between the force sensing tip and the substrate and the sensing tip and ... must be true. (15) However, the results of ... the decay length for tunneling (in most cases), the AFM should .... a range which reflects typical experimental conditions. RESULTS ... studied a much larger data set, the following points emerge: Firstly, we ...
Nucleic Acids Research, 1993, Vol. 21, No. 5 1117-1123
Atomic force microscopy of DNA and bacteriophage in air, water and propanol: the role of adhesion forces Y.L.Lyubchenko12, P.l.Oden1, D.Lampner1, S.M.Lindsay1 * and K.A.Dunker3
Departments of 'Physics and 2Microbiology, Arizona State University, Tempe, AZ 85287 and 3Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164, USA Received December 22, 1992; Revised and Accepted January 29, 1993
ABSTRACT We have developed a chemical treatment for the mica surface which allows biopolymers to be held in place for atomic force microscopy, even under water, using conventional, untreated force sensing tips. We illustrate the procedure with images of X DNA and fd phage. The phage adheres well enough to permit in situ imaging of the adsorption process in water. These experiments yield a mean length for the phage of 883 + 72nm. This compares with a measured length of 883-4-33nm when the phage are imaged after drying following adsorption from water, showing that the effect of dehydration is quite small. Adhesion forces between the force sensing tip and the substrate and the sensing tip and the biomolecules are very different in the three media (air, water and propanol). The apparent height of the phage and the width and height of the DNA depends upon these adhesion forces quite strongly. In contrast, changing the Hookean spring force exerted by the scanning tip makes little difference. These results suggest that the chemical factors involved in adhesion can dominate atomic force images and that the composition of the scanning tip is at least as important a factor as its geometry. INTRODUCTION The Atomic Force Microscope (AFM) has been shown to be an important tool for structural biology with the recent demonstration of a number of reliable methods for imaging nucleic acids. (1-10) Early work (11) was limited by motion of the molecules under the scanning tip. A significant development occurred when the ionic treatment, first tried by Weisenhorn et al., (12) was perfected by Vesenka, Bustamante and co-workers. (3,4) In this approach the mica surface is treated with multivalent ions to increase its affinity for DNA which is held in place strongly enough to permit reliable imaging in an AFM. The first images produced by these methods yielded relatively poor resolution and dramatic improvements were obtained by using specially made tips and imaging under propanol. (1,9) Yang et al. have developed a different approach. They use a modification of the *
To whom correspondence should be addressed
well-known Kleinschmidt procedure, spreading the DNA onto a carbon coated mica substrate using cytochrome C that has been denatured at an air-water interface. (6) Rather remarkably, this approach yielded high resolution with conventional (untreated) tips operated in air. We have worked on a third, quite different approach. We use aminopropylytriethoxy silane (APTES) to functionalize the mica surface with amine groups which protonate at neutral pH. (2,7,8,10) We have also used a subsequent treatment with methyl iodide which places methyl groups on the surface. (7,10) We have not used specially formed tips and the resolution in the work we have reported to date has been poor. On the other hand, our method does hold molecules in place for AFM imaging even under water, using conventional tips. (13) The ionic method appears to require the use of specially treated tips in order to function under water (14) while the cytochrome C spreading method (6) does not work under water (Z.Shao, personal communication). Thus, it would appear that the use of a chemically functionalized surface offers some advantages. In particular, it appears to work in a wide variety of ionic conditions and over a wide range of temperatures (10) so that it may be a particularly flexible surface for bonding delicate (i.e. nucleoprotein) complexes to. It is therefore important to understand what factors limit resolution and if they are intrinsic to the APTES treated substrates. It is commonly supposed that the radius of the force sensing tip is the primary factor in determining resolution and, for images of large objects, this must be true. (15) However, the results of Yang et al. (6) demonstrate that the sharp asperities on the surface of otherwise blunt tips can yield very high resolution on small molecules such as DNA. It is such asperities that are responsible for atomic resolution in the scanning tunneling microscope (STM) and, because the range of the contact force is even shorter than the decay length for tunneling (in most cases), the AFM should be capable of even higher resolution than the STM if the sample is both flat enough and completely rigid. (16) However, with the exception of the results obtained by Yang et al., (6) the macroscopically determined radius of the tip appears to be the dominant factor in determining resolution in AFM images so that the best resolution has been obtained with special tips and has shown DNA strands with widths that are as small as 4 to 5nm
1118 Nucleic Acids Research, 1993, Vol. 21, No. S in some places (1) (although Yang and Shao (20) have recently
demonstrated similar resolution with conventional tips). In contrast, the STM produces such resolution rather frequently. (17) We have even been able to resolve individual bases in STM images of polymeric stacked aggregates of purines. (18) The difference between the resolution of the AFM and the STM is an important and, at present, unsolved problem. It has long been clear that forces need to be minimiz for best results with the AFM (4,19) but Yang and Shao (20) have recently made significant progress in quantifying and clarifying the role of forces, and, in so doing, shed some light on the factors that control resolution in the AFM. They have studied many AFM images of DNA made with the cytochrome C spreading procedure and discovered a correlation between the width of the image and the measured adhesion force between the tip and the substrate. They found that an externally applied force does not have the same effect as adhesion forces of the same magnitude. They propose that the adhesion force is generated by contamination which sticks the tip to the substrate and that this contact is essentially rigid (we will refer to it as the adhesion complex). In the simplest case, the total adhesion force (FA )would be just proportional to the cross sectional area of the adhesion complex and the width of the image (W) proportional to the radius of the adhesion complex, so that W=BIFA where B is a constant. A plot of the image width against the adhesion force as measured in many runs exhibits precisely this behavior. (20) Therefore, it appears that contamination plays a major role in limiting resolution in AFM images. In order to obtain high resolution in STM images, we have to use electrochemical procedures to obtain and maintain a clean sample. (17,18) Although we have referred to the material in the gap as contmination, we recognize the possibility the the adhesion complex might be comprised of solvent molecules that are introduced deliberately. We have also investigated the role of forces in AFM images, exploiting the ability of the APTES-mica surface to hold molecules in place in various environments in order to control the adhesion force. We have used fd phage as well as DNA because these bigger particles are somewhat easier to work with and permit probing of adhesion phenomena on a different length scale. We have mesured both the width and height of the images. Our results for the width of the images of DNA molecules are consistent with the findings of Yang and Shao. (20) We also find a strong and unexpected effect on the height of the images. We have measured the effects of changing the Hookean spring force and find iat they cannot account for the height anomaly. Finally, we have found that the silane treatnent allows the adsorption of phage to be examined in situ permitting an investigation of the effects of drying on the images. The same procedure does not work with DNA (at least, using conventional, unitated tips). An intermediate drying stage is required for the images to be stable under water.
MATERIALS AND METHODS Substrates Freshly cleaved slabs of ruby mica (Unimica Inc.) where exposed to an APTES atmosphere by suspending them in a 21 glass dessicator which contained a small pool (301I) of APTES (Aldrich) for 2-3 hours. They where exposed to solutions of the biopolymers (described below) rinsed with deionized water from a Bioresearch Grade NanoPure system (Barnstead Inc.) and blotted at a
corner.
The substrates
were then vacuum
dried at
room temperature in the vacuum produced by a roughing pump (a few ,tm Hg) for periods that varied from 0.5hr. to several days. The length of drying did not appear to affect the AFM
images. DNA Fragments from the Hind III digest of X DNA and whole X DNA (New England Biolabs) were dissolved into Tris-HCl buffer solution (pH7) containing 10mM Tris-HCI, 10mM NaCl and 5mM EDTA. The final DNA concentration was 0. 1lg/ml. APTES treated substrates were left in contact with this solution for one hour. The samples were then dried and rinsed as described above. fd phage Filamentous phage were grown on male E. coli and purified by repeated rounds of polyethylene glycol phase separation followed by banding on KBr gradients as described previously, (21) yielding pure preparations containing 10 (14) particles per ml. Phage were diluted into 10mM Tris-Acetic acid buffer (pH 7.5) containing 20mM NaCl to a final concentration of about 10 (10) particles per ml. Individual phage were immobilized onto APTES-mica as described above. Atomic Force Microscopy We used a commercial instrument (NanoScope II from Digital Instruments Inc. of Sanata Barbara, CA.). The force sensors were Microlevers from Park Scientific Inc. We used the triangular sensors with 13jsm wide legs and an overall length of 100lm. The stated force constant (of 0.21N/m) was checked to within 20% by measuring the deflection of the cantilever when pressed against a quartz fiber of known stiffness. The scanning head was calibrated in the x,y and z directions using polystyrene-latex standads as described elsewhere. (22) There is some uncontrolled variation from run to run which may be associated with hysterysis in the scanners and this introduces a systematic error in
I1
I
ZT
Ls
kH
kA .
-IA S
AZH
'AZ
(A)
-I 2rA
(B)
(C)
Figure 1. Interaction forces in the atomic force microscope: (A) shows a 'force curve' which plots the tip displacement signal (ZT) versus the sample displacement signal (Zs). The adhesion and Hookean forces may be extracted from AZA and AZH as explained in the text. (B): A model for the tip held onto the substrate by an 'adhesion complex'. The attractive interactions (FA) may come from all parts of the complex but only the 'rigid part' (shaded block) contributes to resolution giving a point spread width of 4rA. (C) is a spring model for (B) where kA represents the spring constant of the adhesion complex and kH is the Hookean spring constant of the cantilever. ZT and Z. are the deflection of the tip and sample respectively.
Nucleic Acids Research, 1993, Vol. 21, No. 5 1119 dimensions of up to + 2.5%. Experiments were carried out in the liquid cell supplied by Digital Instruments using air, water (from the Nanopure system) or reagent grade propanol (Fluka). The latter probably contained some contamination which resulted in degradation of the surface after a few hours of scanning. Heights and widths were measured using the software available on a NanoScope Im workstation. At least 20 molecules were measured at 5 points along their contours for each datum reported here. The results were transferred to KaleidaGraph for analysis. For contour length measurements, TIFF files were transferred to a Macintosh where the public domain software NIH Image 1.40 was used to measure and store data which was transferred to KaleidaGraph for final analysis. Force measurements were made from the force display on the Nanoscope. This is not a force versus distance curve, but a plot of the voltage applied to the sample height control (related to sample position, Zs, via the known height calibration) versus the deflection signal (related to the force via tip deflection, ZT, and the spring constant of the cantilever, kH). A schematic of this display is shown in Figure 1A. When the sample is far from the tip, the deflection is zero (ine connecting 1 and 2) until the tip contacts at 2 (it may jump into contact-not shown- because of attractive forces between the tip and substrate). The tip is then pushed up by the sample (2 to 3). On retracting the sample, the tip usually remains in contact (3-4-5) beyond the initial point of contact (2) because of adhesion between the tip and substrate. With the instrument set to maintain deflection at ZS, the Hookean spring force applied to the sample, FH, is kHAZH and the adhesion force, FA, is kHAZA. Adhesion can vary considerably because it is a function of both the uncontrolled tip geometry and of contamination. We quote a median value and a range which reflects typical experimental conditions.
RESULTS AND DISCUSSION DNA and phage images in air, water and propanol Representative images are shown in Figure 2. Figures 2A, B and C show fragments of X DNA in air (A), under water (B) and under propanol (C). We have made studies of this material in air (2,7,10) and under water (13) and the results we show here are similar to those reported earlier. However, now that we have studied a much larger data set, the following points emerge: Firstly, we have found imaging under water to be quite difficult. In some runs the DNA sticks well and in others it does not. We find that the DNA cannot be imaged unless it is dried following the adsorption process. Radio-label assays show that the DNA does stick to the substrate and survives extensive rinsing (10) so it must be that the adhesion is inadequate to withstand the interaction with the scanning probe. This conclusion was confirmed by imaging the same substrates in air after drying. The were found to be covered with DNA molecules at the density anticipated from the radio-label asay, even though molecules were not found during the imaging under water. In principle, drying the sample should worsen the adhesion since one might expect the amine groups to de-protonate. However, the methylated surfaces (which should be permanently 'sticky') produce similar results. Thus, it appears that the tethering of the DNA occurs during the drying process itself. A similar conclusion has been reached by Hansma et al. (14) who studied DNA held onto the surface after an ionic treatment. We have suggested elsewhere (23) that drying might work by embedding the molecule in a layer of tightly packed small ions. Some evidence for such an embedding layer is discussed by Vesenka et al. (30) The larger phage particles adhere more strongly (see below) so the difficulty with DNA may reflect an inadequate number of attachment points (in the absence of drying).
Figure 2. Images of DNA on APTES-mica in air (A), water (B) and propanol (C). The scale bar in (A) applies to B and C also. Images of fd phage are shown in air (D), water (E) and under propanol (F). The scale bar in D applies to E and F also.
1120 Nucleic Acids Research, 1993, Vol. 21, No.
5
Figure 3. High magnification mage of region pointed to by an arrow in Fig. 2C. The arrow heads indicate a feature of 4nm full width. (The faint vertical stripes are caused by mechanical noise).
The second point concerns the dimensions of the images, particularly the apparent height of the molecule. We summarze the results of many experiments in Table I. The large standard deviations reflect the enormofis variations in the data, even in a given run. Even here, we have selected cross sections in as much as we have avoided taking them across points on the molecule that appear to be obviously contminated (e.g., showing a bright spot on an otherwise fairly uniform image). A more serious problem, however, lies with the artifacts produced by buckling of the cantilever as the tip sticks to a molecule. This can even result in negative contrast (24) (the DNA appears to lie below the substrate). We observe similar effects to those reported by Thundat et al. (24) They may be minimized by orienting the scan so that buckling produces the smallest effect on the deflection signal. Nonetheless, the height data will still be incorrect. The buckling produces an enhanced contrast in one direction and a reduced contrast in the opposite direction (the effect is the basis of the lateral force microscope (25)). We have chosen the scan direction that gives the maximum contrast with minimum overall distortion (that is the tallest trace from a pair selected to have the least distortion). Thus, the reported heights represent an upper limit to the true sample deflection. Even so, the heights (particularly in air) are much too small. Indeed, as the distribution suggests, we fairly often find images in which the height of the DNA image is less than 1A. Note that the height increases quite dramatically (five-fold) as the adhesion force is reduced. We will discuss this phenomenon further in a later section. Vesenka et al. (30) have noted similar anomalies in images of DNA and of small gold particles. There is some indication that the image width narrows when the molecule is imaged under water or propanol, where the adhesion forces are smaller, consistent with the findings of Yang and Shao. (20) Under water or propanol, the images can become very narrow indeed in places. Figure 3 shows a high magnification scan over the area pointed to by an arrow in Figure 2C. The vertical stripes are caused by mechanical noise, but the fine structure along the backbone of the molecule is reproducible from scan to scan. The feature marked by arrowheads has a full width of 4nm. This demonstrates that, while not common (this width is 7 standard deviations from the mean) high resolution is at leastpossible using conventional tips on APTES-mica substrates. fd phage images are shown in Figure 2D (air), 2E (water) and 2F (propanol). They are all quite broad and a survey of a number of images (Table I) shows that the image width does not appear
Figure 4. Adsorption of fd phage from Tris-aceate buffer in situ. A is a control experiment showing the surface under buffer alone. Buffer containing 1010 particles per ml is injected into the AFM cell at t=0. B, C and D are scans over the substrate at 6, 15 and 40 minutes after injection of the phage. The histogram inset in A shows a distribution of fragment lengths measured after 12 minutes. The distribution covers 200 to 1200 nm in bins of 200m width. The scale bar on A applies to B, C and D also. Table I. Adhesion force for tip-substrate interactions in air, water and propanol together with widths and heights for images of DNA and phage. Air
Water
Propanol
Median FA (nN) Range (nN)
15 10-25
3 2-5
0 0-1
DNA Width (nm) Height (nm)
60.4k 15
36.7k 19 0.51 0.2
35.5X4.4 0.67 0.13
PHAGE Width (nm) Height (nm)
45.5 5.3
62.6X10 0.68w0.24
53X17 0.59 0.2
0.13 0.06
0.3X0.08
Widths are full widths where the baseline is defined as the point where the trace becomes indistinguishable from the background noise. Forces are quoted as the median value with an accompanying range. Other data are shown as mean with standard deviation.
to change as the solvent is changed. The difference between these images and the DNA images probably lies in the size distribution of the asperities on the tip. These larger samples probably sense the macroscopic radius of curvature of the tip. We have determined this to be typically 20nm (from images of whisker crystals (25)) so that the corresponding full width of the image width would be about 80n, close to what is observed in all conditions (Table I). In the case of fd phage, the height anomaly is even more pronounced. The diameter of the phage is known to vary from about 8.5nm (wet) to about 5.5nm (dry) as
Nucleic Acids Research, 1993, Vol. 21, No. 5 1121 and then imaged under water. The active role of the APTES treated surface was confirmed by repeating the experiment with an untreated mica surface. No adsorption of phage was observed. This experiment permits a direct check of the effects of drying on the images. The contour length of phage measured in air is 883 A 33nm, which is in coincidentally close agreement with the values of 880 + 30nm determined by standard electron microscopy (27) and 883 : 24nm (28) determined by scanning transmission electron microscopy. (29) Images obtained under water show a wide distribution of lengths. The histogram inset in Figure 4A shows data for phage under water after 12 minutes of adsorption. It covers lengths from 200 to 1200nm in bins of 200nm (the highest bin contains 35 samples) and the data yield a length of 658 186nm. Comparison of Fig. 4 and Fig.2 shows that the discrepancy is a consequence of the presence of many short fragments on the surface when imaging is carried out in situ. This is presumably a consequence of the presence of many particles which are only partially attached. However, the distribution has only one particle that appears to exceed 1000nm in length, suggesting that the length of intact particles does not differ much in the two cases. If we select only those molecules that appear to be intact (by the somewhat subjective criterion of choosing images with well defined 'ends') we obtain a length of 883 i 72nm where the data set spans lengths from 831 to 946nm. This indicates that any shrinking upon drying is likely to less than 10%, less than, for example, the difference between A and B-form DNA. The less than 10% shrinkage during drying is supported by earlier fiber x-ray diffraction studies which show that a strong meridional reflection, which should scale with the overall length of the phage, decreased by only 4% as the fd phage were dried (e.g., from about 1.62nm at 100% relative humidity to about 1.55nm at 0% relative humidity). (26) -
Figure 5. Images of X DNA fragments obtained on APTES-mica under propanol. The adhesion forces are approximately zero over the mica and about 2nN over the DNA. The Hookean force was varied from InN (A) to 8.1nN (D).
determined from the lattice spacings in x-ray diffraction from fd fibers as a function of relative humidity (26) and yet the observed heights are less than about one tenth of these values in all circumstances (Table I). In the case of DNA, one plausible explanation of the height anomaly is that the molecule is 'buried' in the dried salt layer that embeds it (see above). This is implausible for the phage. We have measured the dimensions of phage in water immediately following adsorption (see below) and we obtain an average height of 0.49 0.2nm, consistent with the results obtained from samples that were first dried and then imaged in water (Table I). The phage adsorbed and imaged in situ are unlikely to be buried in an embedding layer that is similar to that formed on drying and (coincidentally) almost equal to the phage diameter. Once again, the height increases as adhesion is reduced, although the effect (a two-fold increase) is less dramatic than that observed with DNA. -
In situ imaging of phage adsorption Real-time attachment of the phage to the substrate was observed by injecting a solution of phage particles into the AFM liquid cell and making periodic scans of the surface at intervals thereafter. Some results are shown in Figure 4. Fig. 4A shows a control experiment taken under buffer alone. At zero time, a solution containing 10 (10) phage particle per ml was injected and particles can be seen on the surface in a scan taken after 6 minutes (4B). Scans taken at 15 minutes (4C) and 40 minutes (4D) show an increasing density of particles on the surface. The images are a little streaky, suggesting that the attachment is not as good as for particles that have undergone a drying step. Analysis of an image obtained after 12 minutes yielded full widths of 106.5=37nm and heights 0.49=0.2nm, compared with 62.6 lOnm and 0.68 0.24nm for samples that had been dried
The effects of force on images While the adhesion forces can be only rather crudely controlled through the use of different solvents, the Hookean spring force (kHZT in Fig. 1) can be controlled over a wide range. We took images of both X DNA and phage under propanol over a wide range of Hookean forces (encompassing the range of adhesion forces encountered in air, water and propanol). We found that the adhesion force was quite repeatably too small to be measured for the commercial tips over APTES mica in propanol. Significant forces were encountered over attached particles. These were rather variable and were characterized by measuring adhesion over a dense aggregate patch. For DNA we obtained 2.8 i 2nN with a similar value (and wide range) for phage. Images were taken by first minimizing the Hookean force over the APTESmica (operating near a total vertical force of OnN) and then obtaining a series of images at increasing values of Hookean force. A series of images obtained over X DNA is shown in Figure 5 for FH between 1 and 8. InN. The width and height do not appear to vary significantly but there is a noticeable 'smoothing' of the background (which might reflect the removal of small particles at higher forces). Data for the width and height of images obtained from many such experiments are shown in Figure 6A (phage) and 6B (DNA). The lines are regression fits to the data points. The large standard deviations on each data point preclude a definite result but there is some indication of a trend to smaller heights and increased widths at increased forces in both cases. The most common explanation offered for these anomalous heights is that the sample is being compressed. Associating a
1122 Nucleic Acids Research, 1993, Vol. 21, No. 5 1.2
140 120
lr
1
100 80 60 40 20
LL.O
A
I
0.8 fi
0.6
__
O.4 3
0.2
T
r
A
d
lo 1
2b 215
A
3
_V
Hookean Force (nN)
(A) 1.2
E 0.8 0.6
%-
~0-
0.4 0.2
(26) This apparent loss of volume is even more ridiculous when one considers the likely dominant role of the tip radius in broadening the image. A proper understanding of these effects requires a detailed model for what we have called the 'adhesion complex'. The work of Yang and Shao (20) indicates that the 'rigid' part of this complex (represented by the shaded block in Fig. iB) extends laterally for distances on the order of tens of nm. The anomalous values for both the phage and DNA heights indicate that its vertical extent may be several nm, even in propanol. If this is indeed the case, then image contrast could be difficult to interpret when there is adhesion between the tip and the substrate and/or the molecule being imaged because it depends upon the structural details of the changes in the adhesion complex as it is swept over the molecule. These effects may be minimized by using a tip that sticks to neither the substrate nor the molecule being imaged. In the case of DNA on a hydrophilic substrate, a hydrophobic tip would minimize these effects. The commercial Si3N4 tips contain SiO groups on their surface which will make them hydrophilic. Hansma et al. (14) report that a coating of carbon improves them considerably. Yang and Shao (20) report that touching the commercial tips to carbon coated substrate results in substantial improvement in resolution.
0
10 15 Hookean Force (nN) 0
5
(B) Figure 6. Data for the width (squares) and height (circles) of fd-13 phage (A) and X DNA as a function of Hookean force. The error bars are the standard deviation of the data from which the mean for each point was calculated. The solid lines are linear regression fits.
spring constant, kA, with the adhesion complex (see FigurelB; kA includes the effects of the sample embedded within the complex) permits this effect to be quantified. The gradients of the regression fits yield kA=58N/m (r=0.7) for DNA and kA= 120N/m (r=0.5) for phage. Both these values greatly exceed the spring constant of the cantilever (0.2 lN/m). Modeling the AFM tip-substrate complex (Fig. 1B) by the series spring model shown in Fig. 1C indicates that the effect of the compressibility of the material that constitutes the adhesion complex on the difference between ZT and Zs is negligible. Thus, direct compression as a consequence of the adhesive force cannot account for the overall discrepancy in height. Furthermore, other groups (1,4,14) who have obtained images at similar Hookean forces but with a tip of a different chemical composition do not observe such height anomalies. Another possibility lies in plastic or non-linear elastic deformation of the sample. The measured heights could reflect a large initial deformation on first contact of the tip and sample. However, this appears to be unlikely, based on the molecular volume of the images. If we model the molecule as having an elliptical cross section and make the extreme assumption that all of the image width is attributable to a 'flattened' molecule (rather than the finite radius of the tip) then the cross sectional area of the phage images in air is_ 10-17m (2) compared with 3x1O-17m (2) determined by x-ray fiber diffraction patterns.
CONCLUSIONS We have shown that adhesion plays a significant role in the contrast of images of DNA and fd phage, and, to some extent, in the resolution of images of DNA (the effect on resolution is better documented by Yang and Shao (20)). The effects appear to be chemical, reflecting the difference in the manner in which the tip sticks to the substrate compared with the manner in which it sticks to the molecule being imaged. While changes in the Hookean force with which images are taken has some effect on contrast, it is small compared with the effects associated with adhesion. These conclusions are consistent with the recent findings by Yang and Shao (20) that adhesion dominates the resolution in images of DNA made with conventional, untreated AFM force sensing tips. They also offer some insight into the mechanism whereby carbon-coated tips yield improved resolution. These findings suggest that the low resolution obtained on APTES-mica substrates to date is not an intrinsic failure of the APTES-mica, but rather the consequence of the use of a hydrophilic tip on a hydrophilic surface. The proper choice of tip material and geometry should permit high resolution images to be obtained on this surface also. This is important, because, as we have shown, this chemically activated surface permits the study of certain nucleo-protein complexes to be carried out in situ. We have exploited this capability by carrying out the first direct test of the effects of drying on measurements of the length of images of fd phage.
ACKNOWLEDGMENTS We are grateful to Zhifeng Shao and Jie Yang for many interesting discussions and for showing us reference 20 prior to its submission. Helen Hansma shared reference 14 with us prior to publication. We have benefited from useful discussions with Lyuda Shlyakhtenko and Rodney Harrington. James Vesenka provided useful comments on the manuscript. Rodney Harrington also provided support to one of us (YL) during periods spent
Nucleic Acids Research, 1993, Vol. 21, No. 5 1123 at the University of Nevada, Reno. The work was supported by grants DIR8920053 from the NSF and N0001491J1455 from the ONR. Y.L. is on leave from the Institute of Molecular Genetics of the Russian Academy of Sciences.
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