Problem with your opera calibration plates

[Ghislain Bonamy]

Dear all,

I was wondering if you too have been having problems with your
calibration plates? These plates are an intrinsic part of the
functioning of the Opera. Therefore having good quality plate is
critical. It appears that in the last 2 years, PE has been incapable
of providing us with good quality plates. For instance, there are
often precipitated dyes in the well used to create the Reference image
(or worst dust particles). The wells for the skewAnalysis often have
dried up, beads are not homogenously distributed etc.

If you have also experience similar problems it would be nice to know.
In addition, did anyone experiment with the following ideas (and
combinations thereof):

1) Add water in the empty wells to reduce evaporation of the other
wells
2) Add mineral oil or Paraffin to prevent evaporation.
3) Filter the well containing the dye to remove precipitates/other
particles.
4) Used 37°C / 4°C centrifugation cycles, to force beads to be
homogenously distributed at the well bottom?

I have personally tried some of these ideas with some success on old
plates. Perhaps, you have also some suggestions to bring to PE to fix
this calibration plate issue. If more people can suggest to PE to re-
visit the way they manufacture there calibration plates, perhaps this
prevent some of the frustration when collecting the correction files
(ie. Skewcrop / RefImage).

Best,

Ghislain

[Michelle Ocana]

Finding a quality bead site for skewanalysis is very time consuming. In fact, it takes most of the time (barring any errors) of building an experiment. The real problem with skewanalysis for me is that often the skew will not throw an exception that it failed but will not properly overlay the image due to insufficient information (not enough beads) in a particular portion of the field. I now run the experiment on the bead plate for a few sites to ensure that the pixels are properly aligned. It's time consuming. There should be some kind of visual clue or message to ensure that skew analysis does what it is supposed to consistently in all quadrants of the field. At 60x (our most popular objective) it's a bear to work with because resolutions and proper overlay is critical. I find myself running skew analysis may times until I achieve a good skew.
The reference wells are only slightly less annoying to me. I have found that a 50um focus will sufficiently exclude any precipitate that is near the bottom of the well. I just can't understand why they would create such varying concentrations of dye in those wells. Some adjustment plates I am able to capture a ref image with a 40ms exposure with reduced laser power and others have been 1s or more.
It indicates to me that they don't have very good QC in their adjustment plates. Anyone out there create their own? I was considering it.

Regards,
Michelle

[gbonamy]

Michelle,

I meant to tell you that I had in fact experiment with this. I did a
few years back when I used our 60X W objective experiment with making
my own bead plates, and found that using the 1µm beads where much more
suitable for the 60X objectives that the 2.5µm beads provided.
Although I do not recall what was the best concentration of bead to
use, I can give you the guidelines that I followed to make my plate:

1) Use a 384-well plate (easier to navigate in with the Evoshell) (put
in 42°C Incubator before dispensing beads)
2) Pre-warm centrifuge to as high as possible (42°C)
3) Sonicate beads for 10'
4) Dilute beads in a warm filtered DPBS 10-15% gelatin solution
(determine the concentration based on the number of beads needed per
well area)
5) Dispense 20-30µl of the solution containing the beads
6) Place plate in centrifuge and spin at 1000G for 30' (set for 1Hr)
7) After 30' Change temperature of centrifuge temperature to 4°C
8) After 20' or so, take your plate out and add 20-30µl of mineral oil
(to prevent evaporation)

Hope this helps.

Best,

Ghislain