| ... | @@ -35,44 +35,37 @@ The system will be by default at 27°C to minimize drift. Use the oil for 30°C. |
... | @@ -35,44 +35,37 @@ The system will be by default at 27°C to minimize drift. Use the oil for 30°C. |
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## Dual-Camera Alignment & Colocalization Analysis
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## Dual-Camera Alignment & Colocalization Analysis
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Camera Alignment has to be done before EACH experiment using both cameras and for each filter in the camera link and for each objective individually. It has to be done again if the camera was pushed by accident, or if the cover of the camera link has been opened.
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Camera Alignment has to be checked/done before **each experiment** using both cameras and for **each used filter** in the camera link and for **each used objective** individually. It has to be done again if the camera was pushed by accident, or if the cover of the camera link has been opened.
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### Camera Rotation
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### Camera Rotation
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Use a yellow marker pen slide with a coverslip and a slide with beads (100nm TetraSpeck beads, mounted the same way as your sample). If your lab does not have beads, you can ask the facility staff, which one the purchase (they are stored in the fridge close to the Elyra).
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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's door close to the Elyra).
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1. With the 10x objective (no oil) focus on the marker pen slide
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{width=40%} {width=40%}
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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=30%}
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1. Select the objective that you will use for imaging
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2. Set up camera 2 (TV2) for calibration (camera 1 will not be calibrated): lattice SIM, LBF 405/488/561/642, LP560, use the biggest (42µm) grid
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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=40%}
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3. Focus on the lattice pattern and take a ‘Snap’ (use the ocular for focusing and adjust the image with min/max on the camera)
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{width=100%}
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4. Go to ‘Graphics’ and select the arrow
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5. Draw several lines across the pattern
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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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6. 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=30%} ... {width=27%}
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{width=30%} ... {width=27%}
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{width=50%}
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{width=50%}
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6. In ‘Graphics’ activate ‘M’ (measurement) and check the angle
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7. In ‘Graphics’ activate ‘M’ (measurement) and check the angle
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{width=50%}
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{width=50%}
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{width=40%}
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{width=40%}
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7. Go to ‘Maintain’, and drag out the ‘Dual Camera Adapter Alignment’ tool
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8. Go to ‘Maintain’, and drag out the ‘Dual Camera Adapter Alignment’ tool
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9. Change the ‘Rot [°]’ value.
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{width=40%}
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8. Tick 'Calibration pattern'
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{width=40%}
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8. Change the ‘Rot [°]’ value.
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9. Untick 'Calibration pattern'
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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
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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
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11. Tick 'Calibration pattern' in the ‘Dual Camera Adapter Alignment’ tool and check the rotation again by eye
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12. Untick 'Calibration pattern'
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### Camera xyz-Alignment
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### Camera xyz-Alignment
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| ... | | ... | |
