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Quantitative analysis of sputtering due to ion beam bombardment of solids and biological specimens in high resolution electron microscopy
Micron and Microscopica Acta, Vol. 23, No. 1/2, pp. 4~64, 1992. Printed in Great Britain.
0739~i260/92 $5.00+0.00 © 1992 Pergamon Press Ltd
QUANTITATIVE ANALYSIS OF SPUTTERING DUE TO ION BEAM BOMBARDMENT OF SOLIDS AND BIOLOGICAL SPECIMENS IN HIGH RESOLUTION ELECTRON MICROSCOPY KOICHI KANAYA,* YOSHINORI MURANAKA, t KATSUHISA YONEHARA~ and KOICHI ADACHI* *Electrical Engineering Department, Kogakuin University, 1-24-2, Nishishinjuku, Shinjuku-ku, Tokyo, Japan, tHamamatsu University, School of Medicine 3600, Handa-cho, Hamamatsu city, Japan and SHitachi Naka Works, Ichige, Katsuta-city, Ibaragi-ken, Japan (Received 17 July 1991; revised 10 September 1991)
Abstract--When a positively charged ion beam is used to bombard a solid target, most of the atoms are displaced and sputtered according to the atomic sputtering theory. In the case of biological specimens, most of the bond-breaking molecules in proteins are removed, when based on the molecular sputtering theory. It was found that the thinning rate for solids and the etching rate for biological specimens, when prepared by a normal double fixation and staining method, can be measured from the sputtering yield and density of the specimens. It was also found that the thinning and etching rates depend on the removal weight per sublimation energy and bonding energy, respectively. The angular distribution of sputtering yield, its dependence on incident angle and the secondary electron emission yield were measured, and the optimum etching condition of the incidence was obtained. Experiments showed that the in situ observation of intracellular structures of biological specimens prepared by ion beam etching can be a very effective method in electron microscopy. Index key words: Sputtering yield, displacement, removal rate, etching, protein, sublimation energy, bonding energy.
INTRODUCTION In high resolution electron microscopy, sputtering can be used simply to remove material from the target and thereby p r o d u c e a thinning of solid materials (Boyde, 1974; Howitt, 1984; K a n a y a et al., 1986a), or etching of biological specimens (Lewis et al., 1968; Spector, 1975; K a n a y a et al., 1982a). In addition, it can be used to deposit materials from the target o n t o an adjacent surface ( K a n a y a et al., 1973; Adachi et al., 1976; Peters, 1980; Echlin et al., 1982). In some cases, studies of u n c o a t e d biological specimens and n o n c o n d u c t i v e materials ( K a n a y a et al., 1982b) have shown that specimens can be prepared by a p r e - b o m b a r d m e n t with argon ions to eliminate charging in a scanning electron microscope (S.E.M.). I o n beam application to specimen preparation in electron m i c r o s c o p y has reached a high state of perfection as shown by the review of K a n a y a et al. (1986b) and by the existence of commercially available ion coating and etching apparatus by gas discharge. However, these developments c a n n o t satisfy the recent advances made in high resolution electron microscopic observations at the atomic level. Moreover, theoretical considerations of the interaction p h e n o m e n a of the ion beam with organic substances (especially biological specimens) are required to realize quantitative needs in electron microscopy. 45
K. Kanaya et al.
46
It has been observed that the interesting example of disintegration of casein submicelles, which are scattered around the miceUes using a high energy ion beam of 15 kV and 30 laA, is probably due to the breaking of calcium phosphate bonds (Hojou et al., 1977). Accordingly, molecular sputtering, taking into account the breaking of bonds, can be applied to biological specimens, and the etching rate measured from the removal depth of the specimens. It was confirmed that the sputtering yield for a solid is inversely proportional to the sublimation energy but for an organic substance is inversely proportional to the bonding energy. Moreover, the angular distribution of the sputtering yield and the incident angle dependency were measured, and semi-empirical relationships were obtained. In addition, the angular distribution of secondary electron emissions was calculated by the electronic stopping power. A successful etching example performed by an in situ observation, with the aid of equipment forming an ion beam source attached to an S.E.M. (Muranaka et al., 1990), is presented. ATOMIC SPUTTERING THEORY (a) The sputtering yield The sputtering yield S (atoms/ion), based on the power potential law (Lindhard et al., 1963) and taking into account the weak-screening effect combined with a hard sphere collision which depends on the nuclear stopping cross-section o, the number of atoms per the unit volume N, and the number of primary knock-on atoms per incident particle Vp, also the secondary displaced atoms and recoils ~Yris given in the previous publications of Kanaya et al. (1973, 1986, 1988): S = KNty(Vp + Vr) = KNrca2~- 2/n(Ag/gD)l/n+ 1/2;
A=4M1M2/(M
1 + M 2 ) 2, a = 0.8853 • a H • (Z2~/3 + Z 2/3) -1/2;
= aM2/Z1Z2e2(M1 + M2),
(1)
where Kis the empirical scaling factor, a is the effective screen radius of the atoms, an is the Bohr radius of hydrogen, e is the reduced value of the energy E, A is the momentum of transfer energy, and Z~, M 1 and Z 2 , M 2 are the atomic number and mass for ion and target atoms, respectively. From the condition of the energy dependent parameter n = 2 and reduced energy em which satisfy the constant energy loss d E / d x , the universal yield-energy relationship can be obtained: S / S m = (~/,~m) 1/2 -- 1/n = (E/Em)I/2 - 1/n; S m = 0 . 4 5 p Z 1 Z 2 ( 1 + M 1 / M 2 ) / M 2 E s ( Z 2/3 + Z2/3)1/2; E m = 0.4" Z1Z2e2(M1 + M2)/aM2,
(2)
where Es(eV ) is the sublimation energy, p is the atomic density of the target (g/cm3), K = 6 . 5 9 . 10 - 1 ° and g i n = 0 . 4 a r e used for the experimental results of A+-Cu when S m= 7 at Em = 42.2 keV. The sputtering yield S can be measured by the weight loss Am = p A V of solid targets (Kaminsky, 1965) S = 26.6 " Am(~tg)/Mz(g) . It(~A . h).
(3)
(b) The thinning rate for solid targets When the sputtering yield S is given by the known incident dose lt/e for the removal volume A V= d ' / r r 2, the removal rate for thickness d/jt (nm-cmZ/mA - min) can be
Quantitative Analysis of Sputtering due to Ion Beam B o m b a r d m e n t
47
R = d/(It/nr z) = 6.23" (A2/p)" S.
(4)
obtained
From the incident dose which is measured by a Faraday cup and the sputtering yield measured by the weight loss method, the thinning rate R for solids is measured as shown in Table 1. Table 1 shows the sputtering thinning rate for A + ion beam bombardment at 5 keV for the solid target and W - M o alloy (50:50%). Duoplasmatron type ion beam apparatus with a high brightness was used in the experiment (Kanaya et al. 1974). For the W - M o alloy, the mean values of A 2, S and R were used. Table 1. The sputtering thinning rate for A ÷ ion beam b o m b a r d m e n t at 5 keV
Specimen
Z2
B C Si Cu Mo W Re Au W Mo
5 6 14 29 42 74 75 79
M2 (g)
p (g/cm 2)
Es (eV)
Em (keV)
.sm (atom/ion)
n
S
11 12 28 64 96 184 186 197 140
2.5 2.2 2.4 8.9 10 19 19.5 19 15
5 7.4 2.8 3.56 6.9 8.76 8.2 3.92 8.8
16.2 18.3 26.6 42.2 57.1 98.3 100.5 105 65.5
4.93 3.12 3.18 7 3.2 3.3 4.41 7.16 5.18
2.4 2.5 2.6 2.8 3.1 3.3 3.4 3.6 3.2
4.4 2.6 2.5 4.9 2.1 1.8 2.1 3.9 2.1
R ( n m . cm2/ m A . min) 121 89 181 220 t26 109 124 252 122
(143") (98*) (187") (250*) (128") (115") (118") (250*) (120")
* Experimental value.
M O L E C U L A R S P U T T E R I N G T H E O R Y AND E X P E R I M E N T S (a) The sputtering yield and etching rate For organic substances such as polymers and soft biological specimens, the molecular sputtering yield Su defined by the ratio of removed molecules per incident ion can be obtained and confirmed experimentally for hydroxyapatite of enamel and dentine in human teeth, also other biological specimens: S M = E . (%)A .SA; SA=Srn" (E/Em) 1/2-1/n,
(5)
where E is the summation of molecules considering the number of atoms (°/O)A in molecules and S A the sputtering yield of an individual atom. The mean number of specific reactions (e.g. bond rupture, cross-linking or disappearance of original molecules) that are produced by an energy of 100 eV is known in radiation chemistry as the G_~ value (Reimer, 1984). Following this principle we used the G Mvalue defined by the ratio of removal molecular weight A per bonding energy ER, as shown in Fig. 1. The molecular etching rate can be obtained by
R M= 6.23.(SM/pM).G_M; G_M =A_R/E•,
(6)
where A_R is the removal mass weight of molecules due to bond splitting. For the protein basic amino acids shown in Fig. 1, the G-value can be given as follows, A_H
A NH3
A_C H
A COOH
G_M- EB(_H) -~ EB(C_N ) + EB(C_H)) -~ EB(C_C ) t-
A_CH -'[-A NH3 -Jr-A~:oon
EB(C_R )
, (7)
where - H is the hydrogen bond with bond energy En = 0.2 eV, and C - H , C-N, C-C,
48
K. Kanaya et al. A
+
43m
a
H
O -H(g/eV)
!
2
-- 0 ,
~(
G -coo.=10
/
H.N
--X
G -R=17.
C
/
65
3,8
X
/
6
G-.H.=4,
/ ~
R
C O OH G -c.:2,
(C
0 0"
4
H)
31
21.?
0.2
a
b
¢
e 10
~
21
f
1
g
h
19.7
11
m
n
17.4
10
i
j
14.8
k
25
18,3
0
14.5
11
r
op~ $
b (a) glyeine (Oly), (b) alpine (Ala), (c) phcnylalantne (Ph¢), (d) t~ptophm~ (Trp), (e) tyrosine C£Yr), ~olcucinc (lle), (g) Icucine(Lcu), (h) mcthionlne (Met), (I)
(0
valine (Val), G) asparagine (Ash), (k)
t
OC oH eNOs O0
cystctne (Cys), (I) glutamine (Gin), (m) prollne (Pro), (n) scrinc (Set), (o) thrconine (Thr),(p) arginine (Arg), (q) histidinc (His), (r) lysinc (Ly$), (s) aspartic acid (Asp), (0 glutarnic acid (Glu)
Fig. 1. The molecular sputtering model, and the G-value of a protein basic amino acid and L-amino acid residues.
C O, N - O , and P = O are given by the b o n d breaking energies 5.5, 3.5, 4.2, 4.2, 2.6 and 6 (eV), respectively (Darnell et al., 1986). The hydrogen bond is included mainly in the residue R. (b) The etchin9 rate m e a s u r e m e n t The biological specimens used in the etching rate experiment were prepared by the n o r m a l m e t h o d of double fixation using glutaraldehyde and o s m i u m tetraoxide,
Quantitative Analysis of Sputtering due to Ion Beam Bombardment
49
followed by positive staining with uranyl acetate and lead citrate. The ion beam apparatus was attached to a J E O L S.E.M. JSE-35 for in situ observations of etched intracellular structures. The ion beam source of the K a u f m a n n type was used at 5 keV and a high vacuum of about 10- 5 Torr. The ion beam dosejt was limited by an aperture of 1 x 1 c m 2, and the dry density PM was measured. The etching rate R M was then obtained by the removal depth d. Figure 2 shows the depth measurement for a human tooth specimen b o m b a r d e d with an A + beam at 5 keV through the mesh grids placed on it at a small angle tilted with repect to the ion beam. The increased etching rate due to the incident angle v can be considered to be less than 10% for 15 °. Glutaraldehyde, M = 100, an aliphatic c o m p o u n d ( O H C C H E C H E C H O ) , can be considered to be the residue of the protein basic amino acids and its effect can thus be disregarded. Moreover, the metallic inclusions used as the fixation and stain, such as O s O 4, Pb(NO3) 2 or P b ( C H a C O O ) 2 , are considered to be small compounds which was confirmed by the measurement of dry density PM = 0.434).85. They, like aromatic compounds, have a stable diester of strong double bonds of E R= 6-10 (eV), which we assume will probably remain. The very large values of removal thickness d and the resulting etching rate R Mcan be analysed by the packing densitiy factor 7 (%), considering the inherent gaseous molecules and vacuoles, and the removal mass weight decreases as ~ • A_ u, as analysed in Table 2. The specimens in Table 2 show the ion beam sputtering analysis of biological specimens that are assumed to be the protein basic amino acids based on the molecular sputtering theory. It was assumed that in basic protein amino acids all the bonds are broken, also the NHa-molecule involving the C - N bond, in addition, the H a t o m in C O O H molecules as well as the H a t o m in the hydrogen bond where residues are displaced. The strong bond molecules which are fixed and stained with heavy metal atoms remain as the nuclei. (c) Atomic and molecular sputtering for human tooth enamel and dentine H u m a n tooth enamel and dentine, which are known to be composed of a hexagonal structure of hydroxyapatite [Ca 10(PO4)6(OH)2], as reported in a previous paper by K a n a y a et al. (1984), was examined for the sputtering yield analysis. The specimens of both enamel and dentine were prepared by cuts from a native rod material and polished by emery paper. The mesh grids were mounted on to the specimen, as shown in Fig. 2 and examined by S.E.M. For tooth enamel and dentine, as for solids, the displacement due to the atomic sputtering process for Ca and P ionic atoms, together with molecular sputtering process for the P O 4 and O H covalent molecules, were considered; also both the sputtering yield S A and S Mdepend on the inverse of the sublimation energy E s and the bonding energy E a, respectively. If the content of atoms (%)A and molecules (%)M are expressed, the sputtering rate R M is given as follows: RM= (6'23/pM) " {(%)ca "Sca "G-ca+ ( % ) e ' S v ' G - P + (%)vo4 "Svoa "G-vo4 + (°/O)on "Son" G~H}; G~ca =40/2.1 (g/eV), G_p= 31/1.37 (eV), G_po" = (31 + 64)/6 (g/eV), G on = (16 + 1)/5.5 (g/eV). (8) Table 3 shows the sputtering yield analysis for tooth enamel and dentine as determined by the etching rate measurement for A + ion beam b o m b a r d m e n t at 5 keV, where in the case of enamel the atomic collision process for Ca and molecular sputtering process for PO 4 and O H are considered. But the P O 4 molecules in dentine are assumed to be displaced due to the partially atomic sputtering for P, and mainly the molecular sputtering for P O 4. Since a high level of lattice defects has been observed by electron microscopy, the difference of etching rate between enamel and dentine can be considered to be the result of crystal structure imperfections.
50
K. K a n a y a et al. 15 d e g r e e
Specimen ...... S u r f a c e Grid Mesh Specimen Stage
Fig. 2. Measuring method of removal depth d = d * / c o s v taken with an S.E.M. for tooth enamel and dentine (a), where d* is the observed value when the incident angle v is as shown in (b).
Quantitative Analysis of Sputtering due to Ion Beam Bombardment
51
Table 2. The molecular sputtering analysis of biological specimens obtained by the etching rate for A + ion beam bombardment at 5 keV
Specimen Lung Kidney Liver Cerebellum Muscle Heart muscle Cerebrum
PM (g/cm 3)
RM r a m .cm2~ \ m A . min]
y. R M
SM fmol.'~ \ ion j
0.43* 0.85* 0.75* 0.66* 0.61" 0.58* 0.61"
8800* 2200* 2200* 1500" 1700" 1650" 1300"
5650 1408 1408 960 1088 1056 832
6.5 3.2 2.8 1.7 1.7 1.6 1.46
G _ M
f. removal weight "~ \break bond energy,] 60 60 60 59 61 61 65
(80 + ) (60 + ) (60 + ) (60 + ) (60 +) (60 +) (60 + )
G n = A-n/EB(-H)= 5 (g/eV), G~cn= A~cn/Es (C-H)= 2.4, G_NH3=A_NnJE B (C-N)=2.6, G~cooa= A~:oon/E ( C ~ ) = 10, G R= (A ca +A_Nm + A~coos)/E_s(C~C)= 17.65, E B (-H)=0.2 (eV), E s (C-H)= 5.5, E s (C-N)=3.5, E s (C~C)=4.2, y=0.63. * Experimental value. 60 + value by G M=5G n+G~:n+G_Nn3-G~:ooH+G R.
Table 3. The sputtering yield analysis of tooth enamel and dentine obtained by the etching rate for A + ion beam bombardment at 5 keV Atom
Z2
M2 (g)
PA (g/cm 3)
H O O P P Ca
1 8 8 15 15 20
1 16 16 31 31 40
0.07 1.4 1.4 0.85 0.85 1.54
Es Er~ SM (eV) (keV) (atom/ion) 5.5* 5.5* 0.6t 0.6t 1.37 2.1
10 I0 20 27.4 27.4 33.4
1.62 0.89 1.94 1.8 7.98 2.93
n
SA RA (atom/ion) (nm. cm2/mA • min)
2.3 2.3 2.6 2.7 2.7 2.8
1.5 1.37 1.7 1.4 6.2 2.2
89 155 169 52 227 162
G_M
Element
PM
(%)M
(g/eV)
SM
RM
Ca PO 4 (enamel) PO 4 (dentine) OH
1.5"I" 1.5t 1.5t 1.5t
0.23 0.49 0.36 0.1
4.19 15.8 20.0 2.8
2.2 1.64 2.6 1.43
40 53 79 2
Enamel RM=95 (86.6t) Dentine R u = 121 (123t)
* O-H bonding energy. t Experimental value. :~ P = O bonding energy.
ANGULAR
DISTRIBUTION
OF SPUTTERING
PARTICLES
(a) Solid targets T h e s p a t i a l d i s t r i b u t i o n of s p u t t e r e d particles is of g r e a t i n t e r e s t b o t h f r o m the p o i n t of view of s p u t t e r i n g t h e o r i e s a n d for the v a r i o u s a p p l i c a t i o n s ( K a m i n s k y , 1965, K a n a y a et al., 1982), b u t r e l a t i v e l y few d a t a are c u r r e n t l y a v a i l a b l e ( R o o s e n d a a l a n d S a n d e r s , 1980; B o h d a n s k y et al., 1982; K a n a y a et al., 1986; D o d o r o v et al., 1989). F o r h i g h r e s o l u t i o n s p e c i m e n p r e p a r a t i o n , the e x p e r i m e n t a l d a t a of s p u t t e r i n g yield at v a r i o u s a n g l e s of i n c i d e n c e are r e q u i r e d . T h e d u o p l a s m a t r o n i o n b e a m s o u r c e was u s e d for solid m a t e r i a l e x p e r i m e n t s . F o r b i o l o g i c a l s p e c i m e n s a K a u f m a n n - t y p e s o u r c e (1982) was e m p l o y e d . It was i m p r o v e d in o r d e r to o b t a i n a high b r i g h t n e s s b y f o c u s i n g lens a c t i o n . B a s e d o n the p o w e r p o t e n t i a l law ( K a n a y a et al., 1986a), the differential s p u t t e r i n g
52
K. Kanaya
et al.
yield per solid angle as a function of the nominal angle of incidence v and the observation angle ® measured from the deposition peak density can be obtained as follows: 27r
dS(v,®) d~ -
S(v, 0)"
(COS~)~3/2+ 1/.
\ cos v /
(9)
From the density distribution of deposition, following the mirror symmetry law, the angular distribution can be evaluated on the basis of the incident angle v, the critical angle 0~ showing the peak value, and the incident energy dependent parameter n obtained by the power potential law. Figure 3 shows the method of measuring sputter deposition profiles, which are represented by an anisotropical angular distribution. The deposition profile for Cu extracted from the various solid targets is shown in Fig. 4b. The equal-density lines evaluated by values relative to the maximum are plotted using a light densitometer.
a
b
,oo, ....
pro?ile
Asymme*ry
in the plane tMrough incoming ion beam
and Surface nomol Symmetry in tt~e transverse plane
Fig. 3. Measurement of the angular distribution of ion beam sputter deposition and its angular coordinates.
The reduced value of angular distribution, formulated as follows. In the incident plane as shown in Fig. 4a: _ _ dS - (cos dn
S(v)
dS/S dr2 as a function of 0 can be
(
1 "] 1/2+1/"
0)3/2+1/";S(v)=S(O)'\c~v/
(10)
where ® is the observable angle subtended by the maximum. ® = ( ~ / 2 ) - (0+0c)/{(~/2)+0c}, for O 0 c. In the transverse plane: dS _ (cos ®0 3/2 + 1/,; S(®) = S(®) d n
S(v). (cos 0 ) 3/2 + 1/,.
(11)
When these deposition profiles are theoretically analysed, the angular distribution can be plotted as shown in Fig. 5. It was found that for n = oc, 2, 1, dS/S df~ becomes the following power cosines in a simple form such as (cos ®),/2, corresponding to less than l keV, (cos O) 2,
a Incident plane dS S(Ot)dtq - S(O)
Equal
T
density contour
-----
Transverse
plane
d ~ _ , , S(8t )
e,m
I \ \ ) o.~ \~ 0.5-< \0.2 ~:
I
7.5c
[
/t
-
~ + O e - - - - e c
--~
~+ U-.-45"
• 0©=12.5
Q
A+''~ Cu IJ=65
, 0~=16
U-75
~ , 8e=l~S.5"
6......... 5- .......... ~ ................ z'9
(rad) Fig. 4. Spatial distribution analysis (a) of sputter deposition profiles for the A ÷ Cu combination at 5 keV (b).
54
K. Kanaya et al.
1
o~
o
d$
•
.
' '
I ....
o~ ' '
~1$
F i g . 5. A n g u l a r
distribution
i o15 '
'
'
'
dS
o
• o~ . . . .
i
as
of reduced sputtering yield with bombardment t h e i n c i d e n t a n g l e v.
of A ÷ ion beam at
corresponding to 5-10 keV and (cos ®)5/2, corresponding to over 10 keV, respectively. Figure 6 shows the relationship between the incident angle v and the critical angle 0 c obtained from the equal-density peak. The measured value of 0 c depends on the incident angle and backscattered energy, and are empirically given by sin 0c = sin v/21/4 + 1/2n.
(12)
The important critical angles differ from the previously obtained values (Biersack and Eckstein, 1984; Bohdansky et al., 1982) and are similar to the results obtained by Roosendaal and Sanders (1980) and D o d o r o v et al. (1989). Based on the power cosine law the oblique incident angle dependent on the relative sputtering yield is obtained S(v)/S(O) = [1 - {sin(v - v~)/cos v} z] (COS
,~)1/2+1/n
, V>I)c
- 1/(cos v) 1/2 + 1/,, v < v~.
(13)
The critical angle v¢ of energy attenuation in a transfer energy T = A E cos 2 v is empirically obtained vc = c o s - 1( E J A E ) 1/3,.
(14)
Figure 7 shows the oblique incident angle dependent on the sputtering yield for A + ions at 5 keV. The peak values of Vp can be obtained under the condition of d S { ( v ) / S ( O ) } / d n = O in Eqn (13) as follows, sin(2%-2v~)/cos(2vc - %)-= (2 + n)/2n.
(15)
Figure 8 shows the determinations of Vp as a function of the critical angle vc and the energy dependent parameter n.
Quantitative Analysis of Sputtering due to Ion Beam Bombardment
55
V [r~] 0.5 I ec
25
~
n
•
]:~
12,79" 1P-40" 12.28"
16.49" IE,.97" 15.82"
11.66" tl.58"
15.0;°1 14.90"
Eo=1OkV ; W= W' ~Mo" for E°=SkV_
~
B
~
--
16.49"
3,12" 3.02" ;~.99" 2.90" 2.84" 2,82"
C
I
65" )690.
3.12 o 12.79"
3.2 2.8 2.7 2.4 2.25 2.2
S~
20
4~ • 13,11 •
1.5
I
~.,4- ,z..- ~ o
U-MO*O~LOy 3.2
Re Mo Cu
8c
• 3~9"
1.0
'
~
0.4
~
11,89" 15.31"
m
1.
0,~
2
8c
[ deg ]
0.2 [ red ] 10
0.1
5
0
I
I
I
I
l
15
30
45 P (~g]
60
75
90
Fig. 6. The critical angle 0 c showing the peaks of sputtering yield obtained as a function of the incident angle v.
5
sin(u-~:)
c
0.5
1.0
_~
P(rod)
z
Fig. 7, Oblique incident angle dependence of sputtering yield for A + ion beam at 5 keV.
Up (rod) 90
r
,
~
i
sin (2 v,,-2u,) 70
COS(21A-up)
0,5 [
r
~
r
i
2+n " ~
1.0 1
i
,
T
~
r
2
~(/
n-=oo -~ .
40
ff
50
n=3~)
I
I lO
I
J ' 20
~ ] 30
/ h 40
,
2 ~ / 50
60
7JE3 0
J
20 80
0 5
I
90
Up(~g)
Fig, 8. Analytical determination of the peak angle Vp as a function of the critical angle vc and energy parameter n.
56
K. Kanaya et al.
(b) Biological specimens The angular distribution measurement for biological specimens which was obtained by the Kaufmann-type ion source is shown in Fig. 9. The curves shown in Fig. 9a illustrate the result of the relative density in the incident plane which was measured by thin film chromatography. Figure 9b shows the relative distribution, dS/S(®) dfL by a polar coordinate plot.
a A ° 5keV
Geta?in
e¢=45"
Liver
0¢:4~"
Kidney
~
ds sdQ
Exp.pt - -
|
K
,
-90*
i
,
I LO
, G
O"
1.0
0.5
0
+ 90" (0)
0.5
1.0 sd~)
Fig. 9. Angular distribution of the reduction sputtering yield for biological specimens and organic gelatin (a) measured by thin filmchromatography and their polar coordinate plots (b).
For biological specimens, the basic relationships of sputtering yield characteristics were analysed similarly.
dS/S(v) df~ = (cos ®)3/2 + 1/.; S(v)/S(O)= (cos v)- 1/2 - I/n, y < Vc S(v)/S(O)~__(cos v ) - 1/2
1/n , 0, V~>Vc; I']=
1 - { s i n ( v - v ~ ) / c o s v} 2.
(16)
The large critical angle 0~ = v is assumed to be the molecular sputtering process by molecules of small atomic number. Figure 10 shows an example to find the optimum etching condition for microvilli of the small intestine which was prepared by normal fixation and staining processes. Figure 10a is the relative sputtering yield S(v)/S(O) as a function of the incidence v; Fig. 10c shows the etched surfaces for a large angle of incidence, where the cone-like artifact can be analysed as shown in Fig. 10b. The etched depth d used for the analysis (Yonehara et al., 1989) is given by
d(v)=dorl(cos v ) - l ; do=6.23 • (SM/pM)" G M "jt.
(17)
The m a x i m u m angle vp = 70 ° and d(n/4)/d o = 1.5 are in agreement with the theory. The result shows that biological specimens are etched according to Eqn. (17). In the case of a normal angle of incidence such artifacts can be removed as shown in Fig. 10d.
Quantitative Analysis of Sputtering due to Ion Beam Bombardment
57
a
0
0.5
1.0
/T
v (rad)
b I
/" /
Ion beam
./ ./ "
Fig. 10. The incident angle dependence of the etched surfaces for microvilli and etching mechanism analysis, where (a) is the empirical yield-angle relationship, (b) the etched profile analysis for (c), (c) the experimental result with an angle of incidence, and (d) by normal angle of incidence.
Figure 11 shows a successful result and an example of the rotary etching method for freeze-fractured surfaces of rat liver, which was prepared by the normal fixation and staining process. Figure 11 a shows the rotary etching method, in which the specimen stage is rotated at a variable a n g l e _ Av with respect to the incident angle v, using gimbals capable of universal universal rotation. The original S.E.M. image shown in Figs. 11 b, 11c illustrates the etched patterns bombarded with A ÷ ion beam at 2 keV for 30 min. The embossed nuclei (n), rough endoplasmic reticulum (rer), and mitochondria (m) are clearly revealed. It was considered that the fixed and stained nuclei remained, and their surrounding molecules were removed. The mean optimum angle of the incidence v+Av=(n/4)+_(n/8) at a rotation speed 3 0 r p m was obtained (Muranaka et al., 1986).
58
K. Kanaya et al.
[ Fig. 11. The rotary etching example for the freeze-fractured surface of rat liver. (a) The rotary etching method. (b) The original S.E.M. image taken at 30 kV. (c) Its etched nuclei structure recorded from A ÷ ion beam bombardment at 2 keV during 30 min under the incident rotation angle n/4+ n/8. (d) Subsequent nuclei etched during the 60 min under the same operating conditions.
ANGULAR
DISTRIBUTION OF SECONDARY ELECTRON Y I E L D D U E T O I O N BEAM B O M B A R D M E N T
EMISSION
According to the kinetic theory of secondaries ( K a n a y a et al., 1988a), for less than 100 keV, the emission yield of electronics is given by S =
S m " (/3//3m)1/2;
/~m =
8.4 • 102 = a E / Z l e 2 M 1
", s m = 2 1 . 7 • P 2 Z I Z 2 / I M 2 ( Z
2/3 + Z 2 / 3 ) 1/2
08) where I is the ionization energy (eV). The ratio of electrons yielded to atoms can be calculated as 0.1~3.5 at 1-10 keV. This is similar to the elementary theory of secondary emission for electrons developed by K a n a y a and O n o (1976), where the n u m b e r of secondary electrons due to electronic collision of ion beam b o m b a r d m e n t is p r o p o r t i o n a l to the electronic energy loss ( d E / d x ) e . They arrive at the surface by travelling a distance x cos v/cos 0 t h r o u g h the material, and the secondary yield s is given by
Quantitative Analysis of Sputtering due to Ion Beam Bombardment s(v, O)= k
f
R/2 ~ n / 2
do
o s = 3.7.
l 30
O s COS Y" X
(dE/dx)e " e
cos 0
59
• sin 0 dO dx
p2Z22/3R/M2I,
(19)
where R is the penetration range of the ions (nm), trs is the transmission factor of secondary electrons and k is a constant. Then the anisotropic angular distribution of ds(v, ®)/df~ can be derived from the integration of Eqn. (19) similarly to the angular distribution of sputtering yield as follows, ds(v, O ) / s d ~ = (1/2~) • (cos O / a s cos v)" I-1 - { 1 - e x p ( - a
s cos v/cos O)}
(cos O / a s cos v)],
(20)
where O = (re/2)(0+ 0c)/{(rc/2)_ 0c) and 0¢ = v are considered. Figure 12 shows the angular distribution of secondary electron emission yield in the incident plane, where it is calculated in the normalized form as ds(v)/s dfL The secondary electrons yielded were confirmed by electron exposure experiments. A T S K photoresist (Toyobeam Co.) with a sensitivity of 1.73 (lac/cm 2) was used for detecting the existence of electrons. The oblique angle of incidence of electron emission yield can be integrated from Eqn. (20) as follows, s(v)/s(O) = f ( y ) , for v < v~; s(v)/s(O) = F ( y ) " rl, v > v¢, f ( y ) = (I/y) { 1 - (2/3y) - (1/3)" e x p ( - y)" { 1 - 2 / y ) - y}, y = a s cos v, ~/= 1--{sin(v--v~)/cos v} 2.
a~
S(e) :
Secocdfy electron M~llion yle(d
mctdlmt
•
1
0,8
(21)
O, - 100
O~
1
!
0.6
0
0,8
Fig. 12. Angular distribution of the secondary electron emission yield for A + ion beam at 5 keV.
I
60
K. Kanaya et al.
Figure 13 shows the relative value of the secondary electron emission yield depending on the oblique angle of incidence, which is calculated from Eqn (21 ), where 0 c = v and vc is shown by empirical values. It was shown in a previous paper (Kanaya et al., 1988b), the angular distribution of secondary electron emission has a high value at an early stage of deposition. The secondary electrons were contributed in order to obtain the monolayer films by reducing the surface potential level. In the case of biological specimens, the secondary electron emission yield is considerably smaller than 0.1, and can be disregarded during the applications of ion beam sputtering.
30
O(deg)
60
Re
90 Au
S(u)
s-TN'
0
0.5
1.O
lJ(rad)
2
Fig. 13. Dependence of the secondary electrons on an oblique angle of incidence for an A + ion beam at 5 keV.
U N C O A T E D O B S E R V A T I O N AND E T C H I N G A P P L I C A T I O N S In microscopic studies of the compound eye of the house fly using serial sections, interesting structural details of visual cells forming the retinula have been observed by Fernfindez-Mor~in (1956). As an example of uncoated and subsequent in situ observations of etched surfaces using the ion beam apparatus (Muranaka et al., 1990), different internal structures of the corneal lens in the ommatidium were clearly observed. Figure 14a shows a T.E.M. image of the ommatidium of the house fly (Musca domestica) by microtomy sections, where a-e correspond to the etched surfaces in Fig. 14a-e. Figure 14a shows the uncoated S.E.M. images for the surface of an ommatidium with a hexagonal-shape, bombarded with an A + ion beam at 1 keV for 5 min. Figure 14b shows the etched surface at a depth of about 0.5 gm acting as a dioptic apparatus which is obtained by A + at 2 keV for 20 min. Figure 14c shows the hexagon at a depth of 2 i.tm by 2 keV for 80 min. Figure 14d shows the deep etched hexagon at 20 gm by 2 keV for 140 min. The wide vacuole around the ommatidium and transverse laminated structure was observed. Figure 14e shows the pigment cell (p) in the central cavity and the retinula cell (r) constituting the rhabdomere acting as a light receptor, observed at 2 keV for 200 min. The measured etching rate of the assumed protein specimen was found to depend mostly on the packing density factor ? which is determined by the vacuole and cavity. It was confirmed that uncoated and etching observations are very effective in the internal structural analysis of biological applications.
Fig. 14. Uncoated and etching observations for the ommatidium of the house fly, where (A) is the T.E.M. image of the sectioned compound eye, (a~), (a2), (a3) in (a), (b~(e) S.E.M. images of structural details of the corneal lens in the ommatidium at different depths.
t~
O
.=
OQ
7
J
Quantitative Analysis of Sputtering due to Ion Beam Bombardment
63
CONCLUSIONS (i) Reliable d a t a of s p u t t e r i n g yield for biological specimens a n d solid targets were o b t a i n e d by the m e a s u r e m e n t of the r e m o v a l etching a n d t h i n n i n g rates based o n a t o m i c a n d m o l e c u l a r s p u t t e r i n g theory. (2) T h e spatial d i s t r i b u t i o n of s p u t t e r i n g yield a n d its d e p e n d e n c e o n incident angle were q u a n t i t a t i v e l y analysed by the m e a s u r e m e n t of sputtered d e p o s i t i o n profiles o n the collected stage. (3) T h e a n g u l a r d i s t r i b u t i o n of secondary electron emission yield was theoretically calculated a n d confined by the electron b e a m exposure m e t h o d . (4) It was confirmed that in situ o b s e r v a t i o n s for u n c o a t e d a n d etched specimens of n o n - c o n d u c t i v e materials c a n be very effective for biological a p p l i c a t i o n s in the S.E.M. Acknowledgements--The authors wish to thank Professor R. W. Home, University of East Anglia, for helpful comments and advice in connection with the preparation of this manuscript.
REFERENCES Adachi, K., Hojou, K., Katoh, M. and Kanaya, K., 1976. High resolution shadowing for electron microscopy by sputter deposition. Ultramicroscopy, 2: 17-29. Biersack, J. P. and Eckstein, W., 1984. Sputtering studies with the Monte Carlo program TRIM. SP. Appl. Phys. A, 34: 73-94. Bohdansky, R., Chen, G. L., Eckstein, W., Roth, J., Scherzer, B. M. U. and Bererisch, R., 1982. Light ion sputtering for H.D. and He in the energy range of 25 keV to 100 keV. J. Nuclear Materials, 111, 112: 717-725. Boyde, A., 1962. TEM of ion beam thinned dentine. Cell Tissue Res., 152:543 550. Darnell, J., Lodish, H. and Baltimore, D., 1986. Molecular Cell Biology. ScientificAmerican Books, p. 1192. Dodorov, A. I., Fayazov, I. M., Fedorovich, S. D., Krylova, E. A., Maskova, E. S., Molchanov, V. A. and Eckstein, W., 1989. Experimental and computer study of the spatial distribution of particles sputtered from polycrystals. Appl. Phys. A, 49: 299-304. Echlin, P., Chapman, B. and Stoter, I., Gee, W. and Burgess,A., 1982. Low voltage sputter coating. Scanning Elect. Microsc. 1: 29-38. Ferfiandez-Mor~n, H., 1956. Fine structure of the insect retinula as revealed by electron microscopy. Nature, 21: 742-743. Hojou, K., Oikawa, T., Kanaya, K., Kimura, T. and Adachi, K., 1977. Some applications of ion beam sputtering to high resolution electron microscopy. Micron, 8: 151-170. Howitt, D. G., 1984. Ion milling of materials sciencespecimensfor electron microscopy.J. Electron Microsc. Tech., 1: 405-414. Kaminsky, M., 1965. Atomic and Ionic Impact Phenomena on Metal Surfaces. Springer, New York. Kanaya, K., Hojou, K., Koga, K. and Toki, K., 1973. Consistent theory of sputtering of solid targets by ion bombardment using power potential law. Jap. J. Appl. Phys., 12: 1297-1306. Kanaya, K., Hojou, K., Adachi, K. and Toki, K., 1974. Ion bombardment of suitable target for atomic shadowing for high resolution electron microscopy. Micron, 5:89 119. Kanaya, K., Baba, N., Kai, M., Oho, E. and Muranaka, Y., 1982a. Digital processing method using scanning densitometer and microcomputer for the structural analysis of a scanning electron micrograph. Scanning Elect. Microsc., IV: 61-72. Kanaya, K., Muranaka, Y. and Fujita, H., 1982b. Uncoated observation and etching of non-conductive materials by ion beam bombardment in scanning electron microscopy. Scanning Elec. Microsc., IV: 1379-1394. Kanaya, K., Baba, N., Shinohara, C. and Ichijo, T., 1984. A digital Fourier harmonic superposition method for the structural analysis of human tooth enamel obtained by electron microscopy. Micron Microsc. Acta, 15: 17-35. Kanaya, K., Baba, N., Naka, M., Kitagawa, Y. and Suzuki, K., 1986a. The digital structural analysis of cadium selenide crystals by a method of ion beam thinning for high resolution electron microscopy. Micron Microsc. Acta, 17: 25-43. Kanaya, K., Baba, N., Muranaka, Y. and Adachi, K. 1986b. Applications of ion beam sputtering for highresolution electron microscopy. J. Elect. Microsc. Tech. 4: 1-19. Kanaya, K., Ogihara, A., Oda, T. and Osaki, N., 1988a. Interaction of ion beam with the target in electron microscope applications. Micron Microsc. Acta, 19: 87-103. Kanaya, K., Baba, N., Yamamoto, Y. and Yonehara, K., 1988b. The nucleation and growth of thin films deposited on to carbon substrate by ion beam sputtering. Micron Microsc. Acta, 19: 189-199.
64
K. Kanaya et al.
Kaufmann, H. R., 1982. Technology and application of broad beam ion sources in sputtering. J. Vacuum Sci. Techn., 212:725 736. Lewis, S. M., Osborn, J. S. and Stuart, P. R., 1968. Demonstration of an internal structure within the red blood cells by ion etching and scanning electron microscopy. Nature, 220: 614-616. Lindhard, J., Nielsen, V. and Thomsen, P. V., 1963. Integral equations governing radiation effects. Mat. Fys. Dan. Vid. Selek., 33: 1-42. Muranaka, Y., Shibata, K., Baba, N. and Kanaya, K., 1986. Ion beam etching method for SEM observation of inside structures stepwise from surface in biological specimens. Proceedings of l lth International Congress on Electron Microscopy, Kyoto, I, pp. 357 358. Muranaka, Y., Adachi, K. and Kanaya, K., 1990. Uncoated and etching methods using ion-beam bombardment for scanning electron microscopy and development of ion-beam apparatus for in situ observation. Proceedings of International Conference on SEM, Virginia, 1990. Peters, K.-R., 1980. Penning sputtering of ultra thin films for high resolution electron microscopy. Scannin9 Elect. Microsc., 1:143 154. Roosendaal, H. E. and Sanders, J. B. 1980. On the energy distribution and angular distribution of sputtered particles. Rad. Effects, 52: 137-144. Reimer, L., 1984. Transmission Electron Microscopy. Springer, Berlin, pp. 421-447. Sigmund, P., 1973. A mechanism of surface micro-roughening by ion bombardment, J. Mat. Sci., 8: 1545-1553. Spector, M., 1975. Ion beam etching in a scanning electron microscopy: red blood cell etching. Micron, 5: 263-273. Yonehara, K., Baba, N. and Kanaya, K., 1989. Applications of ion-beam etching techniques to the fine structure of biological specimens as examined with a field emission SEM at low voltage. J. Elect. Microsc. Techn., 12: 71-77.
0739~i260/92 $5.00+0.00 © 1992 Pergamon Press Ltd
QUANTITATIVE ANALYSIS OF SPUTTERING DUE TO ION BEAM BOMBARDMENT OF SOLIDS AND BIOLOGICAL SPECIMENS IN HIGH RESOLUTION ELECTRON MICROSCOPY KOICHI KANAYA,* YOSHINORI MURANAKA, t KATSUHISA YONEHARA~ and KOICHI ADACHI* *Electrical Engineering Department, Kogakuin University, 1-24-2, Nishishinjuku, Shinjuku-ku, Tokyo, Japan, tHamamatsu University, School of Medicine 3600, Handa-cho, Hamamatsu city, Japan and SHitachi Naka Works, Ichige, Katsuta-city, Ibaragi-ken, Japan (Received 17 July 1991; revised 10 September 1991)
Abstract--When a positively charged ion beam is used to bombard a solid target, most of the atoms are displaced and sputtered according to the atomic sputtering theory. In the case of biological specimens, most of the bond-breaking molecules in proteins are removed, when based on the molecular sputtering theory. It was found that the thinning rate for solids and the etching rate for biological specimens, when prepared by a normal double fixation and staining method, can be measured from the sputtering yield and density of the specimens. It was also found that the thinning and etching rates depend on the removal weight per sublimation energy and bonding energy, respectively. The angular distribution of sputtering yield, its dependence on incident angle and the secondary electron emission yield were measured, and the optimum etching condition of the incidence was obtained. Experiments showed that the in situ observation of intracellular structures of biological specimens prepared by ion beam etching can be a very effective method in electron microscopy. Index key words: Sputtering yield, displacement, removal rate, etching, protein, sublimation energy, bonding energy.
INTRODUCTION In high resolution electron microscopy, sputtering can be used simply to remove material from the target and thereby p r o d u c e a thinning of solid materials (Boyde, 1974; Howitt, 1984; K a n a y a et al., 1986a), or etching of biological specimens (Lewis et al., 1968; Spector, 1975; K a n a y a et al., 1982a). In addition, it can be used to deposit materials from the target o n t o an adjacent surface ( K a n a y a et al., 1973; Adachi et al., 1976; Peters, 1980; Echlin et al., 1982). In some cases, studies of u n c o a t e d biological specimens and n o n c o n d u c t i v e materials ( K a n a y a et al., 1982b) have shown that specimens can be prepared by a p r e - b o m b a r d m e n t with argon ions to eliminate charging in a scanning electron microscope (S.E.M.). I o n beam application to specimen preparation in electron m i c r o s c o p y has reached a high state of perfection as shown by the review of K a n a y a et al. (1986b) and by the existence of commercially available ion coating and etching apparatus by gas discharge. However, these developments c a n n o t satisfy the recent advances made in high resolution electron microscopic observations at the atomic level. Moreover, theoretical considerations of the interaction p h e n o m e n a of the ion beam with organic substances (especially biological specimens) are required to realize quantitative needs in electron microscopy. 45
K. Kanaya et al.
46
It has been observed that the interesting example of disintegration of casein submicelles, which are scattered around the miceUes using a high energy ion beam of 15 kV and 30 laA, is probably due to the breaking of calcium phosphate bonds (Hojou et al., 1977). Accordingly, molecular sputtering, taking into account the breaking of bonds, can be applied to biological specimens, and the etching rate measured from the removal depth of the specimens. It was confirmed that the sputtering yield for a solid is inversely proportional to the sublimation energy but for an organic substance is inversely proportional to the bonding energy. Moreover, the angular distribution of the sputtering yield and the incident angle dependency were measured, and semi-empirical relationships were obtained. In addition, the angular distribution of secondary electron emissions was calculated by the electronic stopping power. A successful etching example performed by an in situ observation, with the aid of equipment forming an ion beam source attached to an S.E.M. (Muranaka et al., 1990), is presented. ATOMIC SPUTTERING THEORY (a) The sputtering yield The sputtering yield S (atoms/ion), based on the power potential law (Lindhard et al., 1963) and taking into account the weak-screening effect combined with a hard sphere collision which depends on the nuclear stopping cross-section o, the number of atoms per the unit volume N, and the number of primary knock-on atoms per incident particle Vp, also the secondary displaced atoms and recoils ~Yris given in the previous publications of Kanaya et al. (1973, 1986, 1988): S = KNty(Vp + Vr) = KNrca2~- 2/n(Ag/gD)l/n+ 1/2;
A=4M1M2/(M
1 + M 2 ) 2, a = 0.8853 • a H • (Z2~/3 + Z 2/3) -1/2;
= aM2/Z1Z2e2(M1 + M2),
(1)
where Kis the empirical scaling factor, a is the effective screen radius of the atoms, an is the Bohr radius of hydrogen, e is the reduced value of the energy E, A is the momentum of transfer energy, and Z~, M 1 and Z 2 , M 2 are the atomic number and mass for ion and target atoms, respectively. From the condition of the energy dependent parameter n = 2 and reduced energy em which satisfy the constant energy loss d E / d x , the universal yield-energy relationship can be obtained: S / S m = (~/,~m) 1/2 -- 1/n = (E/Em)I/2 - 1/n; S m = 0 . 4 5 p Z 1 Z 2 ( 1 + M 1 / M 2 ) / M 2 E s ( Z 2/3 + Z2/3)1/2; E m = 0.4" Z1Z2e2(M1 + M2)/aM2,
(2)
where Es(eV ) is the sublimation energy, p is the atomic density of the target (g/cm3), K = 6 . 5 9 . 10 - 1 ° and g i n = 0 . 4 a r e used for the experimental results of A+-Cu when S m= 7 at Em = 42.2 keV. The sputtering yield S can be measured by the weight loss Am = p A V of solid targets (Kaminsky, 1965) S = 26.6 " Am(~tg)/Mz(g) . It(~A . h).
(3)
(b) The thinning rate for solid targets When the sputtering yield S is given by the known incident dose lt/e for the removal volume A V= d ' / r r 2, the removal rate for thickness d/jt (nm-cmZ/mA - min) can be
Quantitative Analysis of Sputtering due to Ion Beam B o m b a r d m e n t
47
R = d/(It/nr z) = 6.23" (A2/p)" S.
(4)
obtained
From the incident dose which is measured by a Faraday cup and the sputtering yield measured by the weight loss method, the thinning rate R for solids is measured as shown in Table 1. Table 1 shows the sputtering thinning rate for A + ion beam bombardment at 5 keV for the solid target and W - M o alloy (50:50%). Duoplasmatron type ion beam apparatus with a high brightness was used in the experiment (Kanaya et al. 1974). For the W - M o alloy, the mean values of A 2, S and R were used. Table 1. The sputtering thinning rate for A ÷ ion beam b o m b a r d m e n t at 5 keV
Specimen
Z2
B C Si Cu Mo W Re Au W Mo
5 6 14 29 42 74 75 79
M2 (g)
p (g/cm 2)
Es (eV)
Em (keV)
.sm (atom/ion)
n
S
11 12 28 64 96 184 186 197 140
2.5 2.2 2.4 8.9 10 19 19.5 19 15
5 7.4 2.8 3.56 6.9 8.76 8.2 3.92 8.8
16.2 18.3 26.6 42.2 57.1 98.3 100.5 105 65.5
4.93 3.12 3.18 7 3.2 3.3 4.41 7.16 5.18
2.4 2.5 2.6 2.8 3.1 3.3 3.4 3.6 3.2
4.4 2.6 2.5 4.9 2.1 1.8 2.1 3.9 2.1
R ( n m . cm2/ m A . min) 121 89 181 220 t26 109 124 252 122
(143") (98*) (187") (250*) (128") (115") (118") (250*) (120")
* Experimental value.
M O L E C U L A R S P U T T E R I N G T H E O R Y AND E X P E R I M E N T S (a) The sputtering yield and etching rate For organic substances such as polymers and soft biological specimens, the molecular sputtering yield Su defined by the ratio of removed molecules per incident ion can be obtained and confirmed experimentally for hydroxyapatite of enamel and dentine in human teeth, also other biological specimens: S M = E . (%)A .SA; SA=Srn" (E/Em) 1/2-1/n,
(5)
where E is the summation of molecules considering the number of atoms (°/O)A in molecules and S A the sputtering yield of an individual atom. The mean number of specific reactions (e.g. bond rupture, cross-linking or disappearance of original molecules) that are produced by an energy of 100 eV is known in radiation chemistry as the G_~ value (Reimer, 1984). Following this principle we used the G Mvalue defined by the ratio of removal molecular weight A per bonding energy ER, as shown in Fig. 1. The molecular etching rate can be obtained by
R M= 6.23.(SM/pM).G_M; G_M =A_R/E•,
(6)
where A_R is the removal mass weight of molecules due to bond splitting. For the protein basic amino acids shown in Fig. 1, the G-value can be given as follows, A_H
A NH3
A_C H
A COOH
G_M- EB(_H) -~ EB(C_N ) + EB(C_H)) -~ EB(C_C ) t-
A_CH -'[-A NH3 -Jr-A~:oon
EB(C_R )
, (7)
where - H is the hydrogen bond with bond energy En = 0.2 eV, and C - H , C-N, C-C,
48
K. Kanaya et al. A
+
43m
a
H
O -H(g/eV)
!
2
-- 0 ,
~(
G -coo.=10
/
H.N
--X
G -R=17.
C
/
65
3,8
X
/
6
G-.H.=4,
/ ~
R
C O OH G -c.:2,
(C
0 0"
4
H)
31
21.?
0.2
a
b
¢
e 10
~
21
f
1
g
h
19.7
11
m
n
17.4
10
i
j
14.8
k
25
18,3
0
14.5
11
r
op~ $
b (a) glyeine (Oly), (b) alpine (Ala), (c) phcnylalantne (Ph¢), (d) t~ptophm~ (Trp), (e) tyrosine C£Yr), ~olcucinc (lle), (g) Icucine(Lcu), (h) mcthionlne (Met), (I)
(0
valine (Val), G) asparagine (Ash), (k)
t
OC oH eNOs O0
cystctne (Cys), (I) glutamine (Gin), (m) prollne (Pro), (n) scrinc (Set), (o) thrconine (Thr),(p) arginine (Arg), (q) histidinc (His), (r) lysinc (Ly$), (s) aspartic acid (Asp), (0 glutarnic acid (Glu)
Fig. 1. The molecular sputtering model, and the G-value of a protein basic amino acid and L-amino acid residues.
C O, N - O , and P = O are given by the b o n d breaking energies 5.5, 3.5, 4.2, 4.2, 2.6 and 6 (eV), respectively (Darnell et al., 1986). The hydrogen bond is included mainly in the residue R. (b) The etchin9 rate m e a s u r e m e n t The biological specimens used in the etching rate experiment were prepared by the n o r m a l m e t h o d of double fixation using glutaraldehyde and o s m i u m tetraoxide,
Quantitative Analysis of Sputtering due to Ion Beam Bombardment
49
followed by positive staining with uranyl acetate and lead citrate. The ion beam apparatus was attached to a J E O L S.E.M. JSE-35 for in situ observations of etched intracellular structures. The ion beam source of the K a u f m a n n type was used at 5 keV and a high vacuum of about 10- 5 Torr. The ion beam dosejt was limited by an aperture of 1 x 1 c m 2, and the dry density PM was measured. The etching rate R M was then obtained by the removal depth d. Figure 2 shows the depth measurement for a human tooth specimen b o m b a r d e d with an A + beam at 5 keV through the mesh grids placed on it at a small angle tilted with repect to the ion beam. The increased etching rate due to the incident angle v can be considered to be less than 10% for 15 °. Glutaraldehyde, M = 100, an aliphatic c o m p o u n d ( O H C C H E C H E C H O ) , can be considered to be the residue of the protein basic amino acids and its effect can thus be disregarded. Moreover, the metallic inclusions used as the fixation and stain, such as O s O 4, Pb(NO3) 2 or P b ( C H a C O O ) 2 , are considered to be small compounds which was confirmed by the measurement of dry density PM = 0.434).85. They, like aromatic compounds, have a stable diester of strong double bonds of E R= 6-10 (eV), which we assume will probably remain. The very large values of removal thickness d and the resulting etching rate R Mcan be analysed by the packing densitiy factor 7 (%), considering the inherent gaseous molecules and vacuoles, and the removal mass weight decreases as ~ • A_ u, as analysed in Table 2. The specimens in Table 2 show the ion beam sputtering analysis of biological specimens that are assumed to be the protein basic amino acids based on the molecular sputtering theory. It was assumed that in basic protein amino acids all the bonds are broken, also the NHa-molecule involving the C - N bond, in addition, the H a t o m in C O O H molecules as well as the H a t o m in the hydrogen bond where residues are displaced. The strong bond molecules which are fixed and stained with heavy metal atoms remain as the nuclei. (c) Atomic and molecular sputtering for human tooth enamel and dentine H u m a n tooth enamel and dentine, which are known to be composed of a hexagonal structure of hydroxyapatite [Ca 10(PO4)6(OH)2], as reported in a previous paper by K a n a y a et al. (1984), was examined for the sputtering yield analysis. The specimens of both enamel and dentine were prepared by cuts from a native rod material and polished by emery paper. The mesh grids were mounted on to the specimen, as shown in Fig. 2 and examined by S.E.M. For tooth enamel and dentine, as for solids, the displacement due to the atomic sputtering process for Ca and P ionic atoms, together with molecular sputtering process for the P O 4 and O H covalent molecules, were considered; also both the sputtering yield S A and S Mdepend on the inverse of the sublimation energy E s and the bonding energy E a, respectively. If the content of atoms (%)A and molecules (%)M are expressed, the sputtering rate R M is given as follows: RM= (6'23/pM) " {(%)ca "Sca "G-ca+ ( % ) e ' S v ' G - P + (%)vo4 "Svoa "G-vo4 + (°/O)on "Son" G~H}; G~ca =40/2.1 (g/eV), G_p= 31/1.37 (eV), G_po" = (31 + 64)/6 (g/eV), G on = (16 + 1)/5.5 (g/eV). (8) Table 3 shows the sputtering yield analysis for tooth enamel and dentine as determined by the etching rate measurement for A + ion beam b o m b a r d m e n t at 5 keV, where in the case of enamel the atomic collision process for Ca and molecular sputtering process for PO 4 and O H are considered. But the P O 4 molecules in dentine are assumed to be displaced due to the partially atomic sputtering for P, and mainly the molecular sputtering for P O 4. Since a high level of lattice defects has been observed by electron microscopy, the difference of etching rate between enamel and dentine can be considered to be the result of crystal structure imperfections.
50
K. K a n a y a et al. 15 d e g r e e
Specimen ...... S u r f a c e Grid Mesh Specimen Stage
Fig. 2. Measuring method of removal depth d = d * / c o s v taken with an S.E.M. for tooth enamel and dentine (a), where d* is the observed value when the incident angle v is as shown in (b).
Quantitative Analysis of Sputtering due to Ion Beam Bombardment
51
Table 2. The molecular sputtering analysis of biological specimens obtained by the etching rate for A + ion beam bombardment at 5 keV
Specimen Lung Kidney Liver Cerebellum Muscle Heart muscle Cerebrum
PM (g/cm 3)
RM r a m .cm2~ \ m A . min]
y. R M
SM fmol.'~ \ ion j
0.43* 0.85* 0.75* 0.66* 0.61" 0.58* 0.61"
8800* 2200* 2200* 1500" 1700" 1650" 1300"
5650 1408 1408 960 1088 1056 832
6.5 3.2 2.8 1.7 1.7 1.6 1.46
G _ M
f. removal weight "~ \break bond energy,] 60 60 60 59 61 61 65
(80 + ) (60 + ) (60 + ) (60 + ) (60 +) (60 +) (60 + )
G n = A-n/EB(-H)= 5 (g/eV), G~cn= A~cn/Es (C-H)= 2.4, G_NH3=A_NnJE B (C-N)=2.6, G~cooa= A~:oon/E ( C ~ ) = 10, G R= (A ca +A_Nm + A~coos)/E_s(C~C)= 17.65, E B (-H)=0.2 (eV), E s (C-H)= 5.5, E s (C-N)=3.5, E s (C~C)=4.2, y=0.63. * Experimental value. 60 + value by G M=5G n+G~:n+G_Nn3-G~:ooH+G R.
Table 3. The sputtering yield analysis of tooth enamel and dentine obtained by the etching rate for A + ion beam bombardment at 5 keV Atom
Z2
M2 (g)
PA (g/cm 3)
H O O P P Ca
1 8 8 15 15 20
1 16 16 31 31 40
0.07 1.4 1.4 0.85 0.85 1.54
Es Er~ SM (eV) (keV) (atom/ion) 5.5* 5.5* 0.6t 0.6t 1.37 2.1
10 I0 20 27.4 27.4 33.4
1.62 0.89 1.94 1.8 7.98 2.93
n
SA RA (atom/ion) (nm. cm2/mA • min)
2.3 2.3 2.6 2.7 2.7 2.8
1.5 1.37 1.7 1.4 6.2 2.2
89 155 169 52 227 162
G_M
Element
PM
(%)M
(g/eV)
SM
RM
Ca PO 4 (enamel) PO 4 (dentine) OH
1.5"I" 1.5t 1.5t 1.5t
0.23 0.49 0.36 0.1
4.19 15.8 20.0 2.8
2.2 1.64 2.6 1.43
40 53 79 2
Enamel RM=95 (86.6t) Dentine R u = 121 (123t)
* O-H bonding energy. t Experimental value. :~ P = O bonding energy.
ANGULAR
DISTRIBUTION
OF SPUTTERING
PARTICLES
(a) Solid targets T h e s p a t i a l d i s t r i b u t i o n of s p u t t e r e d particles is of g r e a t i n t e r e s t b o t h f r o m the p o i n t of view of s p u t t e r i n g t h e o r i e s a n d for the v a r i o u s a p p l i c a t i o n s ( K a m i n s k y , 1965, K a n a y a et al., 1982), b u t r e l a t i v e l y few d a t a are c u r r e n t l y a v a i l a b l e ( R o o s e n d a a l a n d S a n d e r s , 1980; B o h d a n s k y et al., 1982; K a n a y a et al., 1986; D o d o r o v et al., 1989). F o r h i g h r e s o l u t i o n s p e c i m e n p r e p a r a t i o n , the e x p e r i m e n t a l d a t a of s p u t t e r i n g yield at v a r i o u s a n g l e s of i n c i d e n c e are r e q u i r e d . T h e d u o p l a s m a t r o n i o n b e a m s o u r c e was u s e d for solid m a t e r i a l e x p e r i m e n t s . F o r b i o l o g i c a l s p e c i m e n s a K a u f m a n n - t y p e s o u r c e (1982) was e m p l o y e d . It was i m p r o v e d in o r d e r to o b t a i n a high b r i g h t n e s s b y f o c u s i n g lens a c t i o n . B a s e d o n the p o w e r p o t e n t i a l law ( K a n a y a et al., 1986a), the differential s p u t t e r i n g
52
K. Kanaya
et al.
yield per solid angle as a function of the nominal angle of incidence v and the observation angle ® measured from the deposition peak density can be obtained as follows: 27r
dS(v,®) d~ -
S(v, 0)"
(COS~)~3/2+ 1/.
\ cos v /
(9)
From the density distribution of deposition, following the mirror symmetry law, the angular distribution can be evaluated on the basis of the incident angle v, the critical angle 0~ showing the peak value, and the incident energy dependent parameter n obtained by the power potential law. Figure 3 shows the method of measuring sputter deposition profiles, which are represented by an anisotropical angular distribution. The deposition profile for Cu extracted from the various solid targets is shown in Fig. 4b. The equal-density lines evaluated by values relative to the maximum are plotted using a light densitometer.
a
b
,oo, ....
pro?ile
Asymme*ry
in the plane tMrough incoming ion beam
and Surface nomol Symmetry in tt~e transverse plane
Fig. 3. Measurement of the angular distribution of ion beam sputter deposition and its angular coordinates.
The reduced value of angular distribution, formulated as follows. In the incident plane as shown in Fig. 4a: _ _ dS - (cos dn
S(v)
dS/S dr2 as a function of 0 can be
(
1 "] 1/2+1/"
0)3/2+1/";S(v)=S(O)'\c~v/
(10)
where ® is the observable angle subtended by the maximum. ® = ( ~ / 2 ) - (0+0c)/{(~/2)+0c}, for O
S(v). (cos 0 ) 3/2 + 1/,.
(11)
When these deposition profiles are theoretically analysed, the angular distribution can be plotted as shown in Fig. 5. It was found that for n = oc, 2, 1, dS/S df~ becomes the following power cosines in a simple form such as (cos ®),/2, corresponding to less than l keV, (cos O) 2,
a Incident plane dS S(Ot)dtq - S(O)
Equal
T
density contour
-----
Transverse
plane
d ~ _ , , S(8t )
e,m
I \ \ ) o.~ \~ 0.5-< \0.2 ~:
I
7.5c
[
/t
-
~ + O e - - - - e c
--~
~+ U-.-45"
• 0©=12.5
Q
A+''~ Cu IJ=65
, 0~=16
U-75
~ , 8e=l~S.5"
6......... 5- .......... ~ ................ z'9
(rad) Fig. 4. Spatial distribution analysis (a) of sputter deposition profiles for the A ÷ Cu combination at 5 keV (b).
54
K. Kanaya et al.
1
o~
o
d$
•
.
' '
I ....
o~ ' '
~1$
F i g . 5. A n g u l a r
distribution
i o15 '
'
'
'
dS
o
• o~ . . . .
i
as
of reduced sputtering yield with bombardment t h e i n c i d e n t a n g l e v.
of A ÷ ion beam at
corresponding to 5-10 keV and (cos ®)5/2, corresponding to over 10 keV, respectively. Figure 6 shows the relationship between the incident angle v and the critical angle 0 c obtained from the equal-density peak. The measured value of 0 c depends on the incident angle and backscattered energy, and are empirically given by sin 0c = sin v/21/4 + 1/2n.
(12)
The important critical angles differ from the previously obtained values (Biersack and Eckstein, 1984; Bohdansky et al., 1982) and are similar to the results obtained by Roosendaal and Sanders (1980) and D o d o r o v et al. (1989). Based on the power cosine law the oblique incident angle dependent on the relative sputtering yield is obtained S(v)/S(O) = [1 - {sin(v - v~)/cos v} z] (COS
,~)1/2+1/n
, V>I)c
- 1/(cos v) 1/2 + 1/,, v < v~.
(13)
The critical angle v¢ of energy attenuation in a transfer energy T = A E cos 2 v is empirically obtained vc = c o s - 1( E J A E ) 1/3,.
(14)
Figure 7 shows the oblique incident angle dependent on the sputtering yield for A + ions at 5 keV. The peak values of Vp can be obtained under the condition of d S { ( v ) / S ( O ) } / d n = O in Eqn (13) as follows, sin(2%-2v~)/cos(2vc - %)-= (2 + n)/2n.
(15)
Figure 8 shows the determinations of Vp as a function of the critical angle vc and the energy dependent parameter n.
Quantitative Analysis of Sputtering due to Ion Beam Bombardment
55
V [r~] 0.5 I ec
25
~
n
•
]:~
12,79" 1P-40" 12.28"
16.49" IE,.97" 15.82"
11.66" tl.58"
15.0;°1 14.90"
Eo=1OkV ; W= W' ~Mo" for E°=SkV_
~
B
~
--
16.49"
3,12" 3.02" ;~.99" 2.90" 2.84" 2,82"
C
I
65" )690.
3.12 o 12.79"
3.2 2.8 2.7 2.4 2.25 2.2
S~
20
4~ • 13,11 •
1.5
I
~.,4- ,z..- ~ o
U-MO*O~LOy 3.2
Re Mo Cu
8c
• 3~9"
1.0
'
~
0.4
~
11,89" 15.31"
m
1.
0,~
2
8c
[ deg ]
0.2 [ red ] 10
0.1
5
0
I
I
I
I
l
15
30
45 P (~g]
60
75
90
Fig. 6. The critical angle 0 c showing the peaks of sputtering yield obtained as a function of the incident angle v.
5
sin(u-~:)
c
0.5
1.0
_~
P(rod)
z
Fig. 7, Oblique incident angle dependence of sputtering yield for A + ion beam at 5 keV.
Up (rod) 90
r
,
~
i
sin (2 v,,-2u,) 70
COS(21A-up)
0,5 [
r
~
r
i
2+n " ~
1.0 1
i
,
T
~
r
2
~(/
n-=oo -~ .
40
ff
50
n=3~)
I
I lO
I
J ' 20
~ ] 30
/ h 40
,
2 ~ / 50
60
7JE3 0
J
20 80
0 5
I
90
Up(~g)
Fig, 8. Analytical determination of the peak angle Vp as a function of the critical angle vc and energy parameter n.
56
K. Kanaya et al.
(b) Biological specimens The angular distribution measurement for biological specimens which was obtained by the Kaufmann-type ion source is shown in Fig. 9. The curves shown in Fig. 9a illustrate the result of the relative density in the incident plane which was measured by thin film chromatography. Figure 9b shows the relative distribution, dS/S(®) dfL by a polar coordinate plot.
a A ° 5keV
Geta?in
e¢=45"
Liver
0¢:4~"
Kidney
~
ds sdQ
Exp.pt - -
|
K
,
-90*
i
,
I LO
, G
O"
1.0
0.5
0
+ 90" (0)
0.5
1.0 sd~)
Fig. 9. Angular distribution of the reduction sputtering yield for biological specimens and organic gelatin (a) measured by thin filmchromatography and their polar coordinate plots (b).
For biological specimens, the basic relationships of sputtering yield characteristics were analysed similarly.
dS/S(v) df~ = (cos ®)3/2 + 1/.; S(v)/S(O)= (cos v)- 1/2 - I/n, y < Vc S(v)/S(O)~__(cos v ) - 1/2
1/n , 0, V~>Vc; I']=
1 - { s i n ( v - v ~ ) / c o s v} 2.
(16)
The large critical angle 0~ = v is assumed to be the molecular sputtering process by molecules of small atomic number. Figure 10 shows an example to find the optimum etching condition for microvilli of the small intestine which was prepared by normal fixation and staining processes. Figure 10a is the relative sputtering yield S(v)/S(O) as a function of the incidence v; Fig. 10c shows the etched surfaces for a large angle of incidence, where the cone-like artifact can be analysed as shown in Fig. 10b. The etched depth d used for the analysis (Yonehara et al., 1989) is given by
d(v)=dorl(cos v ) - l ; do=6.23 • (SM/pM)" G M "jt.
(17)
The m a x i m u m angle vp = 70 ° and d(n/4)/d o = 1.5 are in agreement with the theory. The result shows that biological specimens are etched according to Eqn. (17). In the case of a normal angle of incidence such artifacts can be removed as shown in Fig. 10d.
Quantitative Analysis of Sputtering due to Ion Beam Bombardment
57
a
0
0.5
1.0
/T
v (rad)
b I
/" /
Ion beam
./ ./ "
Fig. 10. The incident angle dependence of the etched surfaces for microvilli and etching mechanism analysis, where (a) is the empirical yield-angle relationship, (b) the etched profile analysis for (c), (c) the experimental result with an angle of incidence, and (d) by normal angle of incidence.
Figure 11 shows a successful result and an example of the rotary etching method for freeze-fractured surfaces of rat liver, which was prepared by the normal fixation and staining process. Figure 11 a shows the rotary etching method, in which the specimen stage is rotated at a variable a n g l e _ Av with respect to the incident angle v, using gimbals capable of universal universal rotation. The original S.E.M. image shown in Figs. 11 b, 11c illustrates the etched patterns bombarded with A ÷ ion beam at 2 keV for 30 min. The embossed nuclei (n), rough endoplasmic reticulum (rer), and mitochondria (m) are clearly revealed. It was considered that the fixed and stained nuclei remained, and their surrounding molecules were removed. The mean optimum angle of the incidence v+Av=(n/4)+_(n/8) at a rotation speed 3 0 r p m was obtained (Muranaka et al., 1986).
58
K. Kanaya et al.
[ Fig. 11. The rotary etching example for the freeze-fractured surface of rat liver. (a) The rotary etching method. (b) The original S.E.M. image taken at 30 kV. (c) Its etched nuclei structure recorded from A ÷ ion beam bombardment at 2 keV during 30 min under the incident rotation angle n/4+ n/8. (d) Subsequent nuclei etched during the 60 min under the same operating conditions.
ANGULAR
DISTRIBUTION OF SECONDARY ELECTRON Y I E L D D U E T O I O N BEAM B O M B A R D M E N T
EMISSION
According to the kinetic theory of secondaries ( K a n a y a et al., 1988a), for less than 100 keV, the emission yield of electronics is given by S =
S m " (/3//3m)1/2;
/~m =
8.4 • 102 = a E / Z l e 2 M 1
", s m = 2 1 . 7 • P 2 Z I Z 2 / I M 2 ( Z
2/3 + Z 2 / 3 ) 1/2
08) where I is the ionization energy (eV). The ratio of electrons yielded to atoms can be calculated as 0.1~3.5 at 1-10 keV. This is similar to the elementary theory of secondary emission for electrons developed by K a n a y a and O n o (1976), where the n u m b e r of secondary electrons due to electronic collision of ion beam b o m b a r d m e n t is p r o p o r t i o n a l to the electronic energy loss ( d E / d x ) e . They arrive at the surface by travelling a distance x cos v/cos 0 t h r o u g h the material, and the secondary yield s is given by
Quantitative Analysis of Sputtering due to Ion Beam Bombardment s(v, O)= k
f
R/2 ~ n / 2
do
o s = 3.7.
l 30
O s COS Y" X
(dE/dx)e " e
cos 0
59
• sin 0 dO dx
p2Z22/3R/M2I,
(19)
where R is the penetration range of the ions (nm), trs is the transmission factor of secondary electrons and k is a constant. Then the anisotropic angular distribution of ds(v, ®)/df~ can be derived from the integration of Eqn. (19) similarly to the angular distribution of sputtering yield as follows, ds(v, O ) / s d ~ = (1/2~) • (cos O / a s cos v)" I-1 - { 1 - e x p ( - a
s cos v/cos O)}
(cos O / a s cos v)],
(20)
where O = (re/2)(0+ 0c)/{(rc/2)_ 0c) and 0¢ = v are considered. Figure 12 shows the angular distribution of secondary electron emission yield in the incident plane, where it is calculated in the normalized form as ds(v)/s dfL The secondary electrons yielded were confirmed by electron exposure experiments. A T S K photoresist (Toyobeam Co.) with a sensitivity of 1.73 (lac/cm 2) was used for detecting the existence of electrons. The oblique angle of incidence of electron emission yield can be integrated from Eqn. (20) as follows, s(v)/s(O) = f ( y ) , for v < v~; s(v)/s(O) = F ( y ) " rl, v > v¢, f ( y ) = (I/y) { 1 - (2/3y) - (1/3)" e x p ( - y)" { 1 - 2 / y ) - y}, y = a s cos v, ~/= 1--{sin(v--v~)/cos v} 2.
a~
S(e) :
Secocdfy electron M~llion yle(d
mctdlmt
•
1
0,8
(21)
O, - 100
O~
1
!
0.6
0
0,8
Fig. 12. Angular distribution of the secondary electron emission yield for A + ion beam at 5 keV.
I
60
K. Kanaya et al.
Figure 13 shows the relative value of the secondary electron emission yield depending on the oblique angle of incidence, which is calculated from Eqn (21 ), where 0 c = v and vc is shown by empirical values. It was shown in a previous paper (Kanaya et al., 1988b), the angular distribution of secondary electron emission has a high value at an early stage of deposition. The secondary electrons were contributed in order to obtain the monolayer films by reducing the surface potential level. In the case of biological specimens, the secondary electron emission yield is considerably smaller than 0.1, and can be disregarded during the applications of ion beam sputtering.
30
O(deg)
60
Re
90 Au
S(u)
s-TN'
0
0.5
1.O
lJ(rad)
2
Fig. 13. Dependence of the secondary electrons on an oblique angle of incidence for an A + ion beam at 5 keV.
U N C O A T E D O B S E R V A T I O N AND E T C H I N G A P P L I C A T I O N S In microscopic studies of the compound eye of the house fly using serial sections, interesting structural details of visual cells forming the retinula have been observed by Fernfindez-Mor~in (1956). As an example of uncoated and subsequent in situ observations of etched surfaces using the ion beam apparatus (Muranaka et al., 1990), different internal structures of the corneal lens in the ommatidium were clearly observed. Figure 14a shows a T.E.M. image of the ommatidium of the house fly (Musca domestica) by microtomy sections, where a-e correspond to the etched surfaces in Fig. 14a-e. Figure 14a shows the uncoated S.E.M. images for the surface of an ommatidium with a hexagonal-shape, bombarded with an A + ion beam at 1 keV for 5 min. Figure 14b shows the etched surface at a depth of about 0.5 gm acting as a dioptic apparatus which is obtained by A + at 2 keV for 20 min. Figure 14c shows the hexagon at a depth of 2 i.tm by 2 keV for 80 min. Figure 14d shows the deep etched hexagon at 20 gm by 2 keV for 140 min. The wide vacuole around the ommatidium and transverse laminated structure was observed. Figure 14e shows the pigment cell (p) in the central cavity and the retinula cell (r) constituting the rhabdomere acting as a light receptor, observed at 2 keV for 200 min. The measured etching rate of the assumed protein specimen was found to depend mostly on the packing density factor ? which is determined by the vacuole and cavity. It was confirmed that uncoated and etching observations are very effective in the internal structural analysis of biological applications.
Fig. 14. Uncoated and etching observations for the ommatidium of the house fly, where (A) is the T.E.M. image of the sectioned compound eye, (a~), (a2), (a3) in (a), (b~(e) S.E.M. images of structural details of the corneal lens in the ommatidium at different depths.
t~
O
.=
OQ
7
J
Quantitative Analysis of Sputtering due to Ion Beam Bombardment
63
CONCLUSIONS (i) Reliable d a t a of s p u t t e r i n g yield for biological specimens a n d solid targets were o b t a i n e d by the m e a s u r e m e n t of the r e m o v a l etching a n d t h i n n i n g rates based o n a t o m i c a n d m o l e c u l a r s p u t t e r i n g theory. (2) T h e spatial d i s t r i b u t i o n of s p u t t e r i n g yield a n d its d e p e n d e n c e o n incident angle were q u a n t i t a t i v e l y analysed by the m e a s u r e m e n t of sputtered d e p o s i t i o n profiles o n the collected stage. (3) T h e a n g u l a r d i s t r i b u t i o n of secondary electron emission yield was theoretically calculated a n d confined by the electron b e a m exposure m e t h o d . (4) It was confirmed that in situ o b s e r v a t i o n s for u n c o a t e d a n d etched specimens of n o n - c o n d u c t i v e materials c a n be very effective for biological a p p l i c a t i o n s in the S.E.M. Acknowledgements--The authors wish to thank Professor R. W. Home, University of East Anglia, for helpful comments and advice in connection with the preparation of this manuscript.
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