3.4.4. Image acquisition and adjustments

FISH signals can be detected by using an epifluorescence microscope using standard fluorescence microscopy procedure for your instrument. Here is some basic information that might be helpful for beginners.

  1. Frequently, each channel (i.e. each signal from a given probe or DNA counter-stain) is acquired as a black & white image. Each of these images will later be coloured with a specific colour (arbitrarily chosen) and merged, resulting in a single pseudo-coloured image. Therefore, the actual fluorescent colour seen can differ from that in the final images.
  2. The probe will dissociate from the target RNA molecules over time in the mounting media. Thus, after mounting in antifade medium, observation should be conducted as soon as possible.
  3. Minimize exposure of your slides to the excitation light to prevent photobleaching of fluorochromes (i.e. reduction of signal intensity).
  4. Irradiation at higher magnification causes localized photobleaching. This photobleached region will be observed as a dark spot in a lower-magnified image. Therefore, images should be acquired from lower to higher magnifications.