Ultramicroscopy 42-44 (1992) 1243-1249 North-Holland
Substrate preparation for reliable imaging of DNA molecules with the scanning force microscope J. V e s e n k a a, M. G u t h o l d a n d C. B u s t a m a n t e a,,
a
C.L. T a n g
a,
D. K e l l e r b, E. D e l a i n e c
a Institute of Molecular Biology, University of Oregon, Eugene, OR 97405, USA b Chemistry Department, University of New Mexico, Albuquerque, NM 87131, USA c lnstitut Gustave Roussy, Rue Camille Desmoulins, 94805 Villejuif, France Received 12 August 1991
A simple method of substrate preparation for imaging circular D N A molecules with the scanning force microscope (SFM) is presented. These biomolecules are adsorbed onto mica that has been soaked in m a g n e s i u m acetate, sonicated and glow-discharged. The stylus-sample forces that may be endured before sample damage occurs depends on the ambient relative humidity. Images of circular D N A molecules have been obtained routinely using tips specially modified by an electron beam with a radius of curvature, Rc, of about 10 n m [D. Keller and C. Chih-Chung, Surf. Sci. 268 (1992) 333]. The resolution of these adsorbed biomolecules is determined by the R c. At higher forces individual circular D N A molecules can be manipulated with the SFM stylus. Strategies to develop still sharper probes will be discussed.
1. Introduction
Scanning force microscopy (SFM) images are obtained by recording the deflection of a sharp stylus as it scans the surface of interest [1,2]. The measured deflection is proportional to the force between the surface and the tip. SFM has many distinct advantages over electron microscopies, the most important being the ability to work under physiologically relevant conditions. SFM also has an advantage over its sister microscopy, scanning tunneling microscopy (STM), in that the sample need not be conductive since only contact forces are involved. There are many STM papers reporting the observation of naked D N A on graphite [3-7] but the reproducibility of these results is still unsatisfactory. Lindsay et al. [8] have imaged D N A electrodeposited to gold on mica with the STM and, at lower resolution, DNA adsorbed onto glass with the SFM. The SFM has imaged DNA
* Corresponding author.
adsorbed onto a polymerized monolayer on mica [9,10] although reproducibility was poor. The problem of reproducibility stems from: (1) the requirement of a suitably fiat, strongly adsorbing substrate; (2) the use of reproducibly sharp styli; and (3) the need to develop optimal ambient conditions for imaging. In this paper we discuss a simple substrate preparation method that firmly binds plasmid D N A to mica in air at low humidity. Reproducible images are routinely obtained using styli of small radius of curvature, specially modified for these purposes. The combination of these "microtips" and reliable substrate preparation has allowed us to carry out a statistical analysis and comparison to the known lengths, widths and heights of the imaged circular D N A molecules. Differences in the observed lateral dimensions from those of double-stranded D N A are rationalized in term of the finite radius of curvature, Re, and cantilever length. Furthermore, due to the reproducibility of the imaging, it was possible to locally manipulate individual DNA molecules using these sharp microtips.
0304-3991/92/$05.00 © 1992 - Elsevier Science Publishers B.V. All rights reserved
1244
J. Vesenka et al. / A substrate preparation for reliable imaging of DNA molecules
2. Materials and methods The following treatment of mica is a variation of a method previously used to image D N A in transmission electron microscopy [11]. Red mica (Ashville-Schoonmaker Mica Co., Newport New, VA) is freshly cleaved and briefly sonicated in de-ionized/distilled water (DDW). The mica is then soaked in 33mM magnesium acetate overnight and sonicated again in DDW for 30 min. The magnesium is thought to replace potassium ions in the mica surface providing a stronger binding site with the phosphate groups of the DNA. Sonication of the mica removes excess salts
from the surface, providing a smooth substrate for observing the biomolecules. The mica surface is DC glow discharged for 15 s under vacuum between 100 and 200 mTorr, a process that hardens the substrate. As soon as the substrate is brought up to air, a few microliters of 1 0 0 / z g / m l DNA is deposited onto the surface and allowed to adsorb for 2 min. The sample is then rinsed with DDW to remove excess plasmid and finally blown dry with nitrogen. The specimens were imaged with a Nanoscope II (Digital Instruments Inc., Santa Barbara, CA) scanning force microscope and software. All samples were imaged in air at room temperature in a
Microtip
~
P
Cantilever 4ram
lO0~m
Fig. 1. Schematic diagram of progresswe magnifications of a commercially available s u b s t r a t e / c a n t i l e v e r / t i p assembly. The micrograph is an image of microtip generated by electron beam deposition in a scanning electron microscope (Hitachi S-8000) at the "tip" of the above assembly. The focused electron b e a m will deposit a micrometer length structure, composed of carbonaceous material from residual vacuum chamber oils, onto the surface of the tip within minutes.. The radius of curvature of the above microtip is approximately 10 n m with a cone angle of about 15 °.
J. Vesenka et al. / A substrate preparation for reliable imaging of DNA molecules
1245
c h a m b e r with c o n t r o l l e d relative humidity. T o r e d u c e sample d a m a g e , the s u r f a c e - t i p force was m i n i m i z e d by w i t h d r a w i n g the piezo from the s c a n n i n g stylus while i m a g i n g in the attractive force m o d e . Only m i n i m a l filtering, in the form of removal of low-frequency noise in the slow-scan direction (flattening), was applied to the o b t a i n e d images. T h e lateral d i m e n s i o n s of the piezo transd u c e r were c a l i b r a t e d with a 1000 l i n e s / m m gold grating. T h e m e a s u r e d lengths were f o u n d to be accurate within 1% for a 1 . 5 / z m scan, similar to the d i m e n s i o n s typically s c a n n e d in o u r experiments. I n these studies we used sharp styli modified from c o m m e r c i a l tips u n d e r the b e a m of an electron microscope [1]. V a c u u m - p u m p h y d r o c a r b o n s can be selectively d e p o s i t e d o n the tip of commercially available S F M stylus to o b t a i n sharp " m i c r o t i p s " with R c = 10 n m (fig. 1). T h e s e microtips e n a b l e the systematic a n d r e p r o d u c i b l e imaging of p l a s m i d D N A molecules (figs. 2 a n d 3).
3. Results
Fig. 2. Typical low-magnificationview of the surface of treated mica with adsorbed plasmid DNA at a relative humidity of 25% and scanning force of 3 nN. A plasmid DNA at this humidity can be scanned for over an hour, even at higher magnifications, without distortion due to the microtip-sample contact. A 100 /xg/ml solution of plasmid DNA adsorbed onto the mica surface for a few minutes followed by rinsing and drying will yield a plasmid density of about 1/p.m 2. Notice that the plasmid is clearly distinguished from background features, most likely residual salt deposits, on the mica.
Fig. 3. Two typical plasmid DNA images. (a) An image of a 3.1 kilobasepair (kbp) plasmid DNA molecule taken with 100 /zm length cantilever at a relative humidity (RH) of 45% and force of 5 nN. The higher features near the cross-over point are probably due to supercoiling of the plasmid. (b) An image of a 3.0 kbp plasmid taken with a 200/zm length cantilever at RH = 50% and force of about 3 nN. Both images have spurious 20 nm diameter blotches, presumably due to salt deposits. Notice the different height scales, that is, the plasmid in (a) appears much shorter than that of (b). Details of the topological features are discussed under table 1.
R e d mica substrates deposited with purified circular, or plasmid, D N A molecules yielded occasional salt crystals a n d were essentially featureless except for the plasmids (fig. 2). Figs. 3a a n d 3b are higher m a g n i f i c a t i o n images of observed D N A molecules on t r e a t e d mica
1246
J. Vesenka et al. / A substrate preparation for reliable imaging o f DNA molecules
routinely o b t a i n e d using the microtips. A v e r a g e c o n t o u r lengths, widths a n d heights of different plasmids are p r e s e n t e d in table 1. Short cantilevers (100 /xm) with larger spring c o n s t a n t s ( ~ 0.5 n N / n m ) a n d lower noise (0.2-0.3 n m ) gave g r e a t e r image contrast, b u t had large plasmid widths ( ~ 13 n m ) a n d short plasmid heights ( ~ 0.6 nm). Longer, t h i n n e r cantilevers (200 ~ m ) with smaller spring c o n s t a n t s ( ~ 0.1 n N / n m ) revealed n a r r o w e r widths ( ~ 10 n m ) a n d taller height ( ~ 1.9 n m ) b u t at the expense of r e d u c e d image contrast due to g r e a t e r noise levels (0.5-0.8 nm). Force control was m a i n t a i n e d by withdrawing the tip from the sample in the attractive mode. M i n i m a l o p e r a t i n g forces were typically in the
Table 1 Statistical analysis of DNA plasmids Plasmid DNA
Calculated a) length (nm)
Measured length (nm)
D i f f e r - Relative ence humidity (%) (%)
pDL34 b) pVCB5 c) pSK31 d)
1069 1000 1131
1049 _+52 874 _+54 938_+35
2 13 17
Plasmid DNA
Measured height (nm)
Measured width(nm)
Cantilever length (/~m)
pDL34 b) pVCB5 c) pSK31 d)
0.65_+0.26 1.89 _+0.72 1.43_+0.09
13.1_+4.3 10.3 ± 3.4 12.7_+2.2
100 200 200
45 50 25
a) Assuming 0.34 nm per base pair of plasmid DNA. b~ Average of 7 images. c) With 242 bp clone. Average of 5 images. d) Average of 8 measurements. Two of the plasmid contour lengths were shorter than expected and beyond the range of experimental error (+5%). We are confident a piezo calibration error of the lateral scanning dimensions was not the cause. It is possible that the plasmid DNA molecules folded up, without supercoiling, and could not be detected by the relatively wide microtip. We suspect that low humidity might play a role in shrinking the plasmid contour lengths. The vertical heights, 0.6-1.9 nm, are consistent with the anticipated effect of squeezing the DNA with a scanning stylus. The shorter cantilevers are more rigid, less sensitive to vertical motion and can apply greater force to the sample. Consequently they tend to squeeze the DNA more than the longer cantilevers. The D N A widths, 10-13 nm, are consistent with the R c of the microtip (figs. 2 and 3) and are explained in greater detail in fig. 7.
Fig. 4. Plasmid DNA at high humidity. It depicts a plasmid at a relative humidity (RH) of 65% with a stylus force of about 3 nN. This image was taken during the first scan. Within several consecutive scans this plasmid had been completely removed from the mica surface by the stylus. At higher RH the presence of water degrades the image quality, perhaps by interfering with the plasmid/treated-mica surface bonding or by enhancing the hydrostatic forces between the stylus and sample. We have found that the most stable images are obtained at RH below 40%. range of 3 n N in air and, in relative humidities ( R H ) less t h a n 40%, the plasmid could be s c a n n e d up to an h o u r without observable d e g r a d a t i o n . At R H above 60% only fleeting images of plasmid D N A molecules could be o b t a i n e d before they are swept aside by the stylus (fig. 4). O n u n t r e a t e d surfaces (no m a g n e s i u m ) the D N A are swept aside by the s c a n n i n g tip regardless of the relative humidity. A t the lower h u m i d i t y the N a n o s c o p e II software provides two m e a n s of cutting the D N A with the stylus (figs. 5 a n d 6). O n e m e c h a n i s m involves increasing the force between the tip a n d substrate slowly, with excellent control over the desired cutting point, as in fig. 5. T h e second m e t h o d utilizes a s u d d e n increase of the force while arresting the slow-scan direction, resulting in a sharp, crisp scission of the D N A , as in fig. 6.
4. Discussion O n e of the criticisms of s c a n n i n g p r o b e microscopies is that m a n y of the biological images are
J. Vesenka et al. / A substrate preparation for reliable imaging of DNA molecules
1247
unconvincing, looking like "blobs on a screen". The SFM images of plasmid DNA molecules obtained in this work look similar to electron micrographs of plasmids. Furthermore, we were able to routinely observe the DNA using the above sample preparation method and microtips.
Fig. 5. Images of plasmid a D N A cut with the scanning stylus using slow force increase. (a) A 3.0 kbp plasmid prior to dissection with the SFM stylus. First the stylus is placed near the top center of the plasmid and the sample is slowly raised, increasing the force from 3 to 45 nN, until the above pit is carved (b). Raising the sample a vertical distance, ~z, causes a finite lateral movement of the tip, bx, related by: ~x ~ a~z, where a is the average angle between the cantilever and sample. For a 10 ° angle (0.175 rad) and a 1 ~ m rise in the piezo, the lateral traversal of the tip is about 175 nm, as indicated by arrows in (b). Because the force is slowly increased the outcome of the dissection can be easily predicted.
Fig. 6. Images of plasmid D N A cut with the scanning stylus using sudden force increase. (a) An image of a 3.3 kbp plasmid before it was dissected by disabling the slow-scan direction, causing the sample to be scanned at the same point in the y direction. At a force of 3 nN the "neck" of the structure was imaged (beside arrow near the top), followed by a sudden increase of force by raising the sample. After the higher force (45 nN) was applied for 10 s, the force was reduced to 3 nN and the slow-scan direction was enabled. Notice that the region of the sliced plasmid, indicated by an arrow in (b), was below the neck by about 175 nm, for the same reason as discussed in fig. 5. This scission method provides cleaner cuts, but it is more difficult to control the position of the cutting point.
Although imaged in air, a microscopic layer of moisture must be present, evidenced by the fact that observations of plasmids at humidities higher than 60% are extremely difficult. At high humidity the presence of water degrades the image
1248
J. Vesenka et al. / A substrate preparation for reliable imaging of DNA molecules
quality, perhaps by interfering with the plasmid/ treated-mica surface bonding or by enhancing the hydrostatic forces between the stylus and sample. We have found that the most stable images are obtained at R H below 40%. The presence of a water layer on the mica may play an important role in keeping the plasmid hydrated and stabilizing the conformation of the DNA [12]. The initial identification of the observed features of the plasmids was confirmed by making a statistical analysis of the contour lengths of the observed molecules. This treatment of the data was possible because of the reproducibility in the imaging. Plasmid D N A of varying sizes were measured from SFM images and compared to a length assuming 0.34 nm per base pair (table 1). The pDL34 had a deviation of 2% from the expected value, well within the 5% error associated with these measurements. Two double plasmids of the pDL34 were distinguished and the contour lengths from these images were nearly twice that of single plasmids (within 10%). However, pVCB5 had a 13% deviation and pSK31 had a 17% deviation from their anticipated base pair values. A similar result for pVCB5 was obtained from STM images of coated samples [13]. Because identical results were obtained from different instruments having different piezo elements it is unlikely to be a calibration problem. One possibility is that the plasmid molecules have folded upon themselves, without supercoiling. Since the D N A is much narrower than the microtip, folding of the DNA in this manner would be hidden in the width of the scanning stylus. It is possible that reduced relative humidity might play an important role in causing the DNA to contract. All the plasmids observed in this work were five to seven times broader than expected. The "broadening" phenomena [14] is modeled in fig. 7. The key feature is that the microtips we used have a radius of curvature of around 10 nm, larger than the diameter of the DNA molecule of 2 nm. From simple geometric arguments one can show that (cf. fig. 7) W = 4R~RRRR~,
(1)
where W is the estimated width and R m is the radius of the plasmid DNA molecule. This ex-
Fig. 7. As seen in the plasmid images and tabulated in table 1, the width of the DNA is much greater than 2 nm. This is the result of a finite radius of curvature, Re, of the microtip, as shown schematically in fig. 7. For a microtip of Rc, a linear biomolecule of radius R m where R c >>R__Rmz_the anticipated imaged biomolecule width is W=4~/RmR c. For doublestranded DNA and the microtips used in the work, having Rc = 10 nm, the anticipated width is about 12.6 nm, in excellent agreement with our statistical measurements.
pression is valid for large radius of curvature for which the tip can be approximated by a sphere. For DNA and our microtips we estimate a width of 12.6 nm, in excellent agreement with our data. This result shows the crucial factor determining the lateral dimension, W, and therefore the resolution of the surface features is the dimension of the radius of curvature, R c. The ability of the microscopic stylus to manipulate individual molecules of plasmid DNA can be seen in figs. 5 and 6. We clearly can cut the plasmid at any desired location. In order to cut the DNA at specific sites we intend to examine labelled plasmids in future studies. Another system for future examination is the structure of the RNA p o l y m e r a s e / D N A complex. The key issue to improvement of the image resolution is sharper microtips. A program is underway in our laboratory for developing sharper styli. The limiting feature in electron beam microtip construction is the beam spot size. The tips used in this study were from a Hitachi 8000 scanning electron microscope (SEM) with 5 nm spot size. Field-emission SEM can attain 2-3 nm diameter spot size and scanning transmission electron microscopes have about 0.7-2.0 nm spot size in the secondary emission mode. We are presently using these microscopes at the University of Oregon to manufacture sharper tips. In principle, the STM can also be used to fabricate sharp tips through its nanolithography ability [15].
J. Vesenka et al. / A substrate preparation for reliable imaging of DNA molecules
H o w e v e r , t h e r a t e at which t h e s e tips a r e m a d e is very slow a n d t h e m a t e r i a l s a r e p r e s e n t l y l i m i t e d to m e t a l s t h a t m a y b e t o o soft for o b t a i n i n g s h a r p images. W e a r e also p u r s u i n g silicon m i c r o f a b r i c a t i o n t e c h n i q u e s that, in principle, can a t t a i n R c--- 1 nm [16,17]. T h e r e is a q u e s t i o n t h a t n e e d s addressing as to w h e t h e r s h a r p e r styli will l e a d to g r e a t e r s a m p l e d a m a g e d u e to t h e R~-2 p r e s s u r e increase. H o w e v e r , since t h e m a i n attractive force in air is f r o m surface t e n s i o n t h e force should also d e c r e a s e with t h e c o n t a c t area. Thus, in p r i n c i p l e , s h a r p e r tips s h o u l d result in h i g h e r resolution without increased sample damage.
5. Conclusions W e have p r e s e n t e d a reliable, r e p r o d u c i b l e m e a n s of d e p o s i t i n g a n d i m a g i n g D N A with t h e s c a n n i n g force m i c r o s c o p e . T h e key i n g r e d i e n t s to successful i m a g e f o r m a t i o n are: (1) S u b s t r a t e p r e p a r a t i o n - m a g n e s i u m on t h e surface o f mica s e e m s to assist a n c h o r i n g the p l a s m i d D N A m o l e c u l e s to t h e surface. (2) S h a r p stylus - e l e c t r o n - b e a m - d e p o s i t e d mic r o t i p s p r o v i d e convincing i m a g e s o f t h e deposited biomolecules. (3) R e l a t i v e h u m i d i t y - stable i m a g e s can b e o b t a i n e d with the above two i n g r e d i e n t s so long as the relative h u m i d i t y is b e l o w 40%. W e w e r e able to cut the p l a s m i d D N A m o l e c u l e s with t h e stylus, i n d i c a t i n g the p o t e n t i a l o f t h e S F M as a device for m i c r o - m a n i p u l a t i o n . T h e r e s o l u t i o n o f t h e b i o m o l e c u l e s is a f u n c t i o n o f R~c. W e have p r e s e n t e d high resolution, highly r e p r o d u c i b l e i m a g e s o f b i o m o l e c u l e s . W e do not have to rely u p o n the f o r t u i t o u s use o f a c o m m e r cial stylus having a s h a r p d e f e c t on the tip [18]. G r e a t e r a p p l i c a t i o n of the S F M awaits the develo p m e n t of s h a r p e r styli.
Acknowledgments W e t h a n k W i l l i a m R e e s a n d M i k e R e d d y for t h e i r g e n e r o u s supply o f p l a s m i d D N A . This re-
1249
s e a r c h was p a r t i a l l y s u p p o r t e d by N I H g r a n t G M 3 2 5 4 3 a n d N S F g r a n t Dir-8820732 to C. Bust a m a n t e . A d d i t i o n a l s u p p o r t was p r o v i d e d by the M a r k e y F o u n d a t i o n g r a n t to the I n s t i t u t e of M o l e c u l a r Biology at t h e University of O r e g o n .
References [1] D. Keller and C. ChihoChung, Surf. Sci. 268 (1992) 333. [2] G. Binnig, C.F. Quate and C. Gerber, Phys. Rev. Lett. 56 (1986) 930. [3] T.P. Beebe, Jr., T.E. Wilson, D.F. Ogletree, J.E. Katz, R. Balhorn, M.B. Salmeron and W.J. Siekhaus, Science 243 (1989) 370. [4] G. Lee, P.G. Arscott, V.A. Bloomfield and D.F. Evans, Science 244 (1989) 475. [5] D.D. Dunlap and C. Bustamante, Nature 342 (1989) 204. [6] A. Cricenti, S. Selci, A.C. Felici, R. Generosi, E. Gori, W.D. Djaczenko and G. Chiaroni, Science 245 (1989) 1226. [7] R.J. Driscoll, M.G. Youngquist and J.D. Baldeschwieler, Nature 346 (1990) 294. [8] S.M. Lindsay, L.A. Nagahara, T. Thundat, U. Knipping, R.L. Rill, B. Drake, C.B. Prater, A.L. Weisenhorn, S.A.C. Gould and P.K. Hansma, J. Biomol. Struct. Dyn. 7 (1989) 279. [9] A.L. Weisenhorn, H.E. Gaub, H.G. Hansma, R.L. Sinsheimer, G.L. Kelderman and P.K. Hansma, Scanning Microscopy 4 (1990) 511. [10] H.G. Hansma, A.L. Weisenhorn, S.A.C. Gould, R.L. Sinsheimer, H.E. Gaub, G.D. Stucky, C.M. Zaremba and P.K. Hansma, J. Vac. Sci. Technol. B 9 (1991) 1282. [11] J. Sogo, A. Stasiak, W. de Bernardin, R. Losa and T. Koller, in: Electron Microscopy in Molecular Biology, Eds. J. Sommerville and U. Scheer (IRL, Oxford, 1987). [12] B. Drake, C.B. Prater, A.L. Weisenhorn, S.A.C. Gould, T.R. Albrecht, C.F. Quate, D.S. Cannell, H.G. Hansma and P.K. Hansma, Science 243 (1989) 1586. [13] R. Garcia, J. Yiqiu, E. Schabtach and C. Bustamante, Ultramicroscopy 42-44 (1992) 1249. [14] D. Keller, Surf. Sci. 253 (1991) 353. [15] H.J. Mamin, S. Chiang, H. Birk, P.H. Guethner and D. Rugar, J. Vac. Sci. Technol. B 9 (1991) 1398. [16] R.B. Marcus, T.S. Ravi, T. Gmitter, K. Chin, D. Liu, W.J. Orvis, D.R. Ciarlo, J.T. Trujillo and C.E. Hunt, Appl. Phys. Lett. 56 (1990) 236. [17] J.T. Trujillo and C.E. Hunt, Semicond. Sci. Technol. 6 (1991) 223. [18l J.A.N. Zasadzinski, C.A. Helm, M.L. Longo, A.L. Weisenhorn, S.A.C. Gould and P.K. Hansma, Biophys. J. 59 (1991) 755.
Substrate preparation for reliable imaging of DNA molecules with the scanning force microscope J. V e s e n k a a, M. G u t h o l d a n d C. B u s t a m a n t e a,,
a
C.L. T a n g
a,
D. K e l l e r b, E. D e l a i n e c
a Institute of Molecular Biology, University of Oregon, Eugene, OR 97405, USA b Chemistry Department, University of New Mexico, Albuquerque, NM 87131, USA c lnstitut Gustave Roussy, Rue Camille Desmoulins, 94805 Villejuif, France Received 12 August 1991
A simple method of substrate preparation for imaging circular D N A molecules with the scanning force microscope (SFM) is presented. These biomolecules are adsorbed onto mica that has been soaked in m a g n e s i u m acetate, sonicated and glow-discharged. The stylus-sample forces that may be endured before sample damage occurs depends on the ambient relative humidity. Images of circular D N A molecules have been obtained routinely using tips specially modified by an electron beam with a radius of curvature, Rc, of about 10 n m [D. Keller and C. Chih-Chung, Surf. Sci. 268 (1992) 333]. The resolution of these adsorbed biomolecules is determined by the R c. At higher forces individual circular D N A molecules can be manipulated with the SFM stylus. Strategies to develop still sharper probes will be discussed.
1. Introduction
Scanning force microscopy (SFM) images are obtained by recording the deflection of a sharp stylus as it scans the surface of interest [1,2]. The measured deflection is proportional to the force between the surface and the tip. SFM has many distinct advantages over electron microscopies, the most important being the ability to work under physiologically relevant conditions. SFM also has an advantage over its sister microscopy, scanning tunneling microscopy (STM), in that the sample need not be conductive since only contact forces are involved. There are many STM papers reporting the observation of naked D N A on graphite [3-7] but the reproducibility of these results is still unsatisfactory. Lindsay et al. [8] have imaged D N A electrodeposited to gold on mica with the STM and, at lower resolution, DNA adsorbed onto glass with the SFM. The SFM has imaged DNA
* Corresponding author.
adsorbed onto a polymerized monolayer on mica [9,10] although reproducibility was poor. The problem of reproducibility stems from: (1) the requirement of a suitably fiat, strongly adsorbing substrate; (2) the use of reproducibly sharp styli; and (3) the need to develop optimal ambient conditions for imaging. In this paper we discuss a simple substrate preparation method that firmly binds plasmid D N A to mica in air at low humidity. Reproducible images are routinely obtained using styli of small radius of curvature, specially modified for these purposes. The combination of these "microtips" and reliable substrate preparation has allowed us to carry out a statistical analysis and comparison to the known lengths, widths and heights of the imaged circular D N A molecules. Differences in the observed lateral dimensions from those of double-stranded D N A are rationalized in term of the finite radius of curvature, Re, and cantilever length. Furthermore, due to the reproducibility of the imaging, it was possible to locally manipulate individual DNA molecules using these sharp microtips.
0304-3991/92/$05.00 © 1992 - Elsevier Science Publishers B.V. All rights reserved
1244
J. Vesenka et al. / A substrate preparation for reliable imaging of DNA molecules
2. Materials and methods The following treatment of mica is a variation of a method previously used to image D N A in transmission electron microscopy [11]. Red mica (Ashville-Schoonmaker Mica Co., Newport New, VA) is freshly cleaved and briefly sonicated in de-ionized/distilled water (DDW). The mica is then soaked in 33mM magnesium acetate overnight and sonicated again in DDW for 30 min. The magnesium is thought to replace potassium ions in the mica surface providing a stronger binding site with the phosphate groups of the DNA. Sonication of the mica removes excess salts
from the surface, providing a smooth substrate for observing the biomolecules. The mica surface is DC glow discharged for 15 s under vacuum between 100 and 200 mTorr, a process that hardens the substrate. As soon as the substrate is brought up to air, a few microliters of 1 0 0 / z g / m l DNA is deposited onto the surface and allowed to adsorb for 2 min. The sample is then rinsed with DDW to remove excess plasmid and finally blown dry with nitrogen. The specimens were imaged with a Nanoscope II (Digital Instruments Inc., Santa Barbara, CA) scanning force microscope and software. All samples were imaged in air at room temperature in a
Microtip
~
P
Cantilever 4ram
lO0~m
Fig. 1. Schematic diagram of progresswe magnifications of a commercially available s u b s t r a t e / c a n t i l e v e r / t i p assembly. The micrograph is an image of microtip generated by electron beam deposition in a scanning electron microscope (Hitachi S-8000) at the "tip" of the above assembly. The focused electron b e a m will deposit a micrometer length structure, composed of carbonaceous material from residual vacuum chamber oils, onto the surface of the tip within minutes.. The radius of curvature of the above microtip is approximately 10 n m with a cone angle of about 15 °.
J. Vesenka et al. / A substrate preparation for reliable imaging of DNA molecules
1245
c h a m b e r with c o n t r o l l e d relative humidity. T o r e d u c e sample d a m a g e , the s u r f a c e - t i p force was m i n i m i z e d by w i t h d r a w i n g the piezo from the s c a n n i n g stylus while i m a g i n g in the attractive force m o d e . Only m i n i m a l filtering, in the form of removal of low-frequency noise in the slow-scan direction (flattening), was applied to the o b t a i n e d images. T h e lateral d i m e n s i o n s of the piezo transd u c e r were c a l i b r a t e d with a 1000 l i n e s / m m gold grating. T h e m e a s u r e d lengths were f o u n d to be accurate within 1% for a 1 . 5 / z m scan, similar to the d i m e n s i o n s typically s c a n n e d in o u r experiments. I n these studies we used sharp styli modified from c o m m e r c i a l tips u n d e r the b e a m of an electron microscope [1]. V a c u u m - p u m p h y d r o c a r b o n s can be selectively d e p o s i t e d o n the tip of commercially available S F M stylus to o b t a i n sharp " m i c r o t i p s " with R c = 10 n m (fig. 1). T h e s e microtips e n a b l e the systematic a n d r e p r o d u c i b l e imaging of p l a s m i d D N A molecules (figs. 2 a n d 3).
3. Results
Fig. 2. Typical low-magnificationview of the surface of treated mica with adsorbed plasmid DNA at a relative humidity of 25% and scanning force of 3 nN. A plasmid DNA at this humidity can be scanned for over an hour, even at higher magnifications, without distortion due to the microtip-sample contact. A 100 /xg/ml solution of plasmid DNA adsorbed onto the mica surface for a few minutes followed by rinsing and drying will yield a plasmid density of about 1/p.m 2. Notice that the plasmid is clearly distinguished from background features, most likely residual salt deposits, on the mica.
Fig. 3. Two typical plasmid DNA images. (a) An image of a 3.1 kilobasepair (kbp) plasmid DNA molecule taken with 100 /zm length cantilever at a relative humidity (RH) of 45% and force of 5 nN. The higher features near the cross-over point are probably due to supercoiling of the plasmid. (b) An image of a 3.0 kbp plasmid taken with a 200/zm length cantilever at RH = 50% and force of about 3 nN. Both images have spurious 20 nm diameter blotches, presumably due to salt deposits. Notice the different height scales, that is, the plasmid in (a) appears much shorter than that of (b). Details of the topological features are discussed under table 1.
R e d mica substrates deposited with purified circular, or plasmid, D N A molecules yielded occasional salt crystals a n d were essentially featureless except for the plasmids (fig. 2). Figs. 3a a n d 3b are higher m a g n i f i c a t i o n images of observed D N A molecules on t r e a t e d mica
1246
J. Vesenka et al. / A substrate preparation for reliable imaging o f DNA molecules
routinely o b t a i n e d using the microtips. A v e r a g e c o n t o u r lengths, widths a n d heights of different plasmids are p r e s e n t e d in table 1. Short cantilevers (100 /xm) with larger spring c o n s t a n t s ( ~ 0.5 n N / n m ) a n d lower noise (0.2-0.3 n m ) gave g r e a t e r image contrast, b u t had large plasmid widths ( ~ 13 n m ) a n d short plasmid heights ( ~ 0.6 nm). Longer, t h i n n e r cantilevers (200 ~ m ) with smaller spring c o n s t a n t s ( ~ 0.1 n N / n m ) revealed n a r r o w e r widths ( ~ 10 n m ) a n d taller height ( ~ 1.9 n m ) b u t at the expense of r e d u c e d image contrast due to g r e a t e r noise levels (0.5-0.8 nm). Force control was m a i n t a i n e d by withdrawing the tip from the sample in the attractive mode. M i n i m a l o p e r a t i n g forces were typically in the
Table 1 Statistical analysis of DNA plasmids Plasmid DNA
Calculated a) length (nm)
Measured length (nm)
D i f f e r - Relative ence humidity (%) (%)
pDL34 b) pVCB5 c) pSK31 d)
1069 1000 1131
1049 _+52 874 _+54 938_+35
2 13 17
Plasmid DNA
Measured height (nm)
Measured width(nm)
Cantilever length (/~m)
pDL34 b) pVCB5 c) pSK31 d)
0.65_+0.26 1.89 _+0.72 1.43_+0.09
13.1_+4.3 10.3 ± 3.4 12.7_+2.2
100 200 200
45 50 25
a) Assuming 0.34 nm per base pair of plasmid DNA. b~ Average of 7 images. c) With 242 bp clone. Average of 5 images. d) Average of 8 measurements. Two of the plasmid contour lengths were shorter than expected and beyond the range of experimental error (+5%). We are confident a piezo calibration error of the lateral scanning dimensions was not the cause. It is possible that the plasmid DNA molecules folded up, without supercoiling, and could not be detected by the relatively wide microtip. We suspect that low humidity might play a role in shrinking the plasmid contour lengths. The vertical heights, 0.6-1.9 nm, are consistent with the anticipated effect of squeezing the DNA with a scanning stylus. The shorter cantilevers are more rigid, less sensitive to vertical motion and can apply greater force to the sample. Consequently they tend to squeeze the DNA more than the longer cantilevers. The D N A widths, 10-13 nm, are consistent with the R c of the microtip (figs. 2 and 3) and are explained in greater detail in fig. 7.
Fig. 4. Plasmid DNA at high humidity. It depicts a plasmid at a relative humidity (RH) of 65% with a stylus force of about 3 nN. This image was taken during the first scan. Within several consecutive scans this plasmid had been completely removed from the mica surface by the stylus. At higher RH the presence of water degrades the image quality, perhaps by interfering with the plasmid/treated-mica surface bonding or by enhancing the hydrostatic forces between the stylus and sample. We have found that the most stable images are obtained at RH below 40%. range of 3 n N in air and, in relative humidities ( R H ) less t h a n 40%, the plasmid could be s c a n n e d up to an h o u r without observable d e g r a d a t i o n . At R H above 60% only fleeting images of plasmid D N A molecules could be o b t a i n e d before they are swept aside by the stylus (fig. 4). O n u n t r e a t e d surfaces (no m a g n e s i u m ) the D N A are swept aside by the s c a n n i n g tip regardless of the relative humidity. A t the lower h u m i d i t y the N a n o s c o p e II software provides two m e a n s of cutting the D N A with the stylus (figs. 5 a n d 6). O n e m e c h a n i s m involves increasing the force between the tip a n d substrate slowly, with excellent control over the desired cutting point, as in fig. 5. T h e second m e t h o d utilizes a s u d d e n increase of the force while arresting the slow-scan direction, resulting in a sharp, crisp scission of the D N A , as in fig. 6.
4. Discussion O n e of the criticisms of s c a n n i n g p r o b e microscopies is that m a n y of the biological images are
J. Vesenka et al. / A substrate preparation for reliable imaging of DNA molecules
1247
unconvincing, looking like "blobs on a screen". The SFM images of plasmid DNA molecules obtained in this work look similar to electron micrographs of plasmids. Furthermore, we were able to routinely observe the DNA using the above sample preparation method and microtips.
Fig. 5. Images of plasmid a D N A cut with the scanning stylus using slow force increase. (a) A 3.0 kbp plasmid prior to dissection with the SFM stylus. First the stylus is placed near the top center of the plasmid and the sample is slowly raised, increasing the force from 3 to 45 nN, until the above pit is carved (b). Raising the sample a vertical distance, ~z, causes a finite lateral movement of the tip, bx, related by: ~x ~ a~z, where a is the average angle between the cantilever and sample. For a 10 ° angle (0.175 rad) and a 1 ~ m rise in the piezo, the lateral traversal of the tip is about 175 nm, as indicated by arrows in (b). Because the force is slowly increased the outcome of the dissection can be easily predicted.
Fig. 6. Images of plasmid D N A cut with the scanning stylus using sudden force increase. (a) An image of a 3.3 kbp plasmid before it was dissected by disabling the slow-scan direction, causing the sample to be scanned at the same point in the y direction. At a force of 3 nN the "neck" of the structure was imaged (beside arrow near the top), followed by a sudden increase of force by raising the sample. After the higher force (45 nN) was applied for 10 s, the force was reduced to 3 nN and the slow-scan direction was enabled. Notice that the region of the sliced plasmid, indicated by an arrow in (b), was below the neck by about 175 nm, for the same reason as discussed in fig. 5. This scission method provides cleaner cuts, but it is more difficult to control the position of the cutting point.
Although imaged in air, a microscopic layer of moisture must be present, evidenced by the fact that observations of plasmids at humidities higher than 60% are extremely difficult. At high humidity the presence of water degrades the image
1248
J. Vesenka et al. / A substrate preparation for reliable imaging of DNA molecules
quality, perhaps by interfering with the plasmid/ treated-mica surface bonding or by enhancing the hydrostatic forces between the stylus and sample. We have found that the most stable images are obtained at R H below 40%. The presence of a water layer on the mica may play an important role in keeping the plasmid hydrated and stabilizing the conformation of the DNA [12]. The initial identification of the observed features of the plasmids was confirmed by making a statistical analysis of the contour lengths of the observed molecules. This treatment of the data was possible because of the reproducibility in the imaging. Plasmid D N A of varying sizes were measured from SFM images and compared to a length assuming 0.34 nm per base pair (table 1). The pDL34 had a deviation of 2% from the expected value, well within the 5% error associated with these measurements. Two double plasmids of the pDL34 were distinguished and the contour lengths from these images were nearly twice that of single plasmids (within 10%). However, pVCB5 had a 13% deviation and pSK31 had a 17% deviation from their anticipated base pair values. A similar result for pVCB5 was obtained from STM images of coated samples [13]. Because identical results were obtained from different instruments having different piezo elements it is unlikely to be a calibration problem. One possibility is that the plasmid molecules have folded upon themselves, without supercoiling. Since the D N A is much narrower than the microtip, folding of the DNA in this manner would be hidden in the width of the scanning stylus. It is possible that reduced relative humidity might play an important role in causing the DNA to contract. All the plasmids observed in this work were five to seven times broader than expected. The "broadening" phenomena [14] is modeled in fig. 7. The key feature is that the microtips we used have a radius of curvature of around 10 nm, larger than the diameter of the DNA molecule of 2 nm. From simple geometric arguments one can show that (cf. fig. 7) W = 4R~RRRR~,
(1)
where W is the estimated width and R m is the radius of the plasmid DNA molecule. This ex-
Fig. 7. As seen in the plasmid images and tabulated in table 1, the width of the DNA is much greater than 2 nm. This is the result of a finite radius of curvature, Re, of the microtip, as shown schematically in fig. 7. For a microtip of Rc, a linear biomolecule of radius R m where R c >>R__Rmz_the anticipated imaged biomolecule width is W=4~/RmR c. For doublestranded DNA and the microtips used in the work, having Rc = 10 nm, the anticipated width is about 12.6 nm, in excellent agreement with our statistical measurements.
pression is valid for large radius of curvature for which the tip can be approximated by a sphere. For DNA and our microtips we estimate a width of 12.6 nm, in excellent agreement with our data. This result shows the crucial factor determining the lateral dimension, W, and therefore the resolution of the surface features is the dimension of the radius of curvature, R c. The ability of the microscopic stylus to manipulate individual molecules of plasmid DNA can be seen in figs. 5 and 6. We clearly can cut the plasmid at any desired location. In order to cut the DNA at specific sites we intend to examine labelled plasmids in future studies. Another system for future examination is the structure of the RNA p o l y m e r a s e / D N A complex. The key issue to improvement of the image resolution is sharper microtips. A program is underway in our laboratory for developing sharper styli. The limiting feature in electron beam microtip construction is the beam spot size. The tips used in this study were from a Hitachi 8000 scanning electron microscope (SEM) with 5 nm spot size. Field-emission SEM can attain 2-3 nm diameter spot size and scanning transmission electron microscopes have about 0.7-2.0 nm spot size in the secondary emission mode. We are presently using these microscopes at the University of Oregon to manufacture sharper tips. In principle, the STM can also be used to fabricate sharp tips through its nanolithography ability [15].
J. Vesenka et al. / A substrate preparation for reliable imaging of DNA molecules
H o w e v e r , t h e r a t e at which t h e s e tips a r e m a d e is very slow a n d t h e m a t e r i a l s a r e p r e s e n t l y l i m i t e d to m e t a l s t h a t m a y b e t o o soft for o b t a i n i n g s h a r p images. W e a r e also p u r s u i n g silicon m i c r o f a b r i c a t i o n t e c h n i q u e s that, in principle, can a t t a i n R c--- 1 nm [16,17]. T h e r e is a q u e s t i o n t h a t n e e d s addressing as to w h e t h e r s h a r p e r styli will l e a d to g r e a t e r s a m p l e d a m a g e d u e to t h e R~-2 p r e s s u r e increase. H o w e v e r , since t h e m a i n attractive force in air is f r o m surface t e n s i o n t h e force should also d e c r e a s e with t h e c o n t a c t area. Thus, in p r i n c i p l e , s h a r p e r tips s h o u l d result in h i g h e r resolution without increased sample damage.
5. Conclusions W e have p r e s e n t e d a reliable, r e p r o d u c i b l e m e a n s of d e p o s i t i n g a n d i m a g i n g D N A with t h e s c a n n i n g force m i c r o s c o p e . T h e key i n g r e d i e n t s to successful i m a g e f o r m a t i o n are: (1) S u b s t r a t e p r e p a r a t i o n - m a g n e s i u m on t h e surface o f mica s e e m s to assist a n c h o r i n g the p l a s m i d D N A m o l e c u l e s to t h e surface. (2) S h a r p stylus - e l e c t r o n - b e a m - d e p o s i t e d mic r o t i p s p r o v i d e convincing i m a g e s o f t h e deposited biomolecules. (3) R e l a t i v e h u m i d i t y - stable i m a g e s can b e o b t a i n e d with the above two i n g r e d i e n t s so long as the relative h u m i d i t y is b e l o w 40%. W e w e r e able to cut the p l a s m i d D N A m o l e c u l e s with t h e stylus, i n d i c a t i n g the p o t e n t i a l o f t h e S F M as a device for m i c r o - m a n i p u l a t i o n . T h e r e s o l u t i o n o f t h e b i o m o l e c u l e s is a f u n c t i o n o f R~c. W e have p r e s e n t e d high resolution, highly r e p r o d u c i b l e i m a g e s o f b i o m o l e c u l e s . W e do not have to rely u p o n the f o r t u i t o u s use o f a c o m m e r cial stylus having a s h a r p d e f e c t on the tip [18]. G r e a t e r a p p l i c a t i o n of the S F M awaits the develo p m e n t of s h a r p e r styli.
Acknowledgments W e t h a n k W i l l i a m R e e s a n d M i k e R e d d y for t h e i r g e n e r o u s supply o f p l a s m i d D N A . This re-
1249
s e a r c h was p a r t i a l l y s u p p o r t e d by N I H g r a n t G M 3 2 5 4 3 a n d N S F g r a n t Dir-8820732 to C. Bust a m a n t e . A d d i t i o n a l s u p p o r t was p r o v i d e d by the M a r k e y F o u n d a t i o n g r a n t to the I n s t i t u t e of M o l e c u l a r Biology at t h e University of O r e g o n .
References [1] D. Keller and C. ChihoChung, Surf. Sci. 268 (1992) 333. [2] G. Binnig, C.F. Quate and C. Gerber, Phys. Rev. Lett. 56 (1986) 930. [3] T.P. Beebe, Jr., T.E. Wilson, D.F. Ogletree, J.E. Katz, R. Balhorn, M.B. Salmeron and W.J. Siekhaus, Science 243 (1989) 370. [4] G. Lee, P.G. Arscott, V.A. Bloomfield and D.F. Evans, Science 244 (1989) 475. [5] D.D. Dunlap and C. Bustamante, Nature 342 (1989) 204. [6] A. Cricenti, S. Selci, A.C. Felici, R. Generosi, E. Gori, W.D. Djaczenko and G. Chiaroni, Science 245 (1989) 1226. [7] R.J. Driscoll, M.G. Youngquist and J.D. Baldeschwieler, Nature 346 (1990) 294. [8] S.M. Lindsay, L.A. Nagahara, T. Thundat, U. Knipping, R.L. Rill, B. Drake, C.B. Prater, A.L. Weisenhorn, S.A.C. Gould and P.K. Hansma, J. Biomol. Struct. Dyn. 7 (1989) 279. [9] A.L. Weisenhorn, H.E. Gaub, H.G. Hansma, R.L. Sinsheimer, G.L. Kelderman and P.K. Hansma, Scanning Microscopy 4 (1990) 511. [10] H.G. Hansma, A.L. Weisenhorn, S.A.C. Gould, R.L. Sinsheimer, H.E. Gaub, G.D. Stucky, C.M. Zaremba and P.K. Hansma, J. Vac. Sci. Technol. B 9 (1991) 1282. [11] J. Sogo, A. Stasiak, W. de Bernardin, R. Losa and T. Koller, in: Electron Microscopy in Molecular Biology, Eds. J. Sommerville and U. Scheer (IRL, Oxford, 1987). [12] B. Drake, C.B. Prater, A.L. Weisenhorn, S.A.C. Gould, T.R. Albrecht, C.F. Quate, D.S. Cannell, H.G. Hansma and P.K. Hansma, Science 243 (1989) 1586. [13] R. Garcia, J. Yiqiu, E. Schabtach and C. Bustamante, Ultramicroscopy 42-44 (1992) 1249. [14] D. Keller, Surf. Sci. 253 (1991) 353. [15] H.J. Mamin, S. Chiang, H. Birk, P.H. Guethner and D. Rugar, J. Vac. Sci. Technol. B 9 (1991) 1398. [16] R.B. Marcus, T.S. Ravi, T. Gmitter, K. Chin, D. Liu, W.J. Orvis, D.R. Ciarlo, J.T. Trujillo and C.E. Hunt, Appl. Phys. Lett. 56 (1990) 236. [17] J.T. Trujillo and C.E. Hunt, Semicond. Sci. Technol. 6 (1991) 223. [18l J.A.N. Zasadzinski, C.A. Helm, M.L. Longo, A.L. Weisenhorn, S.A.C. Gould and P.K. Hansma, Biophys. J. 59 (1991) 755.
