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 according to the instructions below.

Bleach Settings

Start Bleaching after # scans: This allows you to delay the bleaching event so you can aquire a 'pre-bleach' set of data.

Repeat Bleach after # scans: This is for use in techniques such as Fluorescence Loss in Photobleaching (FLIP).

Iterations: Sometimes a single scan of the region is not enough to bleach or ablate, so you can use this feature to repeated illuminate the region during the bleach phase.

Different scan speed: This allows you to speed up or slow down the scan rate specifically during the bleach process. It can be advantageous to slow down the scan because this increases the dwell time and hence the amount of energy delivered to the region.

Safe bleach for GaAsP: This protects the Gallium Arsenide Phosphide (GaAsP) detectors from intense reflected light by briefly turning them off during the bleach.

Different Z Position (µm): Clicking Mark Z will save your current Z position. The focus will then move to that position to carry out the bleach/ablation event.

Trigger in: Allows a remote event to trigger bleaching.