Setting focus map in Leica LAS X Navigator for manual IBEX: 1 of 2 videos

First of two videos demonstrating how to create 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 (1 of 2) concerns how to create a focus map before acquiring a Cycle 1 image using the Manual IBEX method. The steps detailed in the video titled, “Focus Map Set Up After Cycle 1” are to be followed after imaging Cycle 1, dye inactivating Cycle 1 antibodies, labeling with Cycle 2 antibodies, and before imaging Cycle 2. Together, the workflow detailed here aids with acquiring perfectly aligned Z-stack images using Simple ITK.
Materials for videos:
• Leica TCS SP8 X confocal microscope
• LAS X Navigator software (LAS X 3.5.5.19976)
Procedure:
1. After selecting a region of interest for the tile scan, add focus points using the channel containing the marker found in each cycle (Hoechst or DAPI).
2. Using the fiducial channel (Hoechst or DAPI), examine each focus point to find the optimal Z-slice for the tissue.
3. Set the Z.
4. To double check if the optimal Z-slice is selected, turn on a membrane channel and scroll through the Z and adjust as needed.
5. Move to the next focus point and follow the exact same steps as before and do the same for the rest of the focus points.
6. Important: Be sure to save each focus point.
Tip: It is easier to have an odd number of Z-steps because the focus point will be in the middle of the Z-stack. For example, a 5 µm Z-stack will have a focus point set to Z-slice number 3.
Important: Under “Sequential” sub menu, be sure to select “Between Frames”.