6. Practical Instrumentation

Dynamic force microscopes are available from several manufactures as are liquid cells. Acoustic drive DFM is sold as "Tapping ModeÔ" (Digital Instruments, Santa Barbara, CA) or "Intermittent Contact" (ThermoMicroscopes, Sunnyvale, CA.). Magnetic drive DFM is available as "MACModeÔ (Molecular Imaging, Phoenix, AZ). Systems are available which integrate AFM with an inverted optical microscope, important for large samples such as whole cells. The performance of these systems suffers somewhat from vibrations when compared to the normal compact design of single purpose AFMs.

Figure 15: Showing a typical setup for fluid DFM. The controller workstation is on the right and the microscope is shown sitting on a bungee-cord vibration isolation system inside an acoustic isolation box on the left. The hand-held terminal on the table controls the DFM operating parameters.

The overall layout of a DFM workstation suitable for high-resolution biomolecular imaging is shown in Fig. 15. The microscope sits on a vibration isolation stage with a resonant frequency of ca. 1Hz, enclosed in a heavy wooden box designed to attenuate acoustic noise. This low resonant frequency is adequate to permit atomic resolution with a reasonably-well made SPM even in quite noisy environments (Chen 1993). Our microscopes are located in busy sample preparation laboratory close to noisy fume hoods. Given adequate vibration-isolation, the most severe remaining problem is thermal drift, and a well-controlled environment is of considerable benefit. The problem is acute if reagents are flowed into a liquid cell from outside the microscope chamber, because temperature changes of even a tiny fraction of a degree can cause large bending of the cantilever.

    Figure 16: Top-down view of the liquid flow-through cell used with the microscope shown in Fig 15. It sits underneath the top-down scanning and sample support system which forms the main body of the microscope. The cell is an open Teflon ring into which fluid is gravity fed. The level is kept constant by a drain line attached to a peristaltic pump. The terminal block below the cell is for holding electrodes for electrochemical control.

A liquid cell for a scanning probe microscope is shown in Fig. 16. It is designed for a top-down scanner that dips into the open cell from above. Reagents may be flowed into, and out of the cell using the two fluid tubes. An arrangement for changing reagent flow during imaging is described by Thomson et al. (Thomson, Kasas et al. 1996). Cleanliness, always a problem in high resolution imaging, is a particularly problematic when a flow through system is used. Vigorous cleaning of components is essential between runs. Samples are best prepared in laminar flow hoods using the highest available grade of freshly deionized, filtered and distilled water. Buffer salts should be purified by recrystallization wherever possible. A quick way out is to use low concentrations of the highest available grade of reagent - the concentration of contaminants will be correspondingly lower than in highly concentrated solutions. Repeated column filtration of biopolymer samples is required, particularly if they have been prepared by elution from gels.