Instruments/Zeiss-Elyra-7.md

@@ -167,7 +167,7 @@ Use the yellow marker pen slide with a coverslip (on the desk next to the lens c
 5. Go to 'Live' 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. Focus on the TV1 (camera 1) channel and change the Z value of the TV2 (camera 2) channel, 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])
+8. Take a Z-stack (3um, center) using half of the optimal sampling (double-click on [Opt] and divide by 2)
 9. SIM<sup>2</sup> 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 right in the Ortho View. 
 11. Store current position! Then, acquire another Z-stack and repeat until perfect overlap of all color channels is achieved. 
@@ -189,14 +189,15 @@ Use the yellow marker pen slide with a coverslip (on the desk next to the lens c
 Channel Alignment has to be done before **each colocalization experiment** using multiple color channels and for **each used filter** in the camera link, for **each used objective** and for **each configuration of different cameras/tracks** individually. A bead sample has to be prepared by the user beforehand, the exact same way (same mounting etc.) as the actual sample. If your lab does not have beads, contact the facility staff and they will show you which ones to purchase (Tetraspeck 100nm beads, they are stored in the fridge's door close to the Elyra).
 
 1. Use the 63x 1.4 (SIM) or 1.46 (TIRF) objective, use your own bead slide, and focus on beads. Use the same filters as for your experiment and set up the cameras and tracks the same way as you will use them in your experiment. Set the illumination time to 100ms and use 0.1-1% laser power (when pressing min/max you should have >1000 white/grey values, when ticking 'Show all' in the 'Display' panel). Use the optimal grid pattern.
-2. Take a large Z-stack (50 slices) using optimal sampling (double-click on [Opt])
-3. Go to Processing – Channel alignment
+2. Take a Z-stack (3um, center) using half of the optimal sampling (double-click on [Opt] and divide by 2)
+3. SIM process your bead data
+4. Go to Processing – Channel alignment
 
 ![Channel_alignment](uploads/17ba46853b6143467d8f1913ca8a3b5b/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
-6. You can apply your correction together with the SIM processing
+5. Choose your SIM processed sample-data as input image 1 and your SIM processed beads data as input image 2, activate Fit and Affine, Click apply, save corrected sample-data
+
+
 
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