1
Effects of Relative Humidity and Applied Force on Atomic Force
Microscopy Images of the Filamentous Phage fd
Xiaolong Ji§, Jeong Oh, A. Keith Dunker, and K. W. Hipps*
Departments of Chemistry and Biochemistry
Washington State University, Pullman, WA 99164-4630
IN PRESS: Ultramicroscopy
*Corresponding author: K. W. Hipps, Department of Chemistry, Washington State
University, Pullman, WA 99164-4630 (hipps@wsu.edu)
§Permanent Address: Department of Pathology, People's Liberation Army Hospital,
Beijing, China
ABSTRACT
The filamentous phage fd was studied by both contact and tapping mode atomic
force microscopy under conditions of controlled variations in relative humidity and
changes in the applied tip force. By spin-coating freshly cleaved mica with phage
containing solutions having very low salt content followed by rapid humidity control,
stable and reliable sample preparation was achieved. The apparent height of the phage
varied by about 10-fold with a quadratic dependence on the stabilized relative humidity,
extrapolating to 73% of the accepted x-ray diffraction-based height at 0% relative
humidity. The variation in measured height with relative humidity largely reconciles
previous widely varying atomic force microscopy estimates of this dimension for the
filamentous phage. Our finding that contact mode images of phage are more difficult to
analyze than those acquired in tapping mode are consistent with previously published
results on other biological specimens such as DNA.
INTRODUCTION
Bacteriophage fd is a class I filamentous phage of the Ff family; two other
members of this family are M13 and f1 [1]. Each of these phage contain a circular,
single-strand DNA molecule enclosed in a cylindrical protein sheath constructed of 5
types of proteins [2], with many copies of a highly elongated major coat protein, which
are regularly arranged along the length of the filament [2,3] and with a few copies each
of four different minor coat proteins, two sets of which cap each end [4].
The
asymmetric major coat proteins, also called pVIII because they are encoded by gene 8,
have their long axes nearly parallel to the filament axis [1,5]. The many copies of the
pVIII molecules overlap along the length of the phage like scales on a fish.
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The pVIII subunits constitute about 87% of the total virion mass. The pVIII
sequence is identical in fd and f1 virions and differs by only a single amino acid residue
from that of M13: the carboxyl group of the aspartate at position 12 in the fd and f1 pVIII
protein is replaced by the amide group of the asparagine in M13.
There has been a large body of structural work on the Ff family, including fiber Xray and neutron diffraction [6,7], solid-state NMR spectroscopy [8,9], solution Raman
spectroscopy [10], solution CD spectroscopy [11,12,13], and polarized Raman
spectroscopy on fibers [14]. The results from these studies show that the asymmetric
pVIII subunits are almost entirely α-helical and layered about the filament axis with both
5-fold rotational symmetry and an approximate 2-fold screw axis. Adjacent groups of 5
pVIII molecules are shifted relative to each other by 1.6 nm along the filament axis and
rotated by 33.23° [15] (note that, for a true 2-fold screw with 5 fold symmetry, the twist is
180/5 = 36° which is near the observed value of 33.23). Repeated application of the 5fold and approximate 2-fold screw symmetry operations accounts for the structure of the
phage capsid.
The fd filament is structurally stable, withstanding prolonged incubation at 75 °C
[16] and not exhibiting significant loss in infectivity until about 90 °C [17].
Furthermore, the phage is resistant to exposure to various detergents, high salt
concentrations, 8M urea [18], or various pH values over the range 2 to 11.5 [1,19,20].
Overall, the filamentous phage is one of the more stable nucleo-protein assemblies.
Given the large amounts of material that derive from the ease of growth and
purification of the fd phage and given the exceptional stability of this phage, it has been
possible to achieve remarkable consistency in the various physical and chemical
measurements on this phage. For example, the length of the fd phage has been measured
by three methods: ordinary transmission electron microscopy gave 880 ± 30 nm [21];
scanning transmission electron microscopy gave 883 ± 24 [22,23], and atomic force
microscopy (AFM) of particles in air gave 883 ± 33 nm [24].
A further indication of the consistency of measurements on fd phage is provided
by the observation that the number of pVIII molecules per phage estimated from the
measured lengths coupled with the known phage symmetry is very similar to the number
estimated from physico-chemical solution measurements. That is, from the value of the
length, L, of the phage and the symmetry of the particle, the number, N, of pVIII
molecules/phage is given approximately by N = 0.98 [(L - l')/(1.6)] x 5, where the factor
0.98 is introduced to roughly correct for the minor proteins using their overall mass
contributions, where l' is the projection of the length of one coat protein molecule along
the fiber axis, and where 1.6 and 5 come of course from the symmetry of the particle.
With L = 881± 29 nm obtained by averaging the length estimates given above and l' = 7.0
nm obtained by assuming a 50 amino acid helix inclined by 20° from the filament axis,
then N = 0.98 [(881 ± 29 - 7)/(1.6)] x 5 = 2,680 ± 90; this value is essentially
3
indistinguishable from 2,700 ± 200 pVIII molecules/phage determined from physicochemical measurements [11].
The length measurements by electron microscopy were carried out in vacuum and
the AFM measurements were done in air, whereas the physico-chemical measurements
were carried out in solution. The agreement in the estimates of the number of coat
protein molecules per particle between the methods suggest that the phage length changes
very little upon drying. This is consistent with a relatively small change in the meridional
diffraction spacing as a function of relative humidity (RH) [25] and has been further
supported by comparison of lengths of individual particles in air and under water by
AFM; as noted above, the former gave length estimates of 883 ± 33 nm in air, which was
indistinguishable from the value of 883 ± 72 nm obtained under water [24].
In contrast to the highly consistent results described above, two AFM
measurements of the heights of filamentous phages gave dramatically different values. A
height of 5.4 nm [24] was reported from AFM measurements on M13 phage, whereas a
more than 10-fold smaller value, about 0.5 nm, was reported for fd phage [26]. The
close structural similarity of fd and M13 phage is not consistent with this large disparity
in measured heights. Indeed, the diameter of the fd filament determined from the lattice
spacing in x-ray diffraction experiments on exhaustively dried fibers was 5.5 nm [27];
this value for fd is close to the AFM-measured height for M13. Furthermore, x-ray
diffraction comparisons of fibers of fd and M13 phages yield essentially indistinguishable
diameters for the two phages [4,7]. Thus, the widely differing values for the AFMdetermined heights of the fd and M13 phages represent a substantial disparity.
The purpose of the present work is three-fold: 1. development of sample
preparation techniques that yield isolated and uncontaminated phage; 2. clarification of
factors affecting the measured heights of the filamentous phage; and 3. evaluation of the
reliability of contact versus tapping mode AFM studies of filamentous virus particles.
The results we obtained largely explain the previous differences in measured heights and
suggest that the tapping mode is more reliable than the contact mode for studying the
structure of filamentous phage.
EXPERIMENTAL
Phage and bacterial strains: The fd phage was originally obtained from Don
Marvin, who carried out the original isolation of the fd phage [28]. DNA sequencing of
the pVIII gene confirms that the wild-type phage has been maintained over 20 years
without mutation, at least with regard to the major coat protein. This fd phage was grown
on Escherichia coli MV1190, obtained from cloning kits supplied by Biorad.
Filamentous phage growth and purification: Methods for growth and purification
of phage followed standard procedures, which included centrifugation to remove the host
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cells, 5% polyethylene glycol precipitation from 0.1M NaCl, differential centrifugation,
and equilibrium centrifugation on KBr density gradients. Bands from the density
gradients corresponding to the fd phage were then dialyzed at 4°C into a mixture 0.015 M
boric acid and 0.1M NaCl solution for 2 to 3 days to remove KBr. The resulting phage
was judged to be essentially free of contaminating protein or nucleic acid from the
absorbance ratio at 268 and 243 nm (A268/A243) of 1.33. The purified phage was
stored in a mixture of 0.015 M boric acid and 0.1 M NaCl at pH 8.2 at 4 °C.
AFM sample preparation: In order to reduce the chance of salt crystal formation
upon drying, samples of stored fd phage solutions were diluted about 1000-fold
immediately before use into low concentrations of a ammonium bicarbonate, which is
volatile. Small aliquots of these diluted phage samples, about 10 µL containing 0.001M
NH4HCO3 and 1012 fd phage/mL, were placed on the 1 cm2 surface of a freshly cleaved
piece of mica. The mica substrate had previously been attached to a metal sample puck
and placed on a holder mounted on the shaft of a Dremel Moto-tool. Through variac
control (typically 40 volts) of the Dremel tool, the rotational velocity of the sample (set
off-center, about 2.5 cm from the axis of rotation) was adjusted to rapidly remove the
solvent layer while allowing the fd phage to remain on the surface (about 10,000 rpm).
This speed was found by trial and error. Faster speeds caused the phage to be thrown
from the mica surface; while slower speeds left excess solution that dried to produce
relatively large salt deposits despite our efforts to reduce the salt content in solution.
Thus, by controlling the spin rate and buffer concentration, one can control both the
number of phage per unit area and the relative cleanliness of the sample. Once a sample
had been spun dry, it was immediately placed in a closed container over a given solution
of a salt.
Relative humidity control and measurement: Placement of the sample in the
atmosphere above a salt solution yields a stable relative humidity (RH). Samples were
maintained in such fixed RH environments overnight (e.g. for at least 12 hours), both for
convenience and to assure equilibration. Use of appropriately chosen salt solutions for
different samples gave fixed RH values that spanned the range from 10% to 90% [29].
Samples were not removed from these containers until they were mounted in the AFM.
Humidity control within the AFM was accomplished through the use of a
commercial insert, the "HumPlug" sold by BioForce Laboratory Inc. (Santa Barbara, CA)
and a constant flow of nitrogen gas that had been bubbled through the same solution of
salt [29] used during the equilibration step. The RH just above the sample was measured
by the sensor included with the HumPlug and those values are reported here.
We note that the HumPlug sensor values, the values determined with other
sensors available to us, and also the tabulated values for the equilibrated atmospheres
above the various salt solutions were all in generally good agreement ( ± 5%).
AFM instrumentation: The AFM used was a Digital Instruments, DI, Nanoscope
III with multimode head. Both contact (using Digital Instruments Si3N4 tips) and tapping
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mode (using DI silicon tips) were employed in this study. In contact mode, only the
longest (200 µm) and thinnest cantilevers were used. According to DI literature, this
cantilever has a nominal Hook's law force constant of 0.06 N/m and the tip diameter
ranges from 10 to 40 nm. The tapping mode tips had a cantilever length of 127 µm and
were specified as having a tip diameter in the range from 5 to 10 nm and a force constant
of the order of 50 N/m. The standard DI software was used for image acquisition and for
capturing force calibration curves.
RESULTS AND DISCUSSION
Reproducible sample preparation: One of our earliest concerns centered on the
difficulty of reproducing phage images, especially in the context of variable features that
were not due to phage itself. These latter features, it turned out, could be traced back to
the buffer and very low level impurities in the water used to make the phage suspension.
Dramatic changes in AFM images due to tip force and scanning time variation have
already been documented by others [30,31,32]. These changes were produced by
salt/impurity layers forming on the mica surface when buffer solutions of 10 mM to 100
mM were used. While our buffer concentration was deliberately reduced to 1 mM in an
attempt to avoid this, simple solvent evaporation still results in significant residue.
Figure 1. Typical contact mode images of a portion of two phage taken in Trace and in Retrace modes at
20% relative humidity. The significant difference here is that in one direction the tip is being pushed along
the surface while in the opposite direction it is being pulled along. The difference in contrast is a result of
changes in cantilever bend as a function of surface friction.
In order to further reduce salts and impurities, we employed a spin coating
technique long used in inelastic electron tunneling spectroscopy [33]. This procedure is
described in the Experimental Section. It was found that, with proper application of the
spinning method, and by maintaining total salt and impurity concentrations of the order of
1 mM, mica surfaces having an occasional phage and a few very small salt islands could
6
be formed reproducibly. Moreover, similar small salt islands were present on mica alone
at higher humidity, indicating that they are a result of leaching and/or reactions with
atmospheric carbonate. Figures 1 and 2 are representative of the sample cleanliness that
can be obtained by these procedures in contact and in tapping mode.
Figure 2. Typical Tapping mode images taken in Trace and in Retrace mode. Because the tip is only
touching the surface for a very small part of the total scan time, frictional differences play a very much
smaller role in the contrast obtained.
We also note that we took great pains to use mica that had been cleaved only
seconds before the spin doping occurred. As is well known, freshly cleaved mica is very
hydroscopic but mica exposed to atmosphere for a short time has a finite contact angle (7
to 15°) [34,35,36]. This rapid increase in contact angle is also accompanied by a large
change in surface free energy [34,36] clearly indicating that atmospheric adsorbates have
a marked effect on the mica surface. We only used samples where the solution had
spread freely upon the surface.
Comparison of images obtained by contact and tapping modes: Figure 1 shows
the effect that scan direction has on contact mode images of phage on mica obtained in a
20% relative humidity, RH, environment. As Thundat has proposed and demonstrated,
this scan direction dependence of the apparent height in contact mode is due to changing
flexion, or buckling, of the cantilever as a function of the relative friction between the
sample surface and the tip [32]. The resulting changes in the curvature of the cantilever
are incorrectly reported as changes in height by the AFM. In the remaining contact mode
work reported here, the scan direction was set to give a maximum apparent phage height.
In tapping mode, where the tip only touches the surface for a very small fraction of the
scan time, this effect should be much less significant. As can be seen in Figure 2, this is
in fact the case. The sample in Figure 2 is the same 20% RH phage sample as studied by
contact mode in Figure 1. The phage height in the taping mode image (as determined
7
from scope mode images of trace and retrace), however, is higher and the contrast is not
scan direction dependent.
Effects of tip force on images of fd phage: The apparent height difference
obtained for the same sample in contact mode relative to tapping mode, suggests that the
virus may be partially compressed by the vertical force of the tip. To address this issue,
and to study the relative toughness of the virus to compression as a function of RH, a
series of AFM images were acquired at progressively greater force (set point), starting
with the minimum force necessary to acquire an image reliably. At the end of the series,
the force was reduced to the initial value and the AFM image re-scanned.
In every case, an internal control was used. That is, the initial scan area included
two or three phage, the image region was zoomed to include a single phage and then
images of that phage were acquired at progressively higher set point. At some point,
usually after significant reduction in measured height, the set point was reduced to its
original value and the larger area containing several phage was again scanned.
Figure 3. Before and after AFM images taken at 15% RH. In the initial (A) scan, the applied (Hookean)
tip force was <0.8 nN. Image B was taken (at <0.8 nN) after the phage on the upper right had been scanned
with an applied (Hookean) tip force of about 36 nN. The adhesive force was 25 nN±5nN.
Figure 3 is representative of the results of the experiments described above.
Using the manufacturers published values for the cantilever force constant and the
measured set point-sample displacement curve on a phage free part of the sample, tip
forces in nN can be computed. The reported forces are all Hookian forces. That is, no
correction for the adhesive contribution was made. However, the adhesive force
determined from the Setpoint-distance curve was about 25 nN±5nN for data in the 15% to
60% RH range. In trace A of Figure 3, the Hookean force used was less than 0.8 nN. A
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smaller scan region enclosing only the phage particle in the upper right was then initiated.
For this sample, images were taken at progressively greater force up to a maximum of 36
nN. The force was then reduced to the original (<0.8 nN) value and the original area was
scanned to produce image 3B. Comparison of these images shows that the phage that
experienced the high force scan has been dramatically reduced in height.
Figure 4. Apparent height of phage as a function of maximum applied (Hookean) tip force. Results
obtained from samples prepared and measured at 15% RH and at 60% RH are shown. The adhesive force
was 25 nN±5nN.
This reduction in height does not occur uniformly with increasing tip force.
Figure 4 provides the evolution in measured height with Hookean force observed from
samples prepared and measured at 15% and 60% relative humidity. The height values are
averages taken from at least 5 different cross sectional measurements taken from several
phage. At both low and high humidity it appears that there is an initial small compression
with applied force followed by a plateau. Eventually, this plateau gives way to a rather
sharp drop in measured height with applied force. This last drop in height is irreversible
(as seen in Figure 3), with the structure of the virus suffering permanent change.
The measured width of the phage also changes with humidity, but this effect is
masked by tip-surface interactions and tip width effects that result in measured widths
that are about 10 times too large at low tip force. This kind of broadening is similar to
that observed by Thundat et al [32] for DNA, by Lyubchenko et al for phage and for
DNA [26], and by Yang et al for DNA [37]. There are at least two sources for this
broadening -- convolution of finite tip width into the image and the size of the water
meniscus carried by the tip because of capillary forces. The Si3N4 contact tips used in
this study are known to be from 10 to 40 nm in diameter, and the existence of a 'neck' of
water about the tip contact with the surface is also well studied [32,37,38,39]. Thus, the
width of fd-phage determined in the same experiments as used to define Figure 4 is about
50 nm for forces up to about 35 nN where the width jumps to about 100 nm. While a
width of 50 nm might be explained by the broadening effects discussed above, the jump
9
to 100 nm at 15% RH cannot. Thus, the relatively dry phage appears to be irreversibly
crushed by high tip forces.
One can convert the tip force to pressure by dividing the applied force by contact
area. Assuming that the full tip width bears on a long rectangular section, with the long
direction defined by the tip diameter (ó40 nm) and the short direction defined by the true
phage width (ò5.5 nm [25] ), then the bearing surface is roughly 300nm2. At 36 nN
force, the pressure is 1.0x108 N/m2, or about 1,000 atmospheres. If one includes the
entire adhesive force (25 nN) in the calculation [37], the total possible pressure rises to
about 1,700 atmospheres.
Because the conversion of random coil protein into folded structures usually
involves a positive volume change, high pressures typically induce protein unfolding.
Hydrostatic pressures in the range of 2,000 to 5,000 atmospheres have been shown to
cause such unfolding for a variety of proteins [40,41]. Given the uncertainties in the tip
pressure estimates, the pressures on fd phage in the AFM experiments of figure 4 could
easily approach the values that induce protein unfolding, which in turn would facilitate
the observed morphological changes.
The hydrostatic pressures leading to protein unfolding are isometric, whereas the
tip pressures are localized; thus, the latter include substantial shear forces whereas the
former do not. It seems reasonable to suppose that shifts of proteins relative to one
another should occur at lower pressures as compared to those that induce protein
unfolding. Given that the phage protein sheath is composed of a series of overlapping
rod-like protein subunits, such subunit shifts would lead to overall structural collapse
under extreme pressure. Following such a structural change, the final height would be
expected to be on the order of a single protein, corresponding to about 0.8 nm for the αhelical pVIII molecule.
While a value corresponding to the diameter of an α-helix is roughly what is
observed for the 'crushed' phage at 15% relative humidity, it is much larger than the
observed value at 60% RH. The anomalously small values at higher relative humidity are
probably due to differential changes in cantilever flexion and changes in the adhesion
forces over the mica and phage surfaces.
X-ray diffraction studies indicate that the overall diameter of dry phage is 5.5 nm
[25]. As can be seen from Figure 4, the largest height observed in contact mode, on the
other hand, was about a factor of 4 too small. Further, both the apparent height at low tip
force and the force required to cause the sharp drop in height are strong functions of the
relative humidity in which the phage is equilibrated. These overly small heights might be
due to a number of factors, including: 1) Changes in binding of the phage to mica as a
function of RH, 2) changes in the mechanical properties and/or structure of the phage
associated with adsorption of water, 3) changes in the tip-phage and tip-mica adhesion
forces, 4) buildup of a water layer on the mica surface that the tip somehow fails to
penetrate, and 5) buildup of a salt layer on the entire mica surface that engulfs the phage.
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We immediately discount possibility 5, that of an overall salt covering.
Lyubchenko et al [26], eliminated this on the grounds that their height measurements
under water were not significantly different than observed in dry samples. In our case, we
have used 30 times lower salt concentrations and applied the sample by spin coating.
Thus, the residual solution salts in our samples should be two to four orders of magnitude
less than those present in Lyubchenko's [26] studies. Possibility 4 above also is
extremely unlikely since the high tip forces present in the plateau region of the heightforce curve would certainly have resulted in penetration of any water layer present.
Moreover, as we shall see later, the water layer is too thin at 60% RH to account for the
discrepancy in height.
Figure 5. Tapping mode AFM images obtained from fd phage on mica measured at 25% humidity (RH)
at two extreme values of set point. The set point for image A was 90% of the free amplitude while that for
image B was only 10% of the free amplitude. The gray scale extends over 5 nm in height.
Information regarding possibility 3 can be obtained by comparing tapping and
contact mode data. As shown by comparison of Figures 1 and 2, tapping mode imaging is
less sensitive to changes in adhesion forces than is contact mode. It also turns out that the
normal forces associated with the tapping mode have much less effect on biological
samples than might be expected based solely upon the cantilever spring constant and
tapping amplitude [42] because of visco-elastic effects play a significant role in most
biological samples [42,43]. In fact, it has been shown that cell walls are extremely hard
when tested with impulsive force lasting about 1 millisecond or less [43]. The resistance
of phage to tapping mode forces can be seen from Figure 5, where images of the same
phage are compared at the extreme values of set point. The left hand image was taken
with the set point just at the upper margin of image formation while the right hand image
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was acquired with too much damping - a set point of only 10% of the free oscillation
amplitude. Sectional analysis gives a phage height of 2.8 and 2.2 nm for the left and right
images, respectively. In the broad region of set point values between these obvious
extremes, the height averaged 2.6 nm.
Figure 6. Tapping mode AFM images obtained from fd phage on mica measured at 15% and 98%
relative humidity (RH) respectively.
To provide information regarding possibilities 1 and 2, a systematic study of
phage images as a function of relative humidity was needed. Based on the observations
presented in figures 4, it is expected that tapping mode images will be relatively free of
the set point sensitivity seen in contact mode imaging. We therefore measured the
heights of several (5-10) different phage at 5 positions well-spaced along the filament
lengths under various humidity conditions using tapping mode. Representative gray scale
images of the extremes in humidity conditions are presented in Figure 6. Figure 7 reports
the measured heights at various relative humidity values determined by these tapping
mode measurements.
The smooth curve connecting the points in figure 7 is an empirically fit quadratic
function, 3.95 -0.076(%RH) + 0.00040(%RH)2 nm. The limiting value of height
measured at zero RH is therefore 4.0 nm, which is somewhat less than the X-ray
diffraction value of 5.5 nm. The possibility that the tip does not penetrate a mica
covering water layer in tapping mode must again be considered. However, Beaglehole
[44] has shown that the adsorbed water layer on mica is quite thin. Figure 7 also
reproduces Beaglehole's results for water layer thickness as a function of RH. Even
assuming that the water layer was impenetrable to the tip, there is just not enough water
12
on the mica surface to account for the smaller height value compared to the estimates
from diffraction.
Van Noort has recently demonstrated that mica surfaces in air appear to be
'stickier' than many organic layers [35]. This relative adhesive force can affect the
tapping mode cantilever motion and can produce height anomalies of the order of 1 to 10
nm, with the stickier surface appearing to be relatively higher [35]. Thus even a very thin
water layer might produce some reduction in apparent phage height.
Figure 7. Phage height, as measured by tapping mode AFM, plotted versus relative humidity. The
standard deviation of every point was less than 15% of the plotted value. The smooth curve is a quadratic
function. Also shown is the statistical thickness of adsorbed water on mica based on the work of
Beaglehole and coworkers.
Another source of height reduction is through adhesive forces between the mica
and phage. Consider a cylinder composed of protein subunits on a smooth, flat mica
surface. Certainly there would be some flattening due to adhesive forces between the
surface and the cylinder. Flattening from a perfect cylinder to an ellipsoid with an a/b
ratio of about 1.9 nm would be required to account for a height of 4 rather than 5.5 nm,
assuming no compression during the contact with the tip. This degree of flattening seems
entirely reasonable, and is depicted in Figure 8a, where the idealized circular cross
section and more realistic elliptical section are shown. Also shown in the Figure is the
ionic solution layer discussed below.
20.0 nm
Salt layer
4.0 nm
5.5 nm
Figure 8. Schematic cross sectional view of phage on the mica surface. On the left, is the situation as we
envisage it at very low relative humidity. On the right, is the idealized case of phage unaffected by surface
forces with dimensions based on intra-phage spacing as determined by X-ray diffraction.
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In our several hundred measurements of the height of fd phage at various
humidity values, the largest observed value was 3.6 nm at 15% RH - our lowest
controlled humidity value. This is less than the value of 5.4 nm reported previously for
M13 at "less than 20% RH" [24]. We cannot explain the previous, high value for M13 in
terms of differences in sample or experimental conditions. The differences are especially
curious given the fact that contact mode measurements were used in the case of the M13
study [24], and the current work strongly suggests that contact mode AFM studies on
biological samples generally gives height values that are too low. Given the rapidly
evolving nature of AFM, the difference between our maximum value of 3.6 nm and the
value of 5.4 nm reported for M13 may be due to differences in instrumentation and data
analysis protocols.
The nearly perfect quadratic nature of the drop in height with increasing relative
humidity is difficult to understand. That there should be some decrease in observed
height with RH is consistent with the calculations of Van Noort et al., but their results are
not sufficiently quantitative to predict the form of the variation with RH [35]. Curiously,
there is another example of a nearly quadratic variation with humidity in the phage
literature. Marvin [45] measured the a-axis (phage separation) lattice parameter of the
crystalline regions of fd fibers as a function of relative humidity and found that the lattice
spacing increased with increasing humidity, with rapid rise occurring above about 40%
RH. If that data is fit to a quadratic curve, it fits nicely and is of the form : a(nm) =
0.0125RH2 - 0.39RH + 5.6. The similarity is probably fortuitous, however, since there is
little change in the a lattice parameter below 30%, while the AFM height has dropped by
a factor of 2 in this same range.
To our knowledge, the only published detailed humidity versus image attribute
studies of a biological samples are concerned with DNA [32,37]. Both of these studies
used contact mode exclusively. In the first study [32], the height of DNA was found to be
relatively insensitive to humidity until it exceeded about 30%, after which it dropped
linearly with RH. In the second study [37], the apparent height of DNA decreased
sharply when the humidity increased from 5% to 15%, but then did not change upon
increase to 23%. While our results are in sharp contrast, we are not suggesting that this
previous work is in error. Phage is structurally much more complex than DNA. Its
interaction with water, with the surface of mica, and with the tip are all likely to be
different than occurs with DNA.
The measured widths of phage in tapping mode are more a function of tip choice
than of relative humidity, and average about 45 nm. This value is very close to the 50 nm
measured in contact mode under low force conditions. Thus, tip width effects may be
playing a relatively small role in determining the apparent phage width since tapping
mode tips are generally sharper than contact mode tips. This suggests the possibility of a
meniscus like layer of water containing ions (presumably from the ammonium
bicarbonate buffer, potassium and hydroxyl ions from the mica surface, and carbonate
from the air) surrounding the phage and helping to neutralize the negative surface charge
[1] on the phage. As the relative humidity increases, water would be preferentially
14
adsorbed into this ionic solution, thereby changing the equilibrium configuration of the
phage-mica, phage-solution, and phage-air interfaces. This picture is consistent with the
fact that the phage height decreases to its equilibrium value in only about 8 minutes with
a step increase in humidity (20% to 60% RH); but, it remains at the high RH value 9
hours after being returned to a 20% RH environment. Interestingly, if the same sample is
then heated to 70 °C in a vacuum oven followed by measurement in air at about 20% RH,
the observed height is found to be 2.8 nm, which is close to the value observed before
exposure to higher humidity. In the limit of zero relative humidity, the ionic salt residue
would continue to cause an apparent increased width in each phage. This hypothetical
situation is illustrated in a very schematic form in Figure 8.
While the above picture of an ionic solution meniscus between the sides of the
phage and the mica surface can account for the large observed width and some change in
height as the phage collapses towards the mica surface, it is more difficult to see how it
produces a dramatic height reduction and why that reduction is quadratic. The fact that
vacuum drying restores the measured phage height clearly indicates that the phage is not
being unraveled in some fashion. Further, the measured heights at very high relative
humidity are clearly un-physically small being less than the diameter of the DNA strand
that runs through the phage. It is likely that visco-elastic effects of the type discussed by
Van Noort [35] are playing a significant role in the imaging process.
CONCLUSIONS
Use of AFM for the study of biological molecules is increasing rapidly. Although
relative humidity has been known for some time to alter the appearance of AFM images,
the complex nature of these changes has been poorly appreciated. The studies presented
here on the filamentous phage fd may be the first controlled study on the effects of RH on
AFM images of biological structure as complex as a virus. The demonstration that
humidity changes can result in an approximately 10-fold change in the measured height of
the structurally stable fd phage, and that approximately correct heights are only obtained
after careful extrapolation to zero relative humidity, provides compelling data for the
need to make multiple measurements of height under varying conditions of RH during
investigations of biological structure by AFM. Moreover, the fact that height
measurements on phage are extremely sensitive to RH over the range from below 15% to
100%, while those on DNA appear to be insensitive to humidity values in the range of
15% to 30% strongly suggests that further careful humidity studies on biological samples
are needed.
Fd phage was studied by both contact and tapping mode AFM. It was found that
the height measurements made in contact mode were never reliable, but that reliable
height values could be obtained by extrapolation of tapping mode results. In tapping
mode, the relative humidity in which the sample had equilibrated and was measured is a
critical factor in the accuracy of the measured height. Well dried samples gave the tallest
15
images, and the height extrapolated to zero relative humidity was in satisfactory
agreement with crystallographic results. The apparent height of the phage in tapping
mode varied quadratically with the stabilized relative humidity. The measured height of
the phage in contact mode varied with both relative humidity and applied tip force.
Sample preparation also played a critical role in the quality of results obtained.
Uncoated and undoped, freshly cleaved mica served as an effective substrate provided
that a spin-coating procedure with very low salt content solutions was used. Humidity
control was also a key element in achieving stable and reliable sample preparation.
While the AFM images responded rapidly to increases in relative humidity, decreases in
humidity were not reflected in the AFM for periods of tens of hours. The measured
height reported in the literature for the M13 phage (5.4 nm) [24] is in reasonable
agreement with our determination for fd phage (4.0 nm), as is expected based on the
similarity of their structures.
ACKNOWLEDGMENTS
We thank Miss Jeniffer Uehara for her efforts during early stages of this project,
the National Science Foundation for financial support (Grant 9205197), and Stanley Van
and Molecular Kinetics for their support of X. Ji during his stay at Washington State
University.
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