Figure 2. Action of PLA2 on a DMPC bilayer deposited on mica. 1µm x 0.5µm scans.

Figure 2 is an example of a real-time
enzyme activity study. The successionof images corresponds to thedegradation of adipalmitoylphosphatidylcholine
Langmuir-Blodgett bilayer byphospholipase A23. In this experiment
it is possible to follow the time courseof the hydrolysis of the lipid film.
As with TappingMode in air, important
information can be obtained pertaining
to the physical characteristics of thematerial using PhaseImaging. Phase
information can be recorded
simultaneously with topographic data
in TappingMode on Digital Instruments
MultiMode®, BioScope, andDimension Series systems.
PhaseImaging is a powerful techniquethat can provide information onchanges in viscoelasticity, friction,adhesion or hardness across a sample
surface (see “PhaseImaging: BeyondTopography”, Lit. code: AN11).

Acoustic vs. Magnetic
Actuated Drive
Two drive mechanisms for
TappingMode in fluid are available
to the user on the MultiMode AFM.
The conventional method of driving thecantilever by acoustic excitation has
been joined by a magnetic actuateddrive. The Dimension and BioScopesystems both use the acoustic
excitation method exclusively.
Acoustically driven oscillations of thecantilever in liquid on the MultiMode
AFM occur by excitation of apiezoelectric ceramic element in thecantilever holder (indirect drive). The
acoustic waves induced in the fluid
medium cause the cantilever tooscillate. The Magnetic ActuatedDrive (MAD) mechanism uses anelectromagnet in the fluid cell to createa magnetic field to drive specializedprobes (Figure 3). These probes arecoated with a magnetic film (Co orCo/Cr) on the backside (only) topreserve the tip sharpness. It issomewhat easier to identify theresonant frequency of the cantileverwhen working with the magnetic drive,as the tune shows mainly only theresonant frequency oscillation of theprobe (Figure 4). However, themagnetic coating on the backsideof the cantilever can lead to thepossibility of contamination of sensitive
samples with soluble rare earth andtransition metal ions. Also, theelectromagnet of the magnetic drive
fluid cell may cause undesirable
heating of the sample, which could
induce lateral drift in the system during
scanning. Also worth noting is that theo-ring seal option is only available ona.

the acoustic TappingMode cell. The
MAD fluid cell depends on capillary
forces to keep the liquid at thesurface of the sample. Studies on lipid
bilayers (Figure 5) and DNA molecules(Figure 6) show that the acoustic andmagnetic actuated drives arecomparable in their ability to image
soft samples with minimum
perturbation4. Both achieve highquality resolution with comparable
signal-to-noise ratios, making themagnetic drive equal in every
significant way to the more commonlyused acoustic drive. Since
performance is similar, the user isultimately left to make their choicedepending on factors of ease-of-use,
the few minor disadvantages of MADmode, and cost.

Performing Acoustic
TappingMode in fluid
The first step to successful imaging
in fluid is selecting the appropriate
probe. This choice is largelyb.

Figure 3. Lateral and top views of the fluid cellfor magnetic TappingMode: 1) piezo element,2) electromagnet, 3) permanent magnets,
4) cantilever, and 5) laser beam.

Figure 4. Frequency tune for a silicon nitride
cantilever coated with a Co/Cr film anddriven with a magnetic field.

Figure 5. Phosphatidylserine bilayers imaged in TappingMode in liquid: (a) and (c) were obtainedwith the magnetic drive mode and (b) was obtained with the acoustic drive mode. 6µm scans withZ range = 12nm. The dashed square outlines a characteristic feature visible during the wholeexperiment 4.

dependent on the sample
characteristics (hardness, roughness,etc.). The preferred probes forTappingMode in fluid are the silicon
nitride cantilevers. Generally, the short,narrow cantilever of the “NP” series
or the shorter of the two cantilevers on
the “OTR-4” chip is suggested (see“Choosing AFM probes for BiologicalApplications”, Lit. code: AN44).
For users of the MultiMode AFM,
another decision to be made iswhether or not to use an o-ring when
operating in fluid. The use of an o-ringis recommended when fluid exchange
in the cell is desired or whenevaporation is an issue (e.g., working
with heated fluids or solvents).
Otherwise, capillary forces are strong
enough to ensure that the fluid remains
in between the substrate and the fluid
cell and does not overflow onto thescanner. A small amount of fluid
should be used in that case (typically~100µL), which also presents theadvantage of limiting thermal
drift problems.
Select a peak using the frequency tune
menu. Empirical experience shows usthat relatively low frequencies ofoscillation provide the best conditions
for acquisition of images. We suggest
using a frequency of about 8kHz forthe silicon nitride probes. In any case,apply a drive voltage to the cantileverof about 500mV. Choose a fairly
defined, tall peak from those thatappear in the amplitude line on thesweep. Offset slightly to the left sideof the peak. If there are no peaks inthe expected range, the peaks arepoorly defined, or you require an
unreasonably high drive amplitude to
get large enough peaks, you may beusing the wrong cantilever, a defective
cantilever, or you may simply be too
far from the surface. The TappingMode
response of the BioScope andDimension systems are especiallysensitive to tip-sample distance.
Upon returning to the main imaging
controls, check the RMS amplitudeof the probe oscillation. The optimal
RMS value depends greatly on thesample being studied. A suggested
value would be about 0.8V for
TappingMode imaging of soft samples(and even less for fragile ones likeliving cells).

The “Force Calibration” function may
also be used after engaging to ensurethat the tip is in fact engaged properly
and that a minimal force is being usedon the sample. Use caution, however,
because this procedure can damage
the tip if it is brought too close to thesurface. Refer to the Force Calibrationsection of your microscope manual for
details of this procedure.
It is important to keep in mind that theadjustments are more subtle than in airand it is often preferable to actuallytype the numbers than to use the arrow
keys to change the setpoint and thegains (see “Guidelines for Fluid
Operation with a MultiMode AFM”,
Support Note #PN 013-290-000).

Figure 6. DNA step ladder molecules imaged in TappingMode in liquid. (a) and (b)were obtained using acoustic driven TappingMode. (a’) and (b’) were obtained using
the magnetic driven tapping mode. Z range = 10nm.4