| ... | ... | @@ -19,29 +19,29 @@ Use a yellow marker pen slide with a coverslip and a slide with beads (100nm Tet |
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1. With the 10x objective (no oil) focus on the marker pen slide
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2. Image Setup for camera 2 (TV2) calibration: lattice SIM, LBF 405/488/561/642, LP560, use the biggest (42µm) grid
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{width=75%}
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{width=50%}
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3. Focus on the lattice pattern and take a ‘Snap’
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4. Go to ‘Graphics’ and draw an arrow/line across the pattern
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{width=75%}
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{width=75%}
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{width=50%}
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{width=50%}
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5. Make sure that the ends of the arrow are placed in the same line across the image, switch off the interpolation
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{width=75%}{width=75%}{width=75%}
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{width=75%}{width=50%}{width=50%}
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6. In ‘Graphics’ activate ‘M’ (measurement) and check the angle
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{width=75%}
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{width=50%}
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7. Go to ‘Maintain’, and drag out the ‘Dual Camera Adapter Alignment’ tool
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{width=75%}
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{width=50%}
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8. Tick 'Calibration pattern'
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{width=75%}
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{width=50%}
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8. Change the ‘Rot [°]’ value.
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9. Untick 'Calibration pattern'
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| ... | ... | @@ -53,20 +53,20 @@ Use a yellow marker pen slide with a coverslip and a slide with beads (100nm Tet |
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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:
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11. Go to ‘Maintain’, and drag out the ‘Dual Camera Adapter Alignment’ tool
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{width=50%}
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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.
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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)
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{width=50%}
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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!
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{width=75%}
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{width=50%}
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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.
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{width=75%}{width=75%}
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{width=50%}{width=50%}
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16. Tick 'Calibration pattern' and check XY Overlap. Store current position!
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| ... | ... | @@ -75,7 +75,7 @@ Use a yellow marker pen slide with a coverslip and a slide with beads (100nm Tet |
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2. Take a large Z-stack (50 slices) using optimal sampling
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3. Go to processing – Channel alignment
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{width=75%}
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{width=50%}
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4. Choose the input image, activate Fit and Affine, Click apply
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5. Save the results in order to correct your images later on.
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| ... | ... | |
