High resolution scanning electron microprobe analysis of biological material
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Mrcron. 1980, VoL I I, pp. 403-404 Pergamon Press lad Printed In Great Britain HIGH RESOLUTION SCANNING ELECTRON MICROPROBE ANALYSIS OF BIOLOGICAL MATERIAL* M. STEWART CSIRO Division of Computing Research, PO Box 1800, Canberra City, A.C.T., Australia 2601 A. P. SOMLYO. A. V. SOMLYOand H. SHUMAN Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia PA 19104, U.S.A. W. G. MURRELL and J. A. LINDSAY CSIRO Division of Food Research, PO Box 52, North Ryde, Australia 2113 High resolution X-ray maps of the elemental distribution in biological specimens were obtained with • Philips EM400 microscope fitted with scanning trantrni~on (STEM) attachment, field emi~,6on gun and Kevex Si(Li) detector with a model 7000 multichannel analyser. Using the STEM attachment, the specimen was scanned in a raster pattern with • flue electron beam and electron ~ (bright field or dark field) and up to 3 different X-ray were collected simultaneously. This data was stored in digital form on disc on a PDP 11/34 computer system and subsequently analysed using computer imase p ~ n g in conjunction with • DeAnza VCS000 video terminal. Images containing up to 1024 × 1024 picture elements could he collected and processed in this way. We used tropomyosin paracrystals to test the resolution of the system. These had bands of Hg dslX~ted at 40 rim intervals' and were • very convenient object since they gave • simple one dimensional pattern which could be emily analyled by Fourier methods. Figure I shows a typical result, where the Hg is clear in both the electron and X-ray imalges. The beaded appearance of the Hg bands is probably the result of radiation damage. Fourier analyzis of the X-ray map showed two clear orders and sometimes weak 3rd and 4th orders, indicating • resolution of at least 20 nm and possibly as high as 10 nm. The power of this technique is illustrated by results obtained on the distribution of a number of key elements in Bac///ut cereus T spores. Whe~ whole spores were examined in spot mode, the overall elemental concentrations were in excellent agreement with those determined by atomic absorption analysis of btdk samples. Whole spores were too thick to enable the elemental distribution within the spore to be determined accurately and, for this stage of the analysis, crymections cut from spores frozen in 50% aqueous polyvinyl pyrrofidiue were treed. These sections were freeze dried and coated with • thin layer of carbon prior to examination at 80kV using conditions described elsewhere. Figure 2 shows a typical STEM image and corresponding X-ray maps. Examination of • number of crymections confirmed that divalent cations (Ca, Mg, Mn) were located primarily in the central core region, Si at the periphery aad P and S distributed over the entire particle. The continuum maps were festureleu indicatin~ that the other elemental maps did not contain artefact, asmciated with mass differences. We also examined mutant spores or those grown in low Ca, both of which were ~ t in dipico!i-ic acid. In both the Ca content of the core was about I/4 that in the wild type and, as illustrated in Fig. 3, granules containing Ca, Mn and P could be seen in the core region. In both specimens, the results were confirmed and quantified by spot mode analyses. The elemental distributions determined by this study tead to refute the contracted cortex and exl~mded core theories of spore resistance and support those theories which require high concentrations of divalent cations in the cortex region of the panicle. REFERENCES 1. M.Stewgrt & V.Diakiw, Electron micro~opic location of protein thiol residuei Nature 274, 184 (1978). 2. A.P.Somlyo, A.V.Somlyo, H.Shuman & M.Stewart, Electron probe analysis of mmcle and X-ray mapping of biological specimens with • field emission gun, SoannMgEh~tron Microscopy 1979, 2, 711. *Supported in pan by USPHS HLI5835 to the Pennsylvania Muscle Institute and GM00092. 403
Transcript of High resolution scanning electron microprobe analysis of biological material
Mrcron. 1980, V o L I I , pp. 403-404 Pergamon Press l a d Printed In Great Britain
HIGH RESOLUTION SCANNING ELECTRON MICROPROBE ANALYSIS OF BIOLOGICAL MATERIAL*
M. STEWART
CSIRO Division of Computing Research, PO Box 1800, Canberra City, A.C.T., Australia 2601
A. P. SOMLYO. A. V. SOMLYO and H. SHUMAN
Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia PA 19104, U.S.A.
W. G. MURRELL and J. A. LINDSAY
CSIRO Division of Food Research, PO Box 52, North Ryde, Australia 2113
High resolution X-ray maps of the elemental distribution in biological specimens were obtained with • Philips EM400 microscope fitted with scanning trantrni~on (STEM) attachment, field emi~,6on gun and Kevex Si(Li) detector with a model 7000 multichannel analyser. Using the STEM attachment, the specimen was scanned in a raster pattern with • flue electron beam and electron ~ (bright field or dark field) and up to 3 different X-ray
were collected simultaneously. This data was stored in digital form on disc on a PDP 11/34 computer system and subsequently analysed using computer imase p ~ n g in conjunction with • DeAnza VCS000 video terminal. Images containing up to 1024 × 1024 picture elements could he collected and processed in this way.
We used tropomyosin paracrystals to test the resolution of the system. These had bands of Hg dslX~ted at 40 rim intervals' and were • very convenient object since they gave • simple one dimensional pattern which could be emily analyled by Fourier methods. Figure I shows a typical result, where the Hg is clear in both the electron and X-ray imalges. The beaded appearance of the Hg bands is probably the result of radiation damage. Fourier analyzis of the X-ray map showed two clear orders and sometimes weak 3rd and 4th orders, indicating • resolution of at least 20 nm and possibly as high as 10 nm.
The power of this technique is illustrated by results obtained on the distribution of a number of key elements in Bac///ut cereus T spores. Whe~ whole spores were examined in spot mode, the overall elemental concentrations were in excellent agreement with those determined by atomic absorption analysis of btdk samples. Whole spores were too thick to enable the elemental distribution within the spore to be determined accurately and, for this stage of the analysis, crymections cut from spores frozen in 50% aqueous polyvinyl pyrrofidiue were treed. These sections were freeze dried and coated with • thin layer of carbon prior to examination at 80kV using conditions described elsewhere. Figure 2 shows a typical STEM image and corresponding X-ray maps. Examination of • number of crymections confirmed that divalent cations (Ca, Mg, Mn) were located primarily in the central core region, Si at the periphery aad P and S distributed over the entire particle. The continuum maps were festureleu indicatin~ that the other elemental maps did not contain artefact, asmciated with mass differences. We also examined mutant spores or those grown in low Ca, both of which were ~ t in dipico!i-ic acid. In both the Ca content of the core was about I/4 that in the wild type and, as illustrated in Fig. 3, granules containing Ca, Mn and P could be seen in the core region. In both specimens, the results were confirmed and quantified by spot mode analyses.
The elemental distributions determined by this study tead to refute the contracted cortex and exl~mded core theories of spore resistance and support those theories which require high concentrations of divalent cations in the cortex region of the panicle.
REFERENCES
1. M.Stewgrt & V.Diakiw, Electron micro~opic location of protein thiol residuei Nature 274, 184 (1978).
2. A.P.Somlyo, A.V.Somlyo, H.Shuman & M.Stewart, Electron probe analysis of mmcle and X-ray mapping of biological specimens with • field emission gun, SoannMg Eh~tron Microscopy 1979, 2, 711.
*Supported in pan by USPHS HLI5835 to the Pennsylvania Muscle Institute and GM00092.
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404 M. Stewart, A. P. Somlyo, A. V. Somlyo, H. Shuman, W. G. Murrell and J. A. Lindsay
Figure I. Tropomyosin paracrystals with transverse Hg bands every 40 nm. (a) STEM image using dark field - bright field mode (b) Hg X-ray map. In each case the Hg bands are clear and Fourier analysis indicates a resolution of at least 20 nm. Bar is I00 nm.
q l W W
r .
i
m
a c d e
Figure 2. Elemental distribution in f roze dried cryosections of B. cereus T spores (a) STEM image showing the core and surrounding cortex/coat layers (b) Si (c) Ca (d) P (e) continuum. Bar is I00 nm.
¢ . , --
r !
a b c d
Figure 3. Elemental di~ribution in HWI mutant B. cereus T spores Im~king dipicolinic acid: (a) e l~tron im~qle (b) P (c) Ca (d) Mn. Note the granules present in the core. Bar is I00 nm.
