cantilever holder (indirect drive). Theacoustic waves induced in the fluidmedium cause the cantilever tooscillate. The Magnetic Actuated Drive (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 backside of the cantilever can lead to thepossibility of contamination of sensitivesamples with soluble rare earth andtransition metal ions. Also, theelectromagnet of the magnetic drivefluid cell may cause undesirableheating of the sample, which couldinduce lateral drift in the system duringscanning. Also worth noting is that theo-ring seal option is only available on

the acoustic TappingMode cell. TheMAD fluid cell depends on capillaryforces to keep the liquid at the surface of the sample. Studies on lipidbilayers (Figure 5) and DNA molecules (Figure 6) show that the acoustic andmagnetic actuated drives arecomparable in their ability to imagesoft samples with minimumperturbation4. Both achieve highquality resolution with comparablesignal-to-noise ratios, making themagnetic drive equal in everysignificant way to the more commonlyused acoustic drive. Sinceperformance 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 AcousticTappingMode in fluid

The first step to successful imaging in fluid is selecting the appropriateprobe. This choice is largely

Figure 2 is an example of a real-timeenzyme activity study. The successionof images corresponds to thedegradation of adipalmitoylphosphatidylcholineLangmuir-Blodgett bilayer byphospholipase A23. In this experimentit is possible to follow the time courseof the hydrolysis of the lipid film.

As with TappingMode in air, importantinformation can be obtained pertainingto the physical characteristics of thematerial using PhaseImaging. Phaseinformation can be recordedsimultaneously with topographic datain TappingMode on Digital InstrumentsMultiMode®, BioScope, andDimension Series systems.PhaseImaging is a powerful techniquethat can provide information onchanges in viscoelasticity, friction,adhesion or hardness across a samplesurface (see “PhaseImaging: BeyondTopography”, Lit. code: AN11).

Acoustic vs. Magnetic Actuated Drive

Two drive mechanisms forTappingMode in fluid are available to the user on the MultiMode AFM.The conventional method of driving thecantilever by acoustic excitation hasbeen joined by a magnetic actuateddrive. The Dimension and BioScopesystems both use the acousticexcitation method exclusively.

Acoustically driven oscillations of thecantilever in liquid on the MultiModeAFM occur by excitation of apiezoelectric ceramic element in the

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 nitridecantilever coated with a Co/Cr film anddriven with a magnetic field.

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

a. b.

dependent on the samplecharacteristics (hardness, roughness,etc.). The preferred probes forTappingMode in fluid are the siliconnitride cantilevers. Generally, the short,narrow cantilever of the “NP” series or the shorter of the two cantilevers onthe “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 whenoperating in fluid. The use of an o-ringis recommended when fluid exchangein the cell is desired or whenevaporation is an issue (e.g., workingwith heated fluids or solvents).Otherwise, capillary forces are strongenough to ensure that the fluid remainsin between the substrate and the fluidcell 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 tunemenu. Empirical experience shows usthat relatively low frequencies ofoscillation provide the best conditionsfor acquisition of images. We suggestusing a frequency of about 8kHz forthe silicon nitride probes. In any case,apply a drive voltage to the cantileverof about 500mV. Choose a fairlydefined, tall peak from those thatappear in the amplitude line on thesweep. Offset slightly to the left side

of the peak. If there are no peaks in the expected range, the peaks arepoorly defined, or you require anunreasonably high drive amplitude toget large enough peaks, you may beusing the wrong cantilever, a defectivecantilever, or you may simply be toofar from the surface. The TappingModeresponse of the BioScope andDimension systems are especiallysensitive to tip-sample distance.

Upon returning to the main imagingcontrols, check the RMS amplitude of the probe oscillation. The optimalRMS value depends greatly on thesample being studied. A suggestedvalue would be about 0.8V forTappingMode imaging of soft samples(and even less for fragile ones likeliving cells).

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.

The “Force Calibration” function mayalso be used after engaging to ensurethat the tip is in fact engaged properlyand that a minimal force is being usedon the sample. Use caution, however,because this procedure can damagethe tip if it is brought too close to thesurface. Refer to the Force Calibrationsection of your microscope manual fordetails 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 arrowkeys to change the setpoint and thegains (see “Guidelines for FluidOperation 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 usingthe magnetic driven tapping mode. Z range = 10nm.4