Troubleshooting

Below are several common issues that arise with Fluid Imaging, and their suggested solutions.

Cantilever Tune Plot Looks Poor

The Cantilever Tune plot can be a useful diagnostic tool. Become familiar with the characteristics of the cantilever tune plot when you successfully obtain good images (see Tune the Cantilever (TappingMode Only) for more information). If the plot looks substantially different from previous successful experiments, there may be a problem with the fluid cell or the cantilever may be loose in its holder. Ensure there is good physical coupling between the probe and the fluid cell, and that the laser is positioned correctly at the free end of the cantilever.

Laser Sum Signal Absent or Weak

Remove all air bubbles from the cantilever. Bubbles may attach themselves to the cantilever, causing the laser beam to diffract. Remove bubbles by gently squirting the tip and sample with a stream of fluid, taking care not to squirt or splash fluid into spaces above the protective skirt.

Poor Image Quality

Some types of samples may adhere to the cantilever and tip (e.g., certain proteins). If you suspect tip contamination, you must protect the tip against contamination using either of the following methods:

• If the sample has a tendency to diffuse through solution and adsorb onto the tip surface (e.g., while allowing proteins to diffuse in solution and adsorb onto a mica substrate):
  1. Incubate the sample substance with the substrate, allowing it to adsorb to the substrate.
  2. Gently rinse the substrate with the fluid media to remove unadsorbed sample.
  3. Set the substrate in a bath of media, free of dissolved sample.
  4. Lower the tip into the fluid and image the adsorbed sample.
• If the sample is short-lived and must be imaged quickly, mask the tip against contamination by bringing the tip into gentle contact with an uncontaminated region of the substrate surface:
  1. In the Select Experiment window, set the Experiment Category parameter to Contact.
  2. Engage the substrate surface using a zero scan size.
  3. While keeping the tip gently in contact with the substrate surface, add the sample substance to be imaged to the petri dish and allow the sample substance to settle onto the substrate.
  4. Once diffusion is reached, withdraw the tip from the substrate surface.
  5. Immediately switch to TappingMode and image the sample.

Unable to Locate Particulate Samples

Some particulate samples (e.g., proteins) may prove difficult to find directly beneath a cantilever if the cantilever remains stationary during a diffusion or settling period. This may be due to the fact that some types of particulates are more attracted to the cantilever than to the substrate intended to support them. The result is a “shadow” on the substrate directly beneath the cantilever where fewer sample individuals are located; they are stuck to the cantilever. If you suspect this problem, after allowing the sample to bind to the substrate surface move the probe to a new site, away from the location of the cantilever during diffusion. You should find more individual samples there.

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