Autocalibration failing?

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1

Hi,

I’m trying to get the microscope to auto-calibrate, but I keep getting the error “Moved the stage by [32768,0,0] but saw no motion.”

It’s moving, and it’s in focus, so I’m wondering what else I’m doing wrong.

Thanks!
Dan

2

Hello, try to other slide.

3

Hi Dan, that’s very strange - it suggests the image analysis code that’s responsible for tracking motion of the stage is going wrong. The first thing I’d ask is whether the stage is visibly moving - but you’ve answered that. Trying a different sample might help - and if it does, it would be great if you could post an image of the sample that didn’t work.

Actually, it would be great if you could post a few images, with differing distances between them (e.g. change X by 1, 10, 100, 1000 steps in the “navigate” pane). Then it’s possible to run the camera-stage-mapping image analysis code on it to check what’s failing.

My hunch is that the most likely problem is that something’s gone wrong with the stream and the images that are being used for tracking are all blank. If that’s the case, it won’t work even with a new sample. You can test that by going to http://microscope.local:5000/api/v2/streams/snapshot - if you see an image, it’s working correctly.

4

Hi Richard,

Thanks for getting back to me. I think I figured out the issue. Part of it is PEBKAC, part missing/hidden docs.

Firstly, I was miscalibrating/whitebalancing the camera. There were lots of pretty, mottled colors all over the FOV. Finding the right position for the illumination was a bit tricky, but I’ve got it down to only 3-4 patches of pink and green dapples in the image. I’m sure this was confusing the mapping algorigthm.

Is the camera calibration process described in some docs that I haven’t found, yet? I.e., use a flat, white image, don’t use something with discernible features, etc.?

Secondly, it was NOT actually focussed on the prepared sample (I’m using Amscope prepared slides). Stupid me, I inserted the slide upside down so it was focussing on the dust on the slide. Reversing it allowed for the proper cover glass thickness to be accounted for by the plan 40 objective lens.

My background is astronomy - point the telescope at the white spot in the dome, turn on the quartz lamps, take flat images for image calibration. Objects are always up in the sky - can’t turn those over.

:wink:

Best!
Dan

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5

Actually, I fiddled with the illumination stand a bit more and produced an nice evenly illuminated FOV.

6

Brilliant, well done! We should document how to align the illumination and then calibrate it - Joel started on this with the calibration “tour” that runs when you first start the microscope, but in brief the procedure is:

  • Adjust the illumination (height and X/Y position) to maximise brightness. You probably need to alternate between adjusting height and adjusting lateral position a few times to optimise it, until you get a feel for it. Once it’s set up, you’re more likely to need to tweak Z because it’s been knocked; X and Y should be fairly stable.
  • Place a sample in (coverslip down) and focus on it
  • Remove the sample and calibrate the camera
  • Replace the sample, re-focus, and calibrate the stage

If anybody felt like writing this up for the handbook/instructions I’d be delighted to include it… it’s on our to do list, but that’s always a longer list than we can get through!