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Hi,

I have some questions regarding the quality of microplates for
microscopy, mainly 96- and 384 well SBS-standard layout formats (1536
and 6144 layouts would be interesting too of course).

I have been doing some quality tests for certain types of multiwell
plates which are very popular for use in "readers" with our 40x 0.75
N.A. objective and a 63x 0.8 objective in brightfield and fluorescence.
These objectives have a very good spatial resolution and capture a lot
of light for weak fluorescence (I ~ N.A.^4/M^2). However these
objectives have a short working distance and as such the quality of the
plate bottoms is important.

Our autofocus system only needs about 5 videoframes to gather enough
information to find a focus level, so with a 40 millisec. frametime (PAL
25 fps.) and some time for mechanical movement, it does a complete image
content based autofocus in 0.3 or 0.4 seconds (300 or 400 milliseconds).
But this only is the case, when a focuslevel is found within a certain
range, i.e. the travel range needed for 5 frames, which is in principle
determined by the Nyquist sampling theorem for Z-slicing.

We also do a complete range check, as we want to focus in those
microscopy modes which can cause multiple maxima in a focus algorithm
(patented) in which case we focus on the highest peak/score found (
Geusebroek J.M., Cornelissen F., Smeulders A. W. M., and Geerts H.,
Robust autofocusing in microscopy, Cytometry, 36(1):1-9, 2000 ).

Using an image content based autofocus system, allows us to use very
fine Z-level focusing into cells with a varying offset to the multiwell
plate bottom.

I found out that when you plot the bottom profile of standard multiwell
plates as a 3D landscape, these plates look more than a mountaneous
region than having a flat profile.

I also found some microplate types with very thin plastic bottoms and a
flat plate bottom, but they are of course a bit more expensive. Glass
bottom plates are an alternative in some cases, but I heard that the
glue which is used for these plate-bottoms is not compatible with some
of the solvents used in farmaceutical research (DMSO, etc), so the
bottoms tend to fall off the plate after 24 h. incubation, which would
be rather inconvenient for our time-lapse work :-( ? So using good
quality plastic bottom plates seems to be one possible solution ?

A possible solution for the variability of the plate bottoms is to do a
prescan at lower mag./N.A., either with a plate bottom finding
laser-system or a content-based autofocus and model the plate with a
mathematical model, but this takes time.

Using Long Distance (L.D.) objectives is another possible solution, but
these objectives have a lower N.A. and as such less spatial resolution,
which hampers subcellular imaging and analysis. Also the reduced N.A.
has a very dramatic efect on the amount of light captured by the
objective (I ~ N.A.^4/M^2) and we always do our image capturing in
real-time, we just use very sensitive cameras which allow imaging
without the need to integrate frames. The image capturing must be done
at 40 msec. (PAL 25 fps.), which is important when doing large volumes
of multiwell plates in our screenings. Using the 0.75 and 0.8 N.A.
objective in combination with the ultrasensitive camera allows us to
dilute reagents up to 1000x, which is very good for our assay costs when
doing large screens of subcellular phenomena :-)

I would like to know what is the experience of other researchers with
the quality of multiwell plates on microscopes or similar (automated)
readers ? Does anyone else uses high N.A. objectives on multiwell plates
  for screening ? As far as I understand the SBS-standards define
XY-measures of multiwell plates, but leave the bottom offset and bottom
thickness to the manufactureres of multiwell plates ?

Best regards,

Peter Van Osta

Union Biometrica N.V./S.A.
European Scientific Operations (ESO)
Cipalstraat 3
B-2440 Geel
Belgium
Tel.: +32 (0)14 570 620
Fax.: +32 (0)14 570 621

http://www.unionbio-eu.com/

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