Artifacts, Noise, Background

Avoiding background, artifacts and noise is essential. Such features may disturb the spot detection process: Gel breaking separates spots, sprinkles may mislead the spot detection, noise can cover low intensity spots etc. The weaker the signals the stronger is the effect of artifacts on quantitative analysis. Very low signals may be observed due to faint spots, very low sample amounts or bad incorporation of radioisotopes. Very often it is better and less frustrating to repeat the experiment instead of doing analysis of suboptimal images.

Background may be caused by insufficient destaining, fluorescing glass plates, gel coverings and backings. Using low fluorescence equipment will minimize such problems. Also misusing optical filters for fluorescence imaging may cause background. Reevaluate which light sources and filters are useful for your analysis.

Noise can be produced by high PMT voltages enhancing also random signals. Adapting the PMT-Voltage will solve this problem. Also phosphor screens that had not been used for a longer time accumulate noise. Erasing the screen immediatly before exposure solves this problem. Try to optimize the scanning procedure and avoid post scan manipulation of your images.

Artifacts affect the quantitation process. However, they can often be avoided very easily. The following list shows some problems and the corresponding solution.

• Artifacts on the gel surface
  Try to focus the laser beam inside the gel if your imaging device supports this feature.
• Fingerprints on the gel
  Use (powderfree) gloves and/or clean the glass panels of your scanners.
• Fluorescent sprinkles
  Rinse the gel with destainer or water and avoid evaporation. Use powderfree gloves, dust free experimental equipment in a dust free environment.

  Fluorescent sprinkles can disturb the spot detection.

• Artifacts from phosphorscreens
  Decontaminate or replace phosphorscreens; scan autoexposed phosphorscreens before use to locate and remove contaminations.
• Artifacts and scratches on glas panels
  Handle your imaging devices with care and only use a mild detergent and wood free tissue for cleaning. Keep all glass panels clean and free of dust and fat. Avoid regions containing artifacts during scanning.
• Ghost images or spot shadows
  Do not move the gels during exposure or scan. Be aware of scanner vibrations during the scan process. Fix the gel on the scanner surface (use suction cups) and erase phosphorscreens before using.

  Ghost images are very dangerous problems because they're hard to recognize.

• Air bubbles and water droplets
  Use enough water between glass panel and gel but be aware of water running inside your scanner. Composition rubber may help to seal problematic grooves.

  Bubbles may interfere with quantitation.

• Newtonian rings
  Try to avoid using glass plates. If this is not possible focus the laser beam inside the gel.
• Horizontal or vertical lines
  Sometimes single CCD elements in CCD bars of a desktop scanner fail to work. The corresponding line of the image remains empty. The only way to solve this problem is replacing the CCD element or the whole scanner.
• Gel breakings and gel pieces
  Work carefully and use gel strengthener to give the gel more stability. If necessary try to puzzle the gel pieces to close the breakings before scanning.

  Breaks in the spot cause problems with spot detection. This may be corrected manually.

• Labels on the gel
  Using permanent markers for gel labelling sometimes interferes with the scan. To simplify the subsequent analysis please make sure that the label does not overlay parts of the gel or spot pattern.