... | ... | @@ -166,10 +166,10 @@ Use the yellow marker pen slide with a coverslip (on the desk next to the lens c |
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4. Choose the optimal grid pattern
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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:
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6. Go to ‘Maintain’ and drag out the ‘Dual Camera Adapter Alignment’ tool
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7. Change the Z value 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
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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
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8. Take a small Z-stack (20 slices, center) using optimal sampling (double-click on [Opt])
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9. SIM^2 process Z-Stack in case of SIM (no processing in case of TIRF or WF)
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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 left.
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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.
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11. Store current position! Then, acquire another Z-stack and repeat until perfect overlap of all color channels is achieved.
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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!
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13. Use the 'Profile > Display > Marker > Difference' tool to check the overlap
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