Cell Painting protocol:How to Set Image Acquisition Order on ImageXpress Microscope?

Hi, CellProfiler Team and everyone.
I am trying to reproduce the Cell Painting protocol using ImageXpress Micro Confocal High-Content Imaging System.
In the Cell paniting protocol paper, Bray et al recommended imaging from red to blue to avoid possible photobleaching, and still use the DAPI channel for laser focusing. I tried to import the ImageXpress microscope plate acquisition settings file provided by the paper into ImageXpress Micro Confocal High-Content Imaging System, the imaging channels w1~w5 in the acquisition settings file are DAPI, GFP, Cy3, TxRed, Cy5, and use the DAPI channel for processing Laser focus, but I don’t know how to adjust the imaging order from w5 to w1 (from red to blue). The advice I got from the application engineer of the imaging system is to set the imaging channels w1~w5 to Cy5, TxRed, Cy3, GFP, DAPI, but use the Cy5 channel for laser focus to achieve imaging from red to blue.
Would you mind offer me with more details of setting image acquisition order(imaging from red to blue) on ImageXpress Microscope or the acquisition settings file suitable for ImageXpress Micro Confocal System?@CellProfiler Team and everyone.
I would be very grateful for any suggestions!

It honestly shouldn’t matter much which order you take the channels in, nor which channel you use for focusing (as long as that works). So I would say go with whatever’s easiest to set up on your system; if you DO decide you want to configure your microscope in a certain way and need help with that, it might be a better question for MicroForum (see “Related Forums” at the top of the page)- we mostly deal with analysis, whereas they are more related to the actual staining and microscopy.

Hi, @bcimini
Thank you so much for your suggestion!