Autofocus blues on microplates

Peter Van Osta pvosta_ROMMEL at
Tue Dec 2 04:25:08 EST 2003


I found a plate type with a bottom thickness suitable for the 63x 0.8
N.A. objective, but after storing the plate for one week at room
temperature and filled with 10 micron beads, the shape for the plate
bottom had changed such that the bottom was now on average 50 micron
lowered ???? Of course this would mean recalibration of the system.



In article <1TMyb.23735$sb4.1285 at>, "Kevin
Cunningham" <smskjc at> wrote:

> "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

Met vriendelijke groet,
Best regards,

Peter Van Osta

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

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