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Update Instruments/Zeiss Elyra 7 authored by Clara Bodner's avatar Clara Bodner
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Instruments/Zeiss-Elyra-7.md
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......@@ -64,44 +61,53 @@ Use the yellow marker pen slide with a coverslip (on the desk next to the lens c
![Grid_angle](uploads/fbea05bb3dba5378465ed6d94f36d71a/Grid_angle.jpg){width=40%}
8. Go to ‘Maintain’, and drag out the ‘Dual Camera Adapter Alignment’ tool
9. Change the ‘Rot [°]’ value.
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
11. Tick 'Calibration pattern' in the ‘Dual Camera Adapter Alignment’ tool and check the rotation again by eye
11. Tick 'Calibration pattern' in the ‘Dual Camera Adapter Alignment’ tool and check the rotation again
![calibration_pattern_perfect](uploads/412c4bda0e0caf2cf96075bac5159f62/calibration_pattern_perfect.PNG)
12. Untick 'Calibration pattern'
### Camera xyz-Alignment
1. 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.
2. 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:
3. Go to ‘Maintain’, and drag out the ‘Dual Camera Adapter Alignment’ tool
![Dual_camera_adapter_alignment_z](uploads/baf459b3665bf0259ac6862e6041fa23/Dual_camera_adapter_alignment_z.jpg){width=40%}
1. Use the 63x 1.4 (SIM) or 1.46 (TIRF) objective and the bead slide and focus on beads
2. Use the same imaging method (LASER WF, SIM, Apotome etc.) and the same filter settings as for your experiment!
4. 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.
5. 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)
![cam_z_perfect](uploads/a86788dd6a3694c136010bb0463407bf/cam_z_perfect.PNG)
![z_Stack](uploads/fdd2862a0cfb692cfa79a54091351401/z_Stack.jpg){width=40%}
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 'Continuous' 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. 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
8. Take a small Z-stack (20 slices, center) using optimal sampling (double-click on [Opt])
9. SIM^2 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 left.
11. Store current position! Then, acquire another Z-stack and repeat until perfect overlap of all color channels is achieved.
12. In 'Continuous' 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
6. 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!
![cam_x_lineprofile](uploads/0b92f0c052b16846ceaaf2badf438694/cam_x_lineprofile.PNG)
![Ortho_view](uploads/6019434e0cb0d148dd85d0c02a10e5b8/Ortho_view.jpg){width=40%}
14. Tick 'Calibration pattern' and check XY overlap, fine-tune if necessary. Store current position!
7. 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.
![calibration_pattern](uploads/82161269165ec234399dc008455ae293/calibration_pattern.PNG)
![Dual_camera_adapter_alignment_xy](uploads/1224a127963f3b439bf0547942ef9375/Dual_camera_adapter_alignment_xy.jpg){width=40%}
![beads_xy](uploads/78d4f493776d040aacaddeff9ff0b495/beads_xy.jpg){width=40%}
15. Also check rotation again with the 'Calibration pattern'. Especially the dots in the center should be well aligned
8. Tick 'Calibration pattern' and check XY Overlap. Store current position!
![alignment_perfect](uploads/aa812ef246b98650bcd219c65a3c7b2f/alignment_perfect.PNG)
### Channel Alignment
1. Change to 63x 1.4 (SIM) or 1.46 (TIRF) objective, use your own bead slide, and focus on beads
2. Take a large Z-stack (50 slices) using optimal sampling
1. Use the 63x 1.4 (SIM) or 1.46 (TIRF) objective, use your own bead slide, and focus on beads
2. Take a large Z-stack (50 slices) using optimal sampling (double-click on [Opt])
3. Go to processing – Channel alignment
![Channel_alignment](uploads/c417b9e33f81c34e0d7237a3b287892c/Channel_alignment.jpg){width=40%}
4. Choose the input image, activate Fit and Affine, Click apply
5. Save the results in order to correct your images later on.
5. Save the results in order to correct your images later on
6. You can apply your correction together with the SIM pocessing
## Shutdown
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