3.4.1 The principle of AFM
TMin liquids
The principle of tapping mode AFM in liquids is the same as in air (Knoll, 2003). An oscillating sharp tip mounted to a flexible cantilever tracks the surface of the sample (Figure 8). The cantilever is excited to its resonance oscillation frequency with a piezoelectric driver. The deflection of the cantilever is detected with very high sensitivity by a four-segmented photodiode. The oscillation amplitude is used as a feedback signal that enables the piezoelectric scanner to keep the tip at a constant interaction. The AFM controller supplies the signal for the spatial movement of a piezoelectric positioner with sub-nanometer accuracy resulting in a precise tracking of the sample by AFM scanning tip (Binnig, 1986).
There are advantages to operate AFM with the sample and cantilever immersed in a fluid. To begin with, it fully eliminates impact of the capillary forces and reduces Van der Waals' forces (Hartmann, 1991; Israelachvili, 1997; Weisenhorn, 1989) . For this reason it enables the application of very soft cantilevers with a spring constant of typically 0.1 N/m (compared to tapping mode in air where the cantilevers in the range of 1-100 N/m are used). In our experiments we used oxide sharpened tips with a spring constant of ~0.3 N/m. The cantilevers were purchased from NanoProbesTM (Digital Instruments, Inc., Santa Barbara, CA).
An important disadvantage of AFM operation in liquids is the difficulty of a direct excitation of the cantilever. The piezo is attached to the liquid cell which causes an oscillation of the entire fluid cell in order to couple the oscillation to the cantilever. This is different from the tapping or non-contact operation in air or vacuum where the cantilever is directly attached to the piezoelectric driver. Since the whole cell vibrates, resonances of the entire setup are excited and transduced to the cantilever, which results in the detection of multiple peaks, often refered to as ‘forest of peaks’. In order to find the actual resonance peak of the cantilever, we analysed the Fourier transform of its thermal noise spectrum approximately 1 mm above the surface, which shows a distinct peak at the cantilever’s resonant frequency. Subsequently the drive frequency was set to the resonance frequency and the phase zero was adjusted approximately 20 nm above the surface.
On the other hand, imaging in a fluid medium damps the cantilever's oscillation. In our experiments the drive frequencies ranged from 3.4 to 34 kHz. When an appropriate frequency is selected, the amplitude of the cantilever decreases when the tip begins to tap the sample, similar to TappingMode operation in air.
Metal disc Laser Mirror
Oscillating AFM tip
Piezo
Mica Parafilm Fluid in
Fluid out Segmented photodiode
Fluid
Figure 8. Schema of the MultiMode fluid cell. The cantilever is fixed to the fluid cell with the help of a golden wire. The piezoelectric drive is imbedded into the fluid cell matrix. The position of the tip is recorded by a segmented photodiode tracing the laser reflection form the back side of the cantilever. Scanning of the sample in x,y,z is realized by a piezo- scanner located below the sample. The whole volume of the fluid cell is filled with liquid (approximately 50 µL). An addition or exchange of small amounts of liquid was possible using the ‘in’ and ‘out’ channels.
For the AFM measurements we used a commercial AFM MultiMode (Nanoscope III, Digital Instruments Inc., Santa Barbara, CA) equipped with a 12-µm scanner and a provided fluid cell (Figure 8). Before use, the fluid cell was cleaned with a dish cleaner, gently rinsed with water, ethanol, low warm water and, finally, with Milli-Q ultra pure water (Millipore, USA).
3.4.2 Image processing
All AFM height images presented in this thesis were recorded using the AFMTM. The images were process using Nanoscope software and home-written software ‘Look’ (A.
Knoll, LS Physikalische Chemie II Universität Bayreuth) with a special function of easy flattening of numerous DNA images. The DNA contour length was evaluated using DnaCalc6, which works with inverted TIFF bitmap files. The algorithm automatically recognizes a DNA chain between manually marked ends of the DNA molecule. For the end-to-end distance measurements we used Image Jv1.2, which also works with TIFF files and the ends of the molecules should be marked manually, too. Statistic analysis of the contour and persistence lengths was performed using Origin 7G.
3.4.3 Preparation of mica substrates for AFM measurements in liquids
When operating in liquid the problem of possible spillage of liquid onto the expensive electronics should be considered. Different methods were proposed to overcome this
problem: usage of a silicon ring supplied with the liquid cell by DI (MultiModeTM SPM Instruction Manual, 1999) that seals the liquid cell environment, usage of mica sheets glued onto Teflon (Muller, 1999a), etc.
In our work we developed a routine to fix mica sheets onto a metallic disc with the help of parafilm (American National CanTM). To this extend the Parafilm is stretched onto the metal disc and is heated with the mica sheet on topshortly to approximately 100oC.
After the Parafilm melts, the mica sheet should be gentle pressed into the Parafilm matrix. After cooling down to room temperature the discs are ready for use.
The solidified Parafilm layer holds the mica sheet good enough to be used in the AFM investigations avoiding any undesirable movement. Cleavage of the mica was performed always just before the solution for the AFM investigation is deposited. If the size of the mica sheet is chosen significantly smaller than the parafilm covered disc area, a water drop will reside on the mica surface (Figure 8). In that way prepared mica substrates allow operation of AFM in liquids omitting a usage of a silicon ring. The hydrophobic Parafilm layer protects spillage of the liquid onto the scanner. Additionally this method enables the injection of small amounts of liquid into the AFM liquid cell during scanning without danger of leakage onto the scanner. Omitting the usage of the ring enables free movement of the sample with respect to the tip and therefore increases the resolution of the AFM. And last but not least, such preparation routine allows a multiple usage of the assembled discs.