Submicron elemental mapping with the oxford scanning proton microprobe

Nuclear Instruments and Methods North-Holland, Amsterdam

SUBMICRON ELEMENTAL MICROPROBE G.W.

GRIME,

F. WATT

University of Oxford,

in Physics

Research

MAPPING

and

J.R.

B22 (1987)

109

109-114

WITH THE OXFORD

SCANNING

PROTON

CHAPMAN*

Department of Nuclear Physics, Oxford.

UK

Following recent modifications to the Oxford scanning proton microprobe (SPM) a beam spot diameter of 0.5 Frn has been achieved at a beam current of 20-30pA of 4MeV protons. This has been confirmed by scanning both a copper test grid and microcrystals of barium sulphate. The potential of using high spatial resolutions in microbiology has been explored by scanning a single mouse cell.

1. Introduction As originally described in our previous publications [l-4], the Oxford 1 pm SPM was installed on a beam line of the Nuclear Physics Laboratory’s EN tandem accelerator. Following a major reorganisation within the laboratory, however, the EN tandem has now been removed, and after a period of inactivity, the SPM has now been re-sited on the remaining accelerator in the laboratory, the folded tandem (FT). The FT is a large vertical belt-charged Van der Graaff accelerator with a maximum terminal voltage of 10 MV. It has two modes of operation; folded tandem operation in which beams from an external negative ion source at the base of the machine are accelerated up to the terminal, where, after stripping, a 180” magnet returns them to a second accelerating tube, and single ended operation, using a radiofrequency ion source in the terminal to provide beams of gas ions. For work on the SPM we are using the rf source in single ended mode with a terminal voltage of 4 MV to provide protons at 4 MeV. The SPM was reassembled largely in its original form but with a few minor changes (see below) and the resolution has been found to be significantly improved. Preliminary experiments are now under way to assess the potential of the system in various applications in biology and medicine. We present here an example of elemental mapping within a single cell.

2. Modifications

to the SPM

In this section we discuss the modifications made to the SPM during the re-siting process

we have and how

* Nuffield Department Oxford, UK.

Hospital,

0168-583X/87/$03.50

(North-Holland

of Surgery,

0

Physics

John

Radcliffe

Elsevier Science Publishers Publishing Division)

B.V

these would be expected to affect the performance. (a) The current stabilised power supplies for SPM lenses have been replaced with supplies with a much improved stability. The new supplies, which were built in the laboratory, have a measured ripple and long term stability of better than 1 part in lo5 over a period of several days. This contrasts with the original supplies which in certain circumstances could drift out of alignment over a period of hours and which developed a significant ripple component. As discussed in ref. [3], the Oxford SPM is very sensitive to changes in the excitation of the quadrupoles (( Y[&~) = 1300 pmimr %), and so improving these supplies will have a significant effect on the resolution. The output current from supplies is related to a reference voltage which is derived from an oven-stabilised DAC with a resolution of 16k steps. The DAC settings can be controlled remotely at a speed determined by the operator to allow precise current setting. (b) The object distance (from the microslit to the entrance of the first quadrupole) has been increased from 5.78 m in the original system to 6.8 m (all other dimensions remaining constant). This has the effect of increasing the demagnifications in the x and y planes from 70 and 16 to 85 and 19. The chromatic aberration values also increase so that the optimum acceptance remains virtually unchanged. However, the increased magnification allows wider object slits to be used so that problems due to slit scatter are reduced. The object slit sizes required to achieve a 1 pm overall spot size are calculated to be 40 x 10 pm” in the system compared with 3.5 x 6 in the old system. The object slit jaws were reshaped to the Heidelberg geometry [S] and lapped to 0.1 pm diamond finish to further reduce the effect of slit imperfections. (c) The difference in the accelerator beam quality (brightness and energy spread) will affect the resolution of the SPM. No figures are available for the I. PIXE AND MICROPROBE

METHODOLOGY

relative axial brightness of the rf source as compared to the off-axis duoplasmatron used on the EN tandem. although in our case the analysed current is greater (4-5 I_LAanalysed compared with l-l.5 PA). The FT analysing magnet is of similar dimensions to the EN magnet, so the energy spread of the beam should not be significantly different. (d) The quadrupole imbalance deflection system described in ref. [4] has been replaced by a single deflection coil placed before the lens. This was done to avoid degrading the stabilisation of the excitation current supply to the final lens. Although the theoretical performance of this system is inferior to deflection in the final lens, the spot size at the full deflection of t2 mm is still less than the pixel size of the reconstructed elemental maps, while the use of a separate aircored coil leads to a simplification of the data acquisition system.

Fig.

3. Submicron

performance

The resolution of the improved SPM is significantly better than the original assembly. This is demonstrated in fig. I. which shows a copper X-ray map of a 2000 mesh electro-formed copper grid with 5 pm bars and 7 pm square holes. The resolution of the SPM image is sufficient to allow small imperfections on the edge of the grid bars to be distinguished. which implies a probe diameter of less than 1 pm (a SEM image of a representative portion of the grid is shown in fig. 2). The beam current at this resolution was 20-30 pA. At these small diameters, the edges of electroformed or photo-etched grids are not sharp enough to allow them to be used as resolution standards, since the size of the imperfections is comparable with the size of the beam (see fig. 2). To avoid this problem,

copper K,, X-ray map of a 2000 line per inch grid (5 pm bars and 7 km holes) obtained using the improved Oxford SPM. The resolution allows small imperfections on the edges of the bars to be distinguished.

111

G. W. Grime et al. I Submicron elemental mopping

Fig. 3. Electron micrograph of barium sulphate microcryst“bow-tie” shape and the well als, showing the characteristic defined edges. The bar represents 1 Frn. Fig. 2. Electron

micrograph

of the 2000 l/inch grid.

Fig. 4. A sulphur

K, X-ray

resolution

test

map of the crystals

shown

in fig. 3. Scan size is 15 I. PIXE

AND

X

15 pm’.

MICROPROBE

METHODOLOGY

G.W.

112

Grime et al.

/ Submicron elemental mapping

we have used microcrystals of barium sulphate, which can be grown with a well defined “bow-tie” shape and sizes ranging from tens of microns downward. As can be seen from the electron micrograph in fig. 3, the edges of the crystals are well defined. Scanning these crystals with the high resolution beam resulted in the sulphur map shown in fig. 4. This map is a 128 x 128 pixel reconstruction of the X-ray signals from a 1.5 X 1.5 pm2 area and comparison with the SEM clearly indicates submicron resolution. The beam spot size was estimated by extracting the rate of rise of the X-ray signal along a line perpendicular to the sharpest edge of the crystal at the bottom of fig. 4 (indicated by the arrow). Analysis of the graph of X-ray counts as a function of distance shown in fig. 5 yields an upper limit for the spot size of 0.45 pm fwhm assuming that the edge has a sharp cutoff and that the beam profile is a radial Gaussian. It should be noted that the edge is at 45” to the principal directions of the lens system, implying that both the x and y dimensions of the beam spot are within the estimated 0.45 km.

4. Application

to microbiology

The main thrust of the Oxford SPM programme has been toward elemental mapping at the cellular level. To investigate the feasibility of this we have scanned a single mouse cell of 5 pm diameter obtained from a preparation of dendritic cells and macrophages. This was prepared by freeze-drying onto a formvar coated electron microscope grid. The cell scanned is shown in

Fig. 6. Electron

micrograph

,,i 20

I

l

l

,-.+ / / + I

3,

+ ‘\

/

+\ :

41 I I / f

+’ ./ /+

01 0

05

10

15

2.0

25

w

Fig. 5. X-ray counts as a function of distance perpendicular to the edge of the crystal at the point indicated in fig, 4.

the SEM of fig. 6 and the elemental maps are shown in fig. 7. These cover an area of 25 X 25 pm’. The most obvious feature of the elemental maps is the concentration of Al, Si, Ca and Mg at the edge of the cell. This is similar to the small feature in the lower left hand region of the scan, which may be a fragment of a disintegrated cell or an artefact of the preparation. However, the feature in the cell does not appear on the micrograph and one explanation is that this represents a small fragment which has been engulfed by the cell. Careful examination of the maps reveals that the Ca in the concentration is not completely co-localised with the Al and Si and the possibility that it is a component of the cell cannot be ruled out at this stage.

of a mouse

cell. The bar indicates

S pm

G.W. Grime et al. ! Submicron elemental mrrpping

Fig. 7. SPM elemental maps of the ceti shown in fig. 6. Scan size is 25 x 2~ pm?

Apart from the inclusion, the SPM maps indicate that the main inorganic constituents of the cell are, as expected, P, S, Cl and K, which at this magni~cation appear to be distributed fairly uniformly throughout the cytoplasm. While these preliminary results are inconclusive without further work, they indicate the potential of the SPM mapping technique in studying both the endogenous elements of cells and also in identifying regions of possible contamination. Further work with different cell types (mainly germinating yeasts [6]) is now in progress and it is hoped that this

will shed new light on the biochemical ing place within cells.

processes

tak-

5. Discussion In this report we have demonstrated that relatively minor changes to a quadrupole SPM system can reduce the probe diameter below 1 pm. However, the question raised by this work is “what is the limit to the improvement on the spot size in a specially designed I. PIXE AND

MICROPROBE

METHODOLOGY

114

ti.W. Grime et al. / Suhmicrotl

system?“. In all microbeam systems the beam spot can be considered to consist of two components; the geometrical demagnified image of the object slits and a “halo” generated by aberrations dependent on the entrance divergence (these include the intrinsic spherical and chromatic aberrations as well as parasitic aberrations due to misalignment, which also depend on angle). Thus in principle it is possible to reduce the spot size indefinitely by closing the entrance and collimator slits. However. this also reduces the beam current and the lower limit to this process is set by the smallest current which can be tolerated by the analytical reaction in use. This has been well demonstrated by the Melbourne group [7], who have achieved resolutions of the order of 0.2 pm at the very low currents required for transmitted ion energy loss imaging (hundreds of particles per second). For emission spectroscopy (i.e. PIXE) the currents required for useful microbeam analysis are of the order of 50-100 pA using currently available detectors. which with the present generation of focusing systems limits the spot size to around 1 pm. In the light of this discussion we can identify four factors which limit the performance of SPM systems. a) Lens performance. Lenses with a lower ratio of aberration to demagnification will give an increased acceptance for a given spot size. b) Ion source and accelerator beam quality. The brightness of the beam from the accelerator determines the current which will pass into the acceptance of the SPM lens, while the energy spread of the beam affects the magnitude of the chromatic aberration effect. c) Detector efficiency. The beam current required for a given application depends on the efficiency with which the reaction products can be detected. If the detector efficiency or acceptance can be increased, then a smaller current can be tolerated. d) The .stahility and precision of construction and a&pnent. Misalignment and imperfections in the lenses, ripple and drift in the lens power supplies and

elementd

mapphg

vibration of the beam relative to the target will all degrade the resolution. Relatively small improvements in each of these areas can combine to give a significant improvement of the whole system. The achievement of a 0.1 pm probe diameter with sufficient current for elemental mapping will be a project combining theoretical lens studies, ion source and accelerator improvements and detector developments with a high standard of precision engineering.

Acknowledgements The authors wish to acknowledge the contribution of Mr. D. Aitken whose work on the stabilised current supplies for the quadrupoles was instrumental in improving the performance of the SPM. We are also grateful to Dr. CC. Perry and Miss J. Wilcock of the Inorganic Chemistry Laboratory, Oxford, who provided the barium sulphate crystals and the SEM in fig. 3.

References F. Watt. G.W. Grime. G.D. Blower and J. Takacs, Trans. Nucl. Sci. NS-2X (IYXl) 1413.

IEEE

F. Watt, G.W. Grime. G.D. Blower, J. Takacs and D.J.T. Vaux. Nucl. Instr. and Meth. lY7 (1982) 65. F. Watt. G.W. Grime. G.D. Blower. J. Takacs and D.N. Jamieson. Nucl. Instr. and Meth. 197 (1982) 97. G.W. Grime. J. Takacs and F. Watt. Nucl. Instr. and Mcth. B3 (lY84) 589. R. Nobiling. K. Traxcl, F. Bosch. Y. Civelekoglu. B. Martin. B. Povh and D. Schwalm, Nucl. Instr. and Meth. 142 (197.5) 49. G.M. Gadd. C. White, F. Sabic. G.W. Grime and F. Watt. submitted to Trans. Brit. Mycol. Sot. (1986). G.J.F. Lcgge et al.. Nucl. Instr. and Meth. Bli (1986) 66Y.