD
20000
COUNTS
stop
10000
0
0
50
100
m/z
150
6 MeV Cu4+
Forward emission
Backward emission
5 MeV C60+
Forward emission
Backward emission
15
200
6 MeV Cu4+ → phenylalanine
Comparison
Comparison
between
with conventional
forward & backward
SIMS
emission
2000
6 MeV Cu4+
Phe/SiN
backward
1500
COUNTS
Backward
1000
C2NO2H+4
[Phe-COOH]+
Intact phenylalanine
1.3 × 10-3
C6H+5 +
C7H7
500
0
[Phe+H]+
0
2000
50
100
m/z
6 MeV Cu4+
Phe/SiN
Conventional
forward
1500
10
Forward
COUNTS
keV Ar+
200
SIMS
[Phe-COOH]+
Intact
C6H+5
+
C7H7
1.5 ×
C2NO21H+4× 10-5 ions/incident ion
1000
phenylalanine
10-3
[Phe+H]+
500
0
150
0
50
About 2 orders of magnitude
larger than the conventional
SIMS
100
m/z
150
200
Yield is enhanced in the
forward direction (×1.2)
What is the mechanism?
16
What is the mechanism of enhancement in the forward direction?
① Preferential momentum transfer in the forward direction
② Charge state dependence of secondary ion yield
6
The ions transmitted through the sample generally have higher charge states than
q+
Bions may be emittedSSBD
the incident charge state, as a result, moreCu
secondary
from
4+
the exit
surface
in the forward direction.
MeV
Cu
Average exit charge ~ 8.5+
COUNTS
Phenylalanine/SiN
6 MeV Cu4+
charge state distribution of transmitted ions
400 mean charge = 8.5
9+
7+
8+
10+
200
6+
0
4+
0
11+
5+
12+
10
20
30
SSD POSITION (mm)
13+
40
In order to see if the ions of higher
charge states really produce more
secondary ions, we measured
secondary ions in coincidence with
the exit charge state.
17
MOLECULAR ION YIELD (arb. units)
Charge state dependence of secondary ion yield
0.004
6 MeV Cu4+
Si3N4(50nm)/Phe(90nm)
Forward emission
Backward emission
0.003
Cuq+
Backward
emission
0.002
0.001
0
All data points align along the same line
irrespective of the direction of emission
3
4
5
6
7
8
9
q: EXIT
CHARGE
CHARGE
STATESTATE
10
11
12
Enhancement in the forward direction can be explained by the charge state dependence.
Preferential momentum transfer does not play a role.
The origin of charge state dependence → Stopping power dependence
18
Stopping power dependence of 2ndary ion yield
6 MeV Cu4+
a-SiN(50nm)/Phe(90nm)
backward emission
9+
forward emission
SECONDARY ION YIELD
0.003
6+
0.002
8+
10+
4+
0.001
0
7+
11+
Stopping power was calculated using CASP code
0
1
2
ELECTRONIC STOPPING POWER (keV/nm)
In the present case, primary ions lose energy mainly by electronic excitation.
Excited electrons transfer their energy to target atoms/molecules and eventually
secondary ions are emitted.
19
6 MeV Cu4+：forward vs backward (Fragment Ions)
2000
6 MeV Cu4+
Phe/SiN
backward
1500
COUNTS
backward
1000
C2NO2H+4
[Phe-COOH]+
C6H+5 +
C7H7
500
[Phe+H]+
0
2000 0
1500
50
6 MeV Cu4+
Phe/SiN
forward
COUNTS
forward
1000
100
m/z
200
[Phe-COOH]+
C6H+5
C7H+7
+
C2NO2H4
[Phe+H]+
500
0
150
0
50
100
m/z
150
200
Yield enhancement of fragment ions in the forward direction
looks more pronounced than the intact molecular ion
20
Ratio
of forward/backward
yield
YIELD
RATIO
OF FORWARD/BACKWARD
Ratio of forward/backward yield: 6 MeV Cu4+
6 MeV Cu4+
101
10
Fragment ions
55
Intact molecular ion
2
1010
0.55
10-1
0
50
100
M/z (u/e)
150
200
Yield enhancement in the forward direction increases with
decreasing mass number
This behavior can be explained by a simple model
21
Why fragment ions are more enhanced ?
Density distribution
of deposited energy
CentralLarger
regionstopping power
→ density of deposited energy is high
→ fragment ions are emitted
Outer region
→ intact molecular ions can be emitted
position
intact molecule
ion
fragments
ion
The secondary ion yield is enhanced in the forward direction
for the mono-atomic ions. → What happens for C60 ions?
22
Backward emission: 6 MeV Cu4+ vs 5 MeV C60+
2000
6 MeV Cu4+
Phe/SiN
backward
1500
COUNTS
6 MeV Cu4+
1000
C2NO2H+4
C6H+5 +
C7H7
1.3 × 10-3 ions/incident
500
0
S = 2.1 keV/nm
[Phe-COOH]+
[Phe+H]+
0
2000
50
5 MeV
C+60
100
m/z
COUNTS
200
× 0.1
1000
4.7 × 10-3 ions/incident
[Phe+H]+
S = 17 keV/nm
500
0
4 times larger
Phe/SiN backward
5 MeV C60+
1500
150
0
50
100
m/z
150
200
○ Yield of intact molecular ion is enhanced by using C60 ion
× Yields of fragment ions are much more enhanced
23
5 MeV C60+ : forward vs backward
Phe/SiN
5 MeV C+60
backward
20000
forward
[Phe-COOH]+
COUNTS
Forward yield: 3.8 × 10-2 ions/incident
10000
8 times larger than the
[Phe+H] backward emission
C2NO2H+4
+
C6H+5
C7H+7
0
0
50
100
m/z
150
200
○ Yield of intact molecular ion is enhanced by a factor
of 8 in the forward direction
○ Yields of fragment ions are suppressed
24
of forward/backward
yield
YIELDRatio
RATIO
OF FORWARD/BACKWARD
Ratio of forward/backward yield: 5 MeV C60+ vs Cu4+
5 MeV C+60
6 MeV Cu4+
101
55
Cu4+
2
1010
0.55
10-1
C60+
0
50
100
m/z
150
200
In the case of C60 ion bombardment, the ratio of forward/ backward
yield decreases with decreasing mass number contrary to the
monoatomic ion bombardment.
25
Due to multiple scattering, the spatial
distribution of constituent C ions
becomes broader along the ion path.
Width becomes several tens nm at the
exit surface
YIELD RATIO OFCOUNTS
FORWARD/BACKWARD
Passage of C60 ion in the sample
1000
1
10
800
+
FWHM
= 47 Cnm
SiN(20nm)/Phe(90nm)
5 MeV
60
4+
6
MeV
Cu
θ = 45º
Spatial distribution
of C ions at the exit
surface
5
600
100
400
5
200
100-1
-200
0
-100
50
0
100
100
150
DISTANCE FROM
m/z CENTER (nm)
200
200
Distribution of deposited
energy at exit surface
For thicker samples, the distribution is wider and so larger enhancement of
intact molecular ion and larger suppression of fragment ions are expected.
→ Measurements with samples of various thicknesses were performed. 26
FRACTION OF INTACT MOLECULAR ION (%)
Fraction of intact molecular ion vs distribution width
40
Forward emission
Backward emission
Transmission
30
SIMS with MeV C60 ion is
+ → phenylalanine
5 MeV
useful
for Cdetection
of amino acid
60
molecules
20
How about larger biomolecules, such as
peptide?
10
0
0
10
20
30
40
50
WIDTH OF SPATIAL DIST. AT EXIT SURFACE (nm)
Yield of intact ion increases with increasing width as is expected
Maximum yield of intact molecular ion obtained so far is
~ 0.2 ions/incident C60 ion
Can be improved by choosing optimal conditions
27
Result of peptide: prepared by drip-dry of water solution
5
1×10
5 MeV C+60
SiN(50nm)/Leucine-Enkephalin(34nm)
Leucine-enkephalin, m = 555.6
(Tyr-Gly-Gly-Phe-Leu)
80000
M+Na+
5 MeV C60+ → phenylalanine
Forward
Backward
COUNTS
60000
M+H+
M+K+
× 25
40000
20000
0
0
200
m/z
400
600
Intact peptide molecules are observed.
The yield of intact molecular ion is ~ 0.1 ions/incident ion
28
Ratio of forward/backward yield: 5 MeV C60+ vs Cu4+
Forward/Backward Yield Ratio
5 MeV C+60
SiN(50nm)/Leucine-Enkephalin(34nm)
Leucine-enkephalin, m = 555.6
(Tyr-Gly-Gly-Phe-Leu)
1.5
1
0.5
0
0
200
m/z
400
600
Fragmentation is largely suppressed in the forward direction
29
Molecular imaging using transmission SIMS
after Barney L. Doyle
PEEM (FOCUS GmbH)
used as 2ndary electron microscope
→ information of ion impact position
2ndary electrons
5 MeV C60+
No μ-beam is needed
Molecular
imaging
Information of
2ndary ions
30
Just installed in the beam line last week
31
PEEM image: test sample
Resolution = 74 nm
In principle, molecular
imaging can be performed
with a resolution of 74 nm
32
Summary
♦ Transmission SIMS measurements were performed using 6
MeV Cu4+ and 5 MeV C60+ ions.
♦ Under the bombardment of 6 MeV Cu4+ ions, secondary ion
yields of both intact molecules and fragment ions are
enhanced in the forward direction.
♦ Under the bombardment of 5 MeV C60+ ions, secondary ion
yield of the intact molecules is enhanced in the forward
direction while the yield of fragment ions is reduced.
♦ The observed yield enhancement and reduction can be
qualitatively understood by a simple model of secondary ion
emission.
♦ The maximum yield of intact phenylalanine ion is ~ 0.2
ions/incident ion so far and may be improved by choosing
optimal conditions.
33