8. Change the ‘Rot [°]’ value. Take another snap of the marker sample and measure the angle again, change the ‘Rot [°]’ value again. Repeat until you measure a value lower than 0.05°. Store current position in the ‘Dual Camera Alignment’ tool
10. Take another snap of the marker sample and measure the angle again, change the ‘Rot [°]’ value again. Repeat until you measure a value lower than 0.05°. Store current position in the ‘Dual Camera Alignment’ tool
### Camera xyz-Alignment
9. Change to 63x 1.4 (SIM) or 1.46 (TIRF) objective, use the bead slide and focus on beads. Use the same imaging method that you will use for your experiment (LASER WF, SIM, Apotome etc.)! In 'Acquisition' set up the 561nm laser for camera 1 (TV1) and the green laser for camera 2 (TV2) with exposure time 100ms and 1-5% of the laserpower (>1000 white/grey values!). If working in multitrack always keep track 1 with camera 2! Choose the optimal grid pattern.
9. Change to 63x 1.4 (SIM) or 1.46 (TIRF) objective, use the bead slide and focus on beads. Use the same imaging method that you will use for your experiment (LASER WF, SIM, Apotome etc.)! In 'Acquisition' set up the 561nm laser for camera 1 (TV1) and the green laser for camera 2 (TV2) with exposure time 100ms and 1-5% of the laserpower (>1000 white/grey values!). Always keep track 1 with camera 2! Choose the optimal grid pattern.
10. Go to 'Continuous' and focus through the beads. The optimal focus (smallest diameter of the bead) should be the same for both color channels. If not:
11. Go to ‘Maintain’, and drag out the ‘Dual Camera Adapter Alignment’ tool
12. Change the Z value until good overlap of the channels is achieved. Focus up and down, and change the Z value until the beads are perfectly in focus for both channels simultaneously.
13. Take a small Z-stack (20 slices) using optimal sampling, SIM process Z-Stack in case of SIM (no processing in case of TIRF or WF)
14. Go to orthogonal View and, in Dual Camera Adapter Alignment tool, fine-tune the Z value if necessary. Larger Z values correspond to a shit of the green channel to the left. Then, acquire another Z-stack until perfect overlap of channels is achieved. Store current position!
15. In Dual Camera Adapter Alignment tool click XY for automatic alignment, if good overlap of channels is achieved, store current position, if not – move to stored position and in 'Continuous' mode change X and Y in the Dual Camera Adapter Alignment tool until overlap is achieved. Especially the beads in the center of the field of view should nicely overlap. Use the 'Profile > Display > Marker > Difference' tool to check the overlap.
