Difference: NikonTiFocusSurface (10 vs. 11)

Revision 1116 Oct 2012 - Main.EdEng

 
META TOPICPARENT name="NikonTiInstructions"
Contents

How to deal with uneven grid?

1) take a Z-­‐stack between a defined Z range
2) auto focus each image or every so often
3) use a focus surface
4) manually take images in focus at select areas.

Focus surface feature

Focus surface feature is in XYZ Overview tab

fs.png

Workflow:
1) Take a 4x image to use as a map.
2) Change to the 50X and manually select 2 dozen or so points randomly to manually find the correct Z-­‐height. Use course focus ~2 turns to get in focus, then use stage for fine focus.
3) After enough points are selected you can calculate a focus surface. Use those manually entered XYZ points to define the focus surface and define an area to take a large image. In the ND acquisition menu: the manually entered points will form a green line for z-­‐height and calculate an orange line that it'll use as the z-­‐height to capture an image.
4) Take a large image in both BF and GFP using the focus surface.

Examples

20X

42 images stitched together with 15% overlap
Area: ~1.7mm x 1.7mm
Time: SF-­‐5min SF&SBS-­‐7min

   fs20x.png

   fs20x2.png

40X

154 images stitched together with 15% overlap
Area: ~1.7mm x 1.8mm
Time: SF-­‐10min SF&SBS-­‐20min

   fs40x.png

50X

180 images stitched together with 15% overlap
Area: ~1.5mm x 1.6mm
Time: SF-­‐18min SF&SBS-­‐25min

   fs50x.png

   fs50x2.png

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