Instruments/Zeiss-Elyra-7.md

@@ -2,14 +2,48 @@
 
 
 
-## Manuals & Guides
+# Manuals & Guides
 
-### Startup & Shutdown Guide
+## Startup & Shutdown Guide
 
-### Sample Preparation
+## Sample Preparation
+
+## Colocalization Analysis
 
 ### Camera Alignment 
 
+**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.
+
+#### Camera Rotation
+
+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).
+
+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
+3. Focus on the lattice pattern and take a ‘Snap’
+4. Go to ‘Graphics’ and draw an arrow/line across the pattern
+5. Make sure that the ends of the arrow are placed in the same line across the image, switch off the interpolation
+6. In ‘Graphics’ activate ‘M’ (measurement) and check the angle 
+7. Go to ‘Maintain’, and drag out the ‘Dual Camera Adapter Alignment’ tool
+8. Change the ‘Rot [°]’ value. 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
+
+#### Camera xyz-Alignment
+
+9. 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). If working in multitrack always keep track 1 with camera 2!
+10. Go to 'Continuous' and focus through the beads. The optimal focus (smalles 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
+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
+14. Go to orthogonal View and, in Dual Camera Adapter Alignment tool, fine-tune the Z value if necessary. Then, acquire another Z-stack until perfect overlap of channels is achieved. Store current position! 
+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. Store current position!
+
+#### 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
+3. Go to processing – Channel alignment
+4. Choose the input image, activate Fit and Affine, Click apply
+5. Save the results in order to correct your images later on.
+
 ### User Manual
 
 ## Technical Specifications