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Observation of an undecagold cluster compound in the scanning transmission electron microscope
Ultramicroscopy 8 (1982) 397-402 North-Holland Pubfishing Company
397
O B S E R V A T I O N O F AN U N D E C A G O L D C L U S T E R C O M P O U N D IN T H E S C A N N I N G TRANSMISSION ELECTRON MICROSCOPE .J.S. W A L L and J.F. H A I N F E L D Brookhaven National Laboratory, Biology-463, Upton, New York 11973, USA
P.A. B A R T L E T T Department of Chemistry, University of California, Berkeley, California 94720, USA
and S.J. S I N G E R American Cancer Society Research Professor, Department of Biology, University of California at Sun Diego, La Jolla, California 92093, USA
Received 8 March 1982
A water-soluble undecagold cluster compound has been shown to be visible in moderate dose images (10 4 electrons/nm 2) obtained with the scanning transmission electron microscope (STEM). The properties of this compound render it potentially useful as a marker for specific sites within biological molecules.
1. Introduction
Localization of specific sites in electron microscopic images of biological specimens is limited by the size of available labels. Ferritin-labeled antibodies provide a resolution of 15-30 nm [1], while biotin-avidin labeling may provide a resolution of 4 - 1 0 nm [2]. Visualization of single heavy atoms with the scanning transmission electron microscope (STEM) [3] suggested that an ultimate resolution of 0.2-0.5 nm might be possible. Although single heavy atom markers have proven valuable in labeling ordered arrays [4,5], a number of practical problems limit their use on single isolated molecules. The cross section for scattering 40 keV electrons from single heavy atoms onto an annular detector subtending 0.04 to 0.2 rad in a practical microscope is approximately 7 × 10 -4 nm 2, or roughly 10 times the scattering power of a single carbon atom [6]. This means that an incident dose of 105-106 e l e c t r o n s / n m 2 is required to image
single atoms, as compared to the dose of 102-103 e l e c t r o n s / n m 2 which is known to damage biological molecules. This might not be a severe limitation if the heavy atoms remained in position during the damage; however, they are observed to migrate from their original attachment sites [7]. In order to increase the visibility of labels at low dose, cluster compounds having several heavy atoms in a compact array have been investigated. Lipka et al. [8] reported previously on a tetra mercury c o m p o u n d , C(HgO2CCH3)4, which showed good specificity but inadequate stability in the electron beam, being volatilized at a dose of 103- l04 e l e c t r o n s / n m 2, but requiring a calculated dose of 2 × l04 e l e c t r o n s / n m 2 to be visualized. A water-soluble undecagold compound described by Bartlett et al. [9], which was synthesized for its possible use as a label for electron microscopy, was shown to be visible at moderate dose [10]. This structure consists of a central core 0.82 n m in diameter containing eleven gold atoms, sur-
0304-3991/82/0000-0000/$02.75 © 1982 North-Holland
398
J.S. Wall et al. / Observation of undecagoM cluster compound in STEM
I
~2
nrn
J
PAr 3
Ar3P
Ar3P
p
- -
NH 2
Fig. 1. Structure of undecagold compound.
rounded by a 2 nm diameter organic shell with 21 benzylic amino groups on its surface, shown in fig. 1.
2. Materials and methods
Starting with a stable precursor (compound 6, ref. [9]), the gold cluster compound was synthesized shortly before sample preparation. The final complex was stable for several months in 0.05M ammonium bicarbonate buffer, pH 7.7. A 5/~1 drop of a 3 X 10 -7 M solution was applied to a 2 nm thick carbon film, and wicked off after 30 s. The grid was washed with a solution of tobacco mosaic virus (TMV) in distilled water and freeze dried according to a standard protocol [11]. STEM operating parameters are given in the caption of fig. 2.
3. Results and discussion
A typical first scan STEM image of this undecagold compound is shown in fig. 2a. Fig. 2b shows the same area on the eight scan (accumulated dose = 1.3 X 105 electrons/nrn2). Typical
dots in fig. 2a, having nearly equal intensity and identified below as being the gold cluster, are indicated with apparent mass values. In specimens prepared at higher undecagold concentration, bright spots are found in patches with a nearest neighbor spacing of 1.8-2.0 nm (data not shown). Fig. 2c (upper curve) shows measured spot intensity as a function of radius of integration for 50 spots in a 129.7 nm scan, a portion of which is shown in fig. 2a. The lower curve represents a similar set of measurements on 50 background points selected by first choosing random x and y position values, then discarding those points found to be within 5 nm of a gold cluster or a TMV particle. Error bars represent -+-one standard deviation (SD) of the essentially Gaussian distributions obtained. The ratio of cluster intensity to background SD is indicated as S/N (signal-tonoise ratio) at several measuring radii along the top of fig. 2c. Mass measurements were performed as described previously [12] using TMV on the same specimen as a calibration standard. The high visibility of this compound at a dose of 1.2 X 10 4 electrons/nm 2 is confirmed by a signal-to-noise ratio greater than 5 [13] for small radii of integration. Although the mass continues to increase out to R - - 2 nm, the measuring noise (SD) is found to increase faster, resulting in a lower S/N. Measuring noise results from thickness variations in the 1.8 nm thick carbon foil substrate and from statistical fluctuation in the number of detected electrons. Both these factors are well characterized [6,12]. The close agreement between the SD measured on clean background areas and the SD measured on gold spots suggests that the variation in spot intensities in fig. 2a results from measuring noise rather than from heterogeneity of the gold compound itself. Statistical variation in the number of electrons detected (per cent) can be reduced to an arbitrarily small value by increasing the dose used in recording the image. The consequent reduction in measuring noise from this source may be offset by several factors: (1) the biological matrix which is being labeled may be altered, (2) the complex may decompose, (3) the complex may move significantly on the substrate, or (4) the substrate may be altered.
J.S. Wall et al. / Observation of undecagold cluster compound in STEM
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/N = 6.2
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Fig. 2. Undecagold cluster visualized with the STEM using a dose of 1.2X 104 electrons/nm 2 on the first scan (a) and the eighth scan (b, total accumulated dose in b, 13.2X 10 4 electrons/nm2). The field shown is 256X 183 picture elements from a 512×512 scan of width 129.7 rim. Six of the 50 spots measured in the 512X512 field are indicated with apparent mass values in kD (R =0.5 nm). Bright feature in upper left is TMV. The Brookhaven STEM was operated at 40 kV with an incident convergence angle of 0.015 red, resulting in a focused spot <0.3 nm diameter. Specimen temperature was -135°C. An annular detector surrounding the beam collected a dark field signal consisting of electrons elastically scattered in the range from 0.04 to 0.2 tad from the beam axis. Data from each picture element were stored digitally as the beam scanned over the specimen. Stored data were displayed on a cathode ray tube and photographed. Analysis was carried out as described previously [ l l ]. (c) Apparent mass of gold cluster spots and background spots as a function of radius of integration, using TMV on the same specimen as an internal mass standard. See text for selection of background areas. Error bars indicate --+one standard deviation. S / N values above certain radii of integration give the ratio of apparent mass of I l-gold to background standard deviation at that same radius. (d) Apparent mass within a radius of 0.5 and 1.5 nm as a function of accumulated dose on the 1st, 2nd, 4th, 8th and 16th scans of the area, a portion of which is shown in (a). For comparison a dose response curve of TMV on the same specimen is shown. Because of the large mass of TMV (39X 10 6 D), measurements could be extended to < 5 0 electrons/nm 2, giving a value indicated by the horizontal line above the TMV curve.
Fig. 2d shows dose response curves for apparent particle mass using radii of integration of
electrons/nm
2
electrons/nm
2 and
0.5 a n d 1.5 n m . F o r c o m p a r i s o n for TMV in the same specimen
t r o n s / n m 2, a t o t a l d e c r e a s e o f 4 6 % . T h u s , i n c r e a s i n g t h e i m a g i n g d o s e t o 2 X 104 e l e c t r o n s / n m 2
R = 0.5 n m ( n e a r l y o p t i m u m mass drops from 1760
a mass loss curve is a l s o s h o w n . A t
S/N) D
at
the apparent 1.2 X 1 0 4
to
1430 to 930
D D
at
2.4 X
10 4
a t 2.9 x 105 e l e c -
o n l y i m p r o v e s t h e S / N t o 6.8 ( R = 0.25 n m , d a t a not shown) and further increases in dose may
400
J.S. Wall et al. / Observation of undecagoM cluster compound in S T E M
actually decrease the S/N. Recording lower dose pictures with the Brookhaven STEM presently requires use of a coarser scan (0.5 × 0.5 nm picture element size) which may result in undersampling the data. Preliminary measurements using this coarse scan at a dose of 0.5 X 104 e l e c t r o n s / n m 2 give an apparent mass of 2310 D and a S/N of 5.9 (R = 0.5 nm, data not shown), suggesting that the dose response curve for undecagold mass may be roughly proportional to that for T M V at low dose. Assuming that the apparent mass at zero dose and 0.5 nm radius of integration is 2560 D, the cluster may be visible (with a S/N = 5) at a dose as low a s 0 . 2 X 10 4 e l e c t r o n s / n m 2. Experiments are now underway to test this prediction. The gold core does not appear to be appreciably more stable than the molecule as a whole. The motion of this cluster induced by imaging appears to be relatively small. Comparing spots from fig. 2a (dose = 1.2 × l0 g e l e c t r o n s / n m z) with the corresponding spots on a second scan (total d o s e - - 2.4 X l04 electrons/nm2), a root mean square (RMS) displacement of 0.35 nm was found and by the fourth scan (total d o s e - - 5 . 2 × 10 4 e l e c t r o n s / n m 2) an RMS displacement of 0.47 nm was observed. Of this displacement, 0.18 nm probably can be accounted for by measuring error, since the specimen was sampled at 0.25 nm intervals in x and y directions. Background noise appears to increase slightly as the dose increases (SD, fig. 2d). This effect is currently being investigated to determine if it is due to desorption of some component from the surface, etching, recrystallization of the carbon foil or something else. The expected apparent mass may be calculated from the chemical composition by assuming that each gold atom produces a scattered intensity equivalent to that of l0 carbon atoms [6] and C, N, O, and P scatter in proportion to their atomic numbers. In this case the apparent mass of the molecule as a whole [(NHECH2C6H4)3P]7 AUll(CN)3 is approximately 3800, in reasonable agreement with the observed value of 4400 -+ 1900. The 0.82 nm diameter projection (perpendicular to the substrate) containing l 1 gold atoms and the upper and lower surfaces of the 2 nm diameter organic shell should have an apparent mass of
1320 (core) + 400 (shell) = 1720, again in reasonable agreement with the 1730 observed at R = 0.5 n m at a dose of 1.2 X l04 electrons/nm 2. Correction to zero dose on the assumption that the undecagold cluster mass loss curve is parallel to that of T M V would increase the measured values by approximately 18%. Calculation of the expected apparent mass of undecagold cluster assumed all amines were protonated. Inclusion of other counter ions could increase this expected value. Application of this undecagold compound as a specific label is complicated by several practical problems. The compound is relatively "sticky" and excess reagent cannot be washed off the substrate. Therefore it is necessary to perform chemical reactions and remove excess reagent prior to application of the sample to the carbon film. The gold cluster may precipitate at p H < 7 and may stick to gel seiving columns and dialysis tubing at low ionic strength. Specific attachment may be accomplished through modification of the amino functions on the outer shell of the cluster incorporated for that purpose. However if more than one amino group per cluster is converted to a specific functionality, problems may be encountered with cross-linking of the target molecules. 4. Conclusion The results we have obtained indicate that the undecagold cluster can function as a highly visible specific label for low dose electron microscopic studies of biological structures at 1-2 nm resolution. The characteristic instability of the compound at high dose may be useful in providing a convenient means to identify gold clusters in a double labeling experiment employing a second, more stable, cluster. Practical conditions for biochemical modification and attachment to obtain adequate labeling without aggregation will be described in subsequent papers.
Acknowledgements We gratefully acknowledge K. Chung, F. Kito and E. Desmond for technical assistance. This
J.S. Wall et al. / Observation of undecagold cluster compound in STEM
work was supported by USDOE (J.S.W.), NIH Biotechnology Resource Grant RR00715, NIH grant GM-21612 (P.A.B.) and NIH grant GM15971 (S.J.S.).
References [1] S.J. Singer, Nature (London) 183 (1959) 1523. [2] N.M. Green, R.C. Valentine, N.G. Wrigley, F. Ahmed, B. Jacobson and H.G. Wood, J. Biol. Chem. 247 (1972) 6284. [3] A.V. Crewe, J. Wall and J. Langrnore, Science 168 (1970) 1338. [4] M. Stewart, Roy. Soc. (London) BI90 (1975) 257. [5] M. Beer, J.W. Wiggins, R. Alexander, R. Schettino, C. Stockert and K. Piez, in: Proc. 37th Ann. EMSA Meeting, 1979, p. 28.
401
[6] J.S. Wall, Chem. Scripta 14 (1978-79) 271. [7] M.D. Cole, J.W. Wiggins and M. Beer, J. Mol. Biol. 117 (1977) 387. [8] J.J. Lipka, S.J. Lippard and J.S. Wall, Science 206 (1979) 1419. [9] P.A. Bartlett, B. Bauer and S.J. Singer, J. Am. Chem. Soc. 100 (1978) 5085. [10] A.V. Crewe, in: Proc. 9th Intern. Congr. on Electron Microscopy, 1978, Vol. 3, p. 197. [11] A.W. Mosesson, J.F. Hainfeld, R.H. Haschemeyer and J.S. Wall, J. Mol. Biol. 153 (1981) 695. [12] J.S. Wall, in: Scanning Electron Microscopy/1979/ll (SEM Inc., A M F O'Hare, IL) p. 291; J. Wall, in: Introduction to Analytical Electron Microscopy, Eds. J.J. Hren, J.I. Goldstein and D.C. Joy (Plenum, New York, 1979) p. 333. [13] A. Rose, Advan. Electron. 1 (1948) 131.
397
O B S E R V A T I O N O F AN U N D E C A G O L D C L U S T E R C O M P O U N D IN T H E S C A N N I N G TRANSMISSION ELECTRON MICROSCOPE .J.S. W A L L and J.F. H A I N F E L D Brookhaven National Laboratory, Biology-463, Upton, New York 11973, USA
P.A. B A R T L E T T Department of Chemistry, University of California, Berkeley, California 94720, USA
and S.J. S I N G E R American Cancer Society Research Professor, Department of Biology, University of California at Sun Diego, La Jolla, California 92093, USA
Received 8 March 1982
A water-soluble undecagold cluster compound has been shown to be visible in moderate dose images (10 4 electrons/nm 2) obtained with the scanning transmission electron microscope (STEM). The properties of this compound render it potentially useful as a marker for specific sites within biological molecules.
1. Introduction
Localization of specific sites in electron microscopic images of biological specimens is limited by the size of available labels. Ferritin-labeled antibodies provide a resolution of 15-30 nm [1], while biotin-avidin labeling may provide a resolution of 4 - 1 0 nm [2]. Visualization of single heavy atoms with the scanning transmission electron microscope (STEM) [3] suggested that an ultimate resolution of 0.2-0.5 nm might be possible. Although single heavy atom markers have proven valuable in labeling ordered arrays [4,5], a number of practical problems limit their use on single isolated molecules. The cross section for scattering 40 keV electrons from single heavy atoms onto an annular detector subtending 0.04 to 0.2 rad in a practical microscope is approximately 7 × 10 -4 nm 2, or roughly 10 times the scattering power of a single carbon atom [6]. This means that an incident dose of 105-106 e l e c t r o n s / n m 2 is required to image
single atoms, as compared to the dose of 102-103 e l e c t r o n s / n m 2 which is known to damage biological molecules. This might not be a severe limitation if the heavy atoms remained in position during the damage; however, they are observed to migrate from their original attachment sites [7]. In order to increase the visibility of labels at low dose, cluster compounds having several heavy atoms in a compact array have been investigated. Lipka et al. [8] reported previously on a tetra mercury c o m p o u n d , C(HgO2CCH3)4, which showed good specificity but inadequate stability in the electron beam, being volatilized at a dose of 103- l04 e l e c t r o n s / n m 2, but requiring a calculated dose of 2 × l04 e l e c t r o n s / n m 2 to be visualized. A water-soluble undecagold compound described by Bartlett et al. [9], which was synthesized for its possible use as a label for electron microscopy, was shown to be visible at moderate dose [10]. This structure consists of a central core 0.82 n m in diameter containing eleven gold atoms, sur-
0304-3991/82/0000-0000/$02.75 © 1982 North-Holland
398
J.S. Wall et al. / Observation of undecagoM cluster compound in STEM
I
~2
nrn
J
PAr 3
Ar3P
Ar3P
p
- -
NH 2
Fig. 1. Structure of undecagold compound.
rounded by a 2 nm diameter organic shell with 21 benzylic amino groups on its surface, shown in fig. 1.
2. Materials and methods
Starting with a stable precursor (compound 6, ref. [9]), the gold cluster compound was synthesized shortly before sample preparation. The final complex was stable for several months in 0.05M ammonium bicarbonate buffer, pH 7.7. A 5/~1 drop of a 3 X 10 -7 M solution was applied to a 2 nm thick carbon film, and wicked off after 30 s. The grid was washed with a solution of tobacco mosaic virus (TMV) in distilled water and freeze dried according to a standard protocol [11]. STEM operating parameters are given in the caption of fig. 2.
3. Results and discussion
A typical first scan STEM image of this undecagold compound is shown in fig. 2a. Fig. 2b shows the same area on the eight scan (accumulated dose = 1.3 X 105 electrons/nrn2). Typical
dots in fig. 2a, having nearly equal intensity and identified below as being the gold cluster, are indicated with apparent mass values. In specimens prepared at higher undecagold concentration, bright spots are found in patches with a nearest neighbor spacing of 1.8-2.0 nm (data not shown). Fig. 2c (upper curve) shows measured spot intensity as a function of radius of integration for 50 spots in a 129.7 nm scan, a portion of which is shown in fig. 2a. The lower curve represents a similar set of measurements on 50 background points selected by first choosing random x and y position values, then discarding those points found to be within 5 nm of a gold cluster or a TMV particle. Error bars represent -+-one standard deviation (SD) of the essentially Gaussian distributions obtained. The ratio of cluster intensity to background SD is indicated as S/N (signal-tonoise ratio) at several measuring radii along the top of fig. 2c. Mass measurements were performed as described previously [12] using TMV on the same specimen as a calibration standard. The high visibility of this compound at a dose of 1.2 X 10 4 electrons/nm 2 is confirmed by a signal-to-noise ratio greater than 5 [13] for small radii of integration. Although the mass continues to increase out to R - - 2 nm, the measuring noise (SD) is found to increase faster, resulting in a lower S/N. Measuring noise results from thickness variations in the 1.8 nm thick carbon foil substrate and from statistical fluctuation in the number of detected electrons. Both these factors are well characterized [6,12]. The close agreement between the SD measured on clean background areas and the SD measured on gold spots suggests that the variation in spot intensities in fig. 2a results from measuring noise rather than from heterogeneity of the gold compound itself. Statistical variation in the number of electrons detected (per cent) can be reduced to an arbitrarily small value by increasing the dose used in recording the image. The consequent reduction in measuring noise from this source may be offset by several factors: (1) the biological matrix which is being labeled may be altered, (2) the complex may decompose, (3) the complex may move significantly on the substrate, or (4) the substrate may be altered.
J.S. Wall et al. / Observation of undecagold cluster compound in STEM
f
/N = 6.2
1
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4.5
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RADIUS OF INTEGRATION (nrn)
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Fig. 2. Undecagold cluster visualized with the STEM using a dose of 1.2X 104 electrons/nm 2 on the first scan (a) and the eighth scan (b, total accumulated dose in b, 13.2X 10 4 electrons/nm2). The field shown is 256X 183 picture elements from a 512×512 scan of width 129.7 rim. Six of the 50 spots measured in the 512X512 field are indicated with apparent mass values in kD (R =0.5 nm). Bright feature in upper left is TMV. The Brookhaven STEM was operated at 40 kV with an incident convergence angle of 0.015 red, resulting in a focused spot <0.3 nm diameter. Specimen temperature was -135°C. An annular detector surrounding the beam collected a dark field signal consisting of electrons elastically scattered in the range from 0.04 to 0.2 tad from the beam axis. Data from each picture element were stored digitally as the beam scanned over the specimen. Stored data were displayed on a cathode ray tube and photographed. Analysis was carried out as described previously [ l l ]. (c) Apparent mass of gold cluster spots and background spots as a function of radius of integration, using TMV on the same specimen as an internal mass standard. See text for selection of background areas. Error bars indicate --+one standard deviation. S / N values above certain radii of integration give the ratio of apparent mass of I l-gold to background standard deviation at that same radius. (d) Apparent mass within a radius of 0.5 and 1.5 nm as a function of accumulated dose on the 1st, 2nd, 4th, 8th and 16th scans of the area, a portion of which is shown in (a). For comparison a dose response curve of TMV on the same specimen is shown. Because of the large mass of TMV (39X 10 6 D), measurements could be extended to < 5 0 electrons/nm 2, giving a value indicated by the horizontal line above the TMV curve.
Fig. 2d shows dose response curves for apparent particle mass using radii of integration of
electrons/nm
2
electrons/nm
2 and
0.5 a n d 1.5 n m . F o r c o m p a r i s o n for TMV in the same specimen
t r o n s / n m 2, a t o t a l d e c r e a s e o f 4 6 % . T h u s , i n c r e a s i n g t h e i m a g i n g d o s e t o 2 X 104 e l e c t r o n s / n m 2
R = 0.5 n m ( n e a r l y o p t i m u m mass drops from 1760
a mass loss curve is a l s o s h o w n . A t
S/N) D
at
the apparent 1.2 X 1 0 4
to
1430 to 930
D D
at
2.4 X
10 4
a t 2.9 x 105 e l e c -
o n l y i m p r o v e s t h e S / N t o 6.8 ( R = 0.25 n m , d a t a not shown) and further increases in dose may
400
J.S. Wall et al. / Observation of undecagoM cluster compound in S T E M
actually decrease the S/N. Recording lower dose pictures with the Brookhaven STEM presently requires use of a coarser scan (0.5 × 0.5 nm picture element size) which may result in undersampling the data. Preliminary measurements using this coarse scan at a dose of 0.5 X 104 e l e c t r o n s / n m 2 give an apparent mass of 2310 D and a S/N of 5.9 (R = 0.5 nm, data not shown), suggesting that the dose response curve for undecagold mass may be roughly proportional to that for T M V at low dose. Assuming that the apparent mass at zero dose and 0.5 nm radius of integration is 2560 D, the cluster may be visible (with a S/N = 5) at a dose as low a s 0 . 2 X 10 4 e l e c t r o n s / n m 2. Experiments are now underway to test this prediction. The gold core does not appear to be appreciably more stable than the molecule as a whole. The motion of this cluster induced by imaging appears to be relatively small. Comparing spots from fig. 2a (dose = 1.2 × l0 g e l e c t r o n s / n m z) with the corresponding spots on a second scan (total d o s e - - 2.4 X l04 electrons/nm2), a root mean square (RMS) displacement of 0.35 nm was found and by the fourth scan (total d o s e - - 5 . 2 × 10 4 e l e c t r o n s / n m 2) an RMS displacement of 0.47 nm was observed. Of this displacement, 0.18 nm probably can be accounted for by measuring error, since the specimen was sampled at 0.25 nm intervals in x and y directions. Background noise appears to increase slightly as the dose increases (SD, fig. 2d). This effect is currently being investigated to determine if it is due to desorption of some component from the surface, etching, recrystallization of the carbon foil or something else. The expected apparent mass may be calculated from the chemical composition by assuming that each gold atom produces a scattered intensity equivalent to that of l0 carbon atoms [6] and C, N, O, and P scatter in proportion to their atomic numbers. In this case the apparent mass of the molecule as a whole [(NHECH2C6H4)3P]7 AUll(CN)3 is approximately 3800, in reasonable agreement with the observed value of 4400 -+ 1900. The 0.82 nm diameter projection (perpendicular to the substrate) containing l 1 gold atoms and the upper and lower surfaces of the 2 nm diameter organic shell should have an apparent mass of
1320 (core) + 400 (shell) = 1720, again in reasonable agreement with the 1730 observed at R = 0.5 n m at a dose of 1.2 X l04 electrons/nm 2. Correction to zero dose on the assumption that the undecagold cluster mass loss curve is parallel to that of T M V would increase the measured values by approximately 18%. Calculation of the expected apparent mass of undecagold cluster assumed all amines were protonated. Inclusion of other counter ions could increase this expected value. Application of this undecagold compound as a specific label is complicated by several practical problems. The compound is relatively "sticky" and excess reagent cannot be washed off the substrate. Therefore it is necessary to perform chemical reactions and remove excess reagent prior to application of the sample to the carbon film. The gold cluster may precipitate at p H < 7 and may stick to gel seiving columns and dialysis tubing at low ionic strength. Specific attachment may be accomplished through modification of the amino functions on the outer shell of the cluster incorporated for that purpose. However if more than one amino group per cluster is converted to a specific functionality, problems may be encountered with cross-linking of the target molecules. 4. Conclusion The results we have obtained indicate that the undecagold cluster can function as a highly visible specific label for low dose electron microscopic studies of biological structures at 1-2 nm resolution. The characteristic instability of the compound at high dose may be useful in providing a convenient means to identify gold clusters in a double labeling experiment employing a second, more stable, cluster. Practical conditions for biochemical modification and attachment to obtain adequate labeling without aggregation will be described in subsequent papers.
Acknowledgements We gratefully acknowledge K. Chung, F. Kito and E. Desmond for technical assistance. This
J.S. Wall et al. / Observation of undecagold cluster compound in STEM
work was supported by USDOE (J.S.W.), NIH Biotechnology Resource Grant RR00715, NIH grant GM-21612 (P.A.B.) and NIH grant GM15971 (S.J.S.).
References [1] S.J. Singer, Nature (London) 183 (1959) 1523. [2] N.M. Green, R.C. Valentine, N.G. Wrigley, F. Ahmed, B. Jacobson and H.G. Wood, J. Biol. Chem. 247 (1972) 6284. [3] A.V. Crewe, J. Wall and J. Langrnore, Science 168 (1970) 1338. [4] M. Stewart, Roy. Soc. (London) BI90 (1975) 257. [5] M. Beer, J.W. Wiggins, R. Alexander, R. Schettino, C. Stockert and K. Piez, in: Proc. 37th Ann. EMSA Meeting, 1979, p. 28.
401
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