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  • Inconsistent Manual Focusing on GAII

    Dear GA users,

    Has anyone had a problem where you already zeroed Z on a tile, you move the objective and return to the same tile and Z is no longer focused? I've been having this problem recently where after zeroing Z on lane 4 column 2 row 25 and then proceeding to align X and the XY drift and going back to that tile to check Z, and it will no longer be in focus. I used to be able to have Z still in focus or at least it is only off by very little (~100) but now, it is always off by at least 1000-3000. I also have problems going through the fine focusing procedure as the lab tracking sheets are set up for (by 100's) because the Z keeps going off by increments in the 1000's.

    Also, when I try to align X and the XY drift, between that and rechecking my Z, I often have to reset my XY drift by the exact same increment as when I zeroed it before going back to check if Z is still in focus as if the origin was never set the first time. And my X and XY drift alignments are set in greater values than before. I used to be able to set X = 3 to maybe 15 but now it's more like 30-55. To me, these differences seem too large. Are your X values going up to the 50's? More troubling is of course that even with a bead flowcell, I have to constantly reset the XY drift by the same increment as if the origin was not set.

    The problem is once I quickly do the fine focus on Z and go straight to first base incorporation, then it will usually look ok and this problem does not reoccur consistently. However, we have had our previous run fail because the Z suddenly went out of focus in the middle of the run. We could see that image A started to get blurry and C went completely out of focus. My hypothesis is something is lose in our machine, but with the inconsistency of reoccurrence, it has been very difficult for us to receive service on our machine that regains our confidence in subsequent runs.

    Does anyone else have a similar experience and do you have any idea what is wrong and how can we fix this?

    Thanks,

    PF

  • #2
    Auto focus

    Hi PF,
    Are you activating the auto focus laser when you z focus?
    -Elwell

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    • #3
      What do you mean by am I "activating" the autofocus laser? I am doing focusing completely manually, as opposed to using FocalAssist, if that's what you're asking. Because I can't seem to get FocalAssist to work on my system. I do attempt to go through the procedure of checking the FQ values for each of the bases while focusing. The only other thing I can think of with "auto" in it is auto calibration. I run that right after manual focusing.

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      • #4
        Auto Scale

        When you manually focus, you have the image of the tile next to your settings. If you right click on the image, highlight "Auto Scale" and click on "Yes", as far as I know, this will turn on the auto focus laser. My FAS told me there is a auto focus laser will use the z-value you tell it and automatically focus on every tile (not all tiles are in focus with the z-value that is set for zero). I hope this helps, let me know if it still doesn't make sense.

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        • #5
          Dear Elwell,

          Hm, that is very interesting. No on has ever explained the autoscale function to me that way before. I was just told that turning it on will often help even out the different intensities between clusters within a given tile so that the end result is you will see more distinct clusters and they will appear brighter. I was unaware of this autofocus laser and would never have thought of "activating one" by turning on autoscale because I can turn it on after taking a picture to see the difference in my image, but I was never under the impression that it re-takes the picture since I've never heard the laser and imaging sounds when I turn on autoscale. I just thought it was a software-dependent function that leads to improved contrast. In any case, I do turn on autoscale and it does help my images look sharper, but it does not help the inconsistency in focusing over time.

          Our new suspicion is that the inconsistency in focusing is temperature dependent. However, I am still skeptical about this being the only cause as I really do think the inconsistency has gotten progressively worse since our upgrade to GAII and if it is just ambient temperatures (which have not changed over this time), then why has the focusing gotten worse?

          Still, thanks for you input!

          PF

          Comment


          • #6
            Plotting the z height from the log files over time might give you some idea as to what's going on. Page 8 of: http://www.genographia.org/portal/to..._download/file has some plots of the Z heights we see.

            My understanding is also that the GA2s are harder to focus, they have a smaller depth of field.

            Comment


            • #7
              Thanks new300. We did plot that with our FSE but did not see anything that seemed concerning from the graphs.

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