Atomic Force Microscopy in Cell Biology Episode 2 Part 10 doc

366 Zuccheri and Samor`ı

between this class of methods and that presented here is that the latter is designed to

specifically adsorb properly functionalized molecules. Often, the protocols also reduce

the non specific adsorption of other molecules in solution (Bamdad, 1998). Such careful

design makes the methods generally complex, involving multistep functionalizations,

and works on a properly designed and modified target molecule (with the exception of

that presented by Shlyakhtenko and co-workers (1999)). For the general application in

biology, a deposition method usually cannot modify the molecules it is interested in,

because it is practically too difficult not to alter their properties. Due to the widespread

interest of DNA attachment to surfaces for many techniques and uses (DNA chip tech￾nology, only to mention one), it is certain that these methods will continue to evolve.

The researcher will soon be offered a variety of methods to employ for the solution of

any research problem.

III. Air Imaging of DNA: Which Present, Which Future?

Imaging dehydrated specimens with the microscope operating in air is the easiest

of the SFM operations. This is, indeed, one of the reasons for the popularity of the

microscope and one of the advantages in its use with respect to the electron microscope,

which must always be used under high vacuum. In our experience, any new user can

master operations in ambient air reasonably well after a few hours of lecture in class

and some hours of hands-on experience with the microscope. In the last few years, the

stability of the microscopes operating, for instance, in tapping mode (see following) and

the constant quality of the commercial probes make imaging in air significantly easier

than it was in the past.

A. The Humidity Issue and SFM under Organic Solvents

The control of the interaction forces between the probe and the specimen is of funda￾mental importance in SFM, especially in contact-mode SFM, the first to be employed

during the years. In contact mode, the probe and the specimen are always in contact,

while the probe is dragged along the surface of the specimen. If the interaction forces are

not minimal, the shear forces generated by the motion are sufficient not only to seriously

damage the soft biological macromolecules irreversibly but also to produce bad quality

images.

Among the possible interactions between the sample and the probe, the most relevant

one in air imaging by contact mode is due to the hydration of the surfaces exposed to

humid air. The layer of water normally present on any hydrophilic surface exposed to

air creates a meniscus around the SFM probe, and this causes the onset of very high

capillary forces that pull the probe toward the specimen.

What is commonly thought in SFM is that the sharper the probe, the better is the

resolution of the images it produces. This concept takes a more subtle meaning in the

presence of capillary forces. Here, a probe with a larger surface entering the hydration

17. SFM of Single DNA Molecules 367

layer would imply the onset of higher capillary forces (Israelachvili, 1992). This argument

in favor of very sharp probes could be balanced by the consideration that the residual

forces that attract the probe toward the specimen would produce higher pressures if they

were applied on the smaller area upon contact with a sharper probe and consequently

be more disruptive for a soft sample. The researchers preferred to use EBD probes in an

attempt to both reduce the effect of capillary forces and improve the resolution of the

early images of DNA. An EBD probe is more hydrophobic than an unmodified Si3N4

probe: this could help in reducing the capillary forces. The reduction of the source

of these forces took two possible paths. Some authors decided to reduce, by imaging

while keeping the microscope with the mounted sample in dry nitrogen, the relative

humidity of the environment in which the probe and the specimen were (Bustamante

et al., 1992; Thundat, Allison et al., 1992; Vesenka et al., 1992). Other authors eliminated

the air–water interface at which the meniscus would form, by submerging the sample

and the probe in propanol (Hansma et al., 1993; Hansma, Sinsheimer et al., 1992;

Hansma, Vesenka et al., 1992; Murray et al., 1993; Samor`ı et al., 1993). For DNA,

this operation also has the side effect of improving the adhesion of the molecules to

the surface, since DNA is insoluble in propanol. At the time this protocol was used, a

stronger adsorption was certainly desired. The images obtained under propanol can still

rival newer techniques as far as resolution is concerned (Hansma et al., 1995).

Several authors studied the effect of both the relative humidity and the applied force

on imaging of DNA with the SFM (Bustamante et al., 1992; Ji et al., 1998; Thundat,

Allison et al., 1992; Thundat, Warmack et al., 1992; Thundat et al., 1993; Vesenka et al.,

1992; Vesenka et al., 1993; Yang et al., 1996; Yang and Shao, 1993) and they generally

found that a lower imaging force at the lowest possible relative humidity was the desired

working condition.

B. AC Modes

The emergence of tapping mode SFM, in which the cantilever is continuously oscil￾lated and in which it contacts the specimen only intermittently, has certainly represented

a great improvement for SFM imaging of DNA (Hansma et al., 1995). The lateral mo￾tion of the probe takes place almost totally when the probe is not in contact with the

sample, so shear forces are virtually eliminated. With this new technique, the control

of ambient humidity is still helpful but not normally necessary to produce high-quality

images. Specimens can be scanned repeatedly in air without any recognizable damage.

The instruments operating in tapping mode are generally very stable, and imaging has

become easier and quicker.

C. In Search of Sharper Probes

A constant theme of research in SFM has always been the search of sharper or more

specific probes. The report of the many advances of the field and the many varieties

of probes available on the market is beyond the scope of this paper. We simply want