Focus Map Set Up After Cycle 1 for manual IBEX: 2 of 2 videos

Second of two videos demonstrating how to use a focus map in LAS X Navigator (LAS X 3.5.5.19976, Leica TCS SP8 X confocal microscope) for pixel-pixel alignment of manual IBEX images. This video (2 of 2) concerns how to set up a focus map after acquiring a Cycle 1 image. See video titled, “Setting focus map in Leica LAS X Navigator for manual IBEX: 1 of 2 videos” for details on how to set up a tile scan and focus map for manual IBEX. Together, the workflow detailed here aids with acquiring perfectly aligned Z-stack images using Simple ITK. The steps detailed here can be repeated after Cycle 2, Cycle 3, Cycle N to acquire highly multiplexed images.
Materials for videos:
• Leica TCS SP8 X confocal microscope
• LAS X Navigator software (LAS X 3.5.5.19976)
Procedure:
1. Open the Cycle 1 image. You will use this image as a reference for setting the focus map and focus points.
2. Find the region corresponding to the first focus map on the Cycle 1 image. For Z-stack images, make sure you are looking at the middle Z as this corresponds to the Z-position used for the focus map.
3. Zoom in and use the Cycle 1 image as a reference to find the correct Z-position for the Cycle 2 image.
4. Use cellular morphology and distinguishing features in the tissue to identify the correct Z-position.
5. Set your new focus point for the Cycle 2 image.
6. Move to the next focus point and repeat above steps.
7. After setting all focus map points, select the first focus map point and then “re-center Z” in the Z-stack panel. See 4:30 mark.
Tip: For future Cycles, use Cycle 2 to reset the focus map instead of Cycle 1 because this image has gone through the bleaching process.
Important: Under “Sequential” sub menu, be sure to select “Between Frames”.