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Update Zeiss Elyra 7 authored by Clara Bodner's avatar Clara Bodner
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Instruments/Zeiss-Elyra-7.md
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...@@ -19,29 +19,29 @@ Use a yellow marker pen slide with a coverslip and a slide with beads (100nm Tet ...@@ -19,29 +19,29 @@ Use a yellow marker pen slide with a coverslip and a slide with beads (100nm Tet
1. With the 10x objective (no oil) focus on the marker pen slide 1. With the 10x objective (no oil) focus on the marker pen slide
2. Image Setup for camera 2 (TV2) calibration: lattice SIM, LBF 405/488/561/642, LP560, use the biggest (42µm) grid 2. Image Setup for camera 2 (TV2) calibration: lattice SIM, LBF 405/488/561/642, LP560, use the biggest (42µm) grid
![Imaging_Setup](uploads/be514fceb9bde8b03521a6bd21101694/Imaging_Setup.jpg){width=75%} ![Imaging_Setup](uploads/be514fceb9bde8b03521a6bd21101694/Imaging_Setup.jpg){width=50%}
3. Focus on the lattice pattern and take a ‘Snap’ 3. Focus on the lattice pattern and take a ‘Snap’
4. Go to ‘Graphics’ and draw an arrow/line across the pattern 4. Go to ‘Graphics’ and draw an arrow/line across the pattern
![Graphics](uploads/a5c85c9914eee4c16a20224af2c69ba5/Graphics.jpg){width=75%} ![Graphics](uploads/a5c85c9914eee4c16a20224af2c69ba5/Graphics.jpg){width=50%}
![Grid](uploads/a1e938d78726d18e1c3285c271ab996e/Grid.jpg){width=75%} ![Grid](uploads/a1e938d78726d18e1c3285c271ab996e/Grid.jpg){width=50%}
5. Make sure that the ends of the arrow are placed in the same line across the image, switch off the interpolation 5. Make sure that the ends of the arrow are placed in the same line across the image, switch off the interpolation
![Grid_left](uploads/a87bec7600fdf31fe9b0065a2f23a0f4/Grid_left.jpg){width=75%}![Grid_right](uploads/662b603921d038da3c9954f49d771493/Grid_right.jpg){width=75%}![Dimensions](uploads/b3c918277fc692ab3a6e923208c0dc5a/Dimensions.jpg){width=75%} ![Grid_left](uploads/a87bec7600fdf31fe9b0065a2f23a0f4/Grid_left.jpg){width=75%}![Grid_right](uploads/662b603921d038da3c9954f49d771493/Grid_right.jpg){width=50%}![Dimensions](uploads/b3c918277fc692ab3a6e923208c0dc5a/Dimensions.jpg){width=50%}
6. In ‘Graphics’ activate ‘M’ (measurement) and check the angle 6. In ‘Graphics’ activate ‘M’ (measurement) and check the angle
![Graphics_measure](uploads/8de9fc67bee34262465186b8022c2c1f/Graphics_measure.jpg)![Grid_angle](uploads/fbea05bb3dba5378465ed6d94f36d71a/Grid_angle.jpg){width=75%} ![Graphics_measure](uploads/8de9fc67bee34262465186b8022c2c1f/Graphics_measure.jpg)![Grid_angle](uploads/fbea05bb3dba5378465ed6d94f36d71a/Grid_angle.jpg){width=50%}
7. Go to ‘Maintain’, and drag out the ‘Dual Camera Adapter Alignment’ tool 7. Go to ‘Maintain’, and drag out the ‘Dual Camera Adapter Alignment’ tool
![Maintain](uploads/4cd5fe821496c21336794064bfb57643/Maintain.jpg){width=75%} ![Maintain](uploads/4cd5fe821496c21336794064bfb57643/Maintain.jpg){width=50%}
8. Tick 'Calibration pattern' 8. Tick 'Calibration pattern'
![Dual_camera_adapter_alignment_Rotation](uploads/412a55c368749394293a088ec4cd79a4/Dual_camera_adapter_alignment_Rotation.jpg){width=75%} ![Dual_camera_adapter_alignment_Rotation](uploads/412a55c368749394293a088ec4cd79a4/Dual_camera_adapter_alignment_Rotation.jpg){width=50%}
8. Change the ‘Rot [°]’ value. 8. Change the ‘Rot [°]’ value.
9. Untick 'Calibration pattern' 9. Untick 'Calibration pattern'
...@@ -53,20 +53,20 @@ Use a yellow marker pen slide with a coverslip and a slide with beads (100nm Tet ...@@ -53,20 +53,20 @@ Use a yellow marker pen slide with a coverslip and a slide with beads (100nm Tet
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: 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:
11. Go to ‘Maintain’, and drag out the ‘Dual Camera Adapter Alignment’ tool 11. 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) ![Dual_camera_adapter_alignment_z](uploads/baf459b3665bf0259ac6862e6041fa23/Dual_camera_adapter_alignment_z.jpg){width=50%}
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. 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.
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) 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)
![z_Stack](uploads/fdd2862a0cfb692cfa79a54091351401/z_Stack.jpg) ![z_Stack](uploads/fdd2862a0cfb692cfa79a54091351401/z_Stack.jpg){width=50%}
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! 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!
![Ortho_view](uploads/6019434e0cb0d148dd85d0c02a10e5b8/Ortho_view.jpg){width=75%} ![Ortho_view](uploads/6019434e0cb0d148dd85d0c02a10e5b8/Ortho_view.jpg){width=50%}
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. 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.
![Dual_camera_adapter_alignment_xy](uploads/1224a127963f3b439bf0547942ef9375/Dual_camera_adapter_alignment_xy.jpg){width=75%}![beads_xy](uploads/78d4f493776d040aacaddeff9ff0b495/beads_xy.jpg){width=75%} ![Dual_camera_adapter_alignment_xy](uploads/1224a127963f3b439bf0547942ef9375/Dual_camera_adapter_alignment_xy.jpg){width=50%}![beads_xy](uploads/78d4f493776d040aacaddeff9ff0b495/beads_xy.jpg){width=50%}
16. Tick 'Calibration pattern' and check XY Overlap. Store current position! 16. Tick 'Calibration pattern' and check XY Overlap. Store current position!
...@@ -75,7 +75,7 @@ Use a yellow marker pen slide with a coverslip and a slide with beads (100nm Tet ...@@ -75,7 +75,7 @@ Use a yellow marker pen slide with a coverslip and a slide with beads (100nm Tet
2. Take a large Z-stack (50 slices) using optimal sampling 2. Take a large Z-stack (50 slices) using optimal sampling
3. Go to processing – Channel alignment 3. Go to processing – Channel alignment
![Channel_alignment](uploads/c417b9e33f81c34e0d7237a3b287892c/Channel_alignment.jpg){width=75%} ![Channel_alignment](uploads/c417b9e33f81c34e0d7237a3b287892c/Channel_alignment.jpg){width=50%}
4. Choose the input image, activate Fit and Affine, Click apply 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.
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Provided by Max Perutz Labs IT-Team