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1. Make sure the lasers you need for bleaching or ablation are powered on in the Zen.
   
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. In contrast, if the MBS 690+ is in place it will block transmission of far red.
   
5. Untick the Chameleon laser wavelength in the track 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 in the multi-dimensional acquisition field. 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. Tick Bleach and, if necessary, Analysis for the region or regions. Analysis is only required when following fluorescence recovery in FRAP and similar experiments.
    
11. Set up the bleach parameters in the Bleach Tool according to the instructions below.
12. Once the bleach parameters, Time Series and Z-Stack settings are configured, click Start Experiment to begin the acquisition.

Bleaching Tool

The Bleaching tool allows you to configure the parameters of the bleach phase of your acquisition. Once you've set the parameters below you can save them for reuse at the top of the tool window.

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