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  1. Make sure the lasers you need for ablation or bleaching are powered on in the software
  2. Configure your light path settings for imaging (i.e. lasers, detectors, mirrors, emission filters). You can use Smart Setup or manually configure them.
  3. If you are going to use the Chameleon multiphoton laser for ablation with infrared light then make sure it is tuned to a suitable wavelength (usually 760 nm).
  4. If you are going to use the Chameleon, then make sure the correct beamsplitting mirror is in place for the invisible laser beam path. For 760 nm this would be the MBS 690+ mirror. If the laser is tuned to a wavelength greater than 760 nm then the MBS 760+ can also be used. The advantage of using the latter is that it is compatible with imaging far red dyes. If the MBS 690+ is in place it will block transmission of far red.
  5. Untick the Chameleon laser wavelength unless you want to image using the multiphoton laser.
  6. Set up a Time Series, and a Z-Stack if needed.
  7. Tick the Bleaching box. The Regions box will be automatically ticked when you tick Bleaching.
  8. Select an ROI shape from Regions.
  9. Draw at least one ROI on a preview image.
  10. Specify Bleach and if necessary Analysis for the ROI or ROIs. Analysis is only required when tracking recovery in FRAP and similar experiments.
  11. Set up the Bleach settings.
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