Autofocus blues on microplates

Kevin Cunningham smskjc at
Mon Dec 1 14:45:01 EST 2003

"Peter Van Osta" <pvosta_ROMMEL at> wrote in message
news:20031201.133934.1884661237.22523 at
> 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/M2) 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

One thing you need to consider is the thickness of the bottom of the
specimen plate.  The objective is designed for a .17 mm thickness in glass.
In fact an old trick question is "What is the first element in a lens that
uses cover glass?".  The answer is the cover glass.  It is critical for your
lenses to have a .17 cover glass, particularly for the NA 0.80 objective.  I
might think of a plate maker that used .17 mm as the standard even if the
item is made of plastic.

Kevin Cunningham

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