Automated Focus Map - Multiwell plate

This option is accessible from the Positions or Tiling dimension using the Set button.

afm_multiwell_intro
  1. Click on Focus Map to configure it.
    afm_multiwell_step1
  2. Select your well where you would like to create your Focus Map then click on Confirm.
    afm_multiwell_step2
  3. Click on Auto to configure the Focus Map.
    afm_multiwell_step3
  4. Choose the step and size of the stack.
  5. You can select an existing pattern from the drop-down list (after that go directly to the step 9) OR you can create a new pattern by clicking on Edit.
    afm_simple_tiling_steps4_5
  6. Select the pattern you want: Grid or Random:
    1. For Grid, indicate the number of image you want to acquire horizontally and vertically, and the Gap (distance between 2 images).
      The offset X and Y allows you to move all your patterns in the well.
    2. If you choose a Random, indicate the position number and the minimal distance between positions.
  7. Save your pattern by clicking on Add.
  8. Come back to the dimension by clicking on Switch to Tiling.
    afm_simple_tiling_steps6-8
  9. Choose the channel to acquire the stack.
  10. You can apply some filters by checking the box Filter unsure position:
    • Filter Radius: filter to smooth the noise by replacing each pixel by the median of the neighbor (0= no smoothing, 1= median on 3x3 square around the pixel, 2= median on 5x5 square around the pixel, …).
    • BackgroundRoi: automatic calculation of Background and BackgroundDelta values by drawing an ROI. Background will be the mean value of the ROI and BackgroundDelta will be the double of the standard deviation.
    • Background: background mean value.
    • Background Delta: amplitude of the noise.
    • Valid pixels: percent of pixel that should be far of the background to validate the position
  11. Click on Start.
    afm_simple_tiling_steps9-11

    System will perform all points to create the Focus Map and you will obtain a list of points. By clicking on Goto or on the image, you can check the focus.

  12. Click on the Confirm, the Focus Map is ready to use.
    afm_simple_tiling_step12
  13. Select the wells where you want to apply the Focus Map then click on Confirm.
    afm_multiwell_step13
  14. Select the wells that you want to acquire and click on Add.
    afm_multiwell_step14
  15. Select if you want to use or not the current Z to adjust the FocusMap.
    afm_multiwell_step15
    After this step, you can continue to configure your acquisition sequence.