- Select the following parameters and click Apply, save the Channel Alignment file.
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### `Camera Rotation (old)`
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Use the yellow marker pen slide with a coverslip (on the desk next to the lens cleaning paper) and the slide with beads (multispec low, they are stored in the fridge close to the Elyra).
8. Go to ‘Maintain’, and drag out the ‘Dual Camera Adapter Alignment’ tool
9. Change the ‘Rot [°]’ value. Store current position in the ‘Dual Camera Alignment’ tool
10. Take another Snap of the marker sample and measure the angle again. Repeat until you measure a value lower than 0.05°. Store current position in the ‘Dual Camera Alignment’ tool
3. In 'Acquisition' set up the same lasers as for your experiment: set up one laser for camera 1 (TV1) and the other laser for camera 2 (TV2) with exposure time 100ms and 1-5% of the laser power (when pressing min/max you should have >1000 white/grey values, when ticking 'Show all' in the 'Display' panel). If you use multiple tracks (in case you want to align more than two colors), always keep track 1 with camera 2!
4. Choose the optimal grid pattern
5. Go to 'Live' and focus through the beads. The optimal focus (smallest diameter of the bead) should be the same for all color channels. If not:
6. Go to ‘Maintain’ and drag out the ‘Dual Camera Adapter Alignment’ tool
7. Focus on the TV1 (camera 1) channel and change the Z value of the TV2 (camera 2) channel, until a good overlap of all color channels is achieved, especially for the beads in the center of your image. Focus up and down, and change the Z value until the beads are perfectly in focus for all color channels simultaneously. Store current position
8. Take a Z-stack (3um, center) using half of the optimal sampling (double-click on [Opt] and divide by 2)
9. SIM<sup>2</sup> process Z-Stack in case of SIM (no processing in case of TIRF or WF)
10. Go to Orthogonal View. Click on one recorded bead in the center of your image and, in 'Dual Camera Adapter Alignment tool', fine-tune the Z value if the different color channels do not overlap perfectly. Larger Z values correspond to a shift of the TV2 channel to the right in the Ortho View.
11. Store current position! Then, acquire another Z-stack and repeat until perfect overlap of all color channels is achieved.
12. In 'Live' mode change X and Y in the 'Dual Camera Adapter Alignment tool' until an XY overlap of beads in all color channels is achieved. Especially the beads in the center of the image should nicely overlap. Store current position!
13. Use the 'Profile > Display > Marker > Difference' tool to check the overlap
Channel Alignment has to be done before **each colocalization experiment** using multiple color channels and for **each used filter** in the camera link, for **each used objective** and for **each configuration of different cameras/tracks** individually. A bead sample has to be prepared by the user beforehand, the exact same way (same mounting etc.) as the actual sample. If your lab does not have beads, contact the facility staff and they will show you which ones to purchase (Tetraspeck 100nm beads, they are stored in the fridge's door close to the Elyra).
1. Use the 63x 1.4 (SIM) or 1.46 (TIRF) objective, use your own bead slide, and focus on beads. Use the same filters as for your experiment and set up the cameras and tracks the same way as you will use them in your experiment. Set the illumination time to 100ms and use 0.1-1% laser power (when pressing min/max you should have >1000 white/grey values, when ticking 'Show all' in the 'Display' panel). Use the optimal grid pattern.
2. Take a Z-stack (3um, center) using half of the optimal sampling (double-click on [Opt] and divide by 2)
5. Choose your SIM processed sample-data as input image and your SIM processed beads data as input image 2, activate Fit and Affine, Click apply, save correction matrix and of course also your corrected image
