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you are not restricted to a Z series and instead you could acquire a XZ image, a
single image that will
give the thickness of the biofilm. An XZ slice is a single linescan that is
repeated as the stage is moved along the
Z axis.  In the final image the Y axis is depth (Z).
For the Z steps in the XZ slice use half the resolution you require - if you
want 1um use a 0.5 micron steps.
theoretically you should base the slice interval on the resolution of objective
in Z and XY but since you are after a fairly unsubtle measurement you can
reasonable ignore this.
for statistical purposes several XZ slices taken at random locations are
advisable.
outstanding problems are converting the Z distances in the XZ slice into microns
since the
refractive index of the film is unknown- however it should be possible to slice
a film in the XZ direction and then image it in the XY plane to get both the
correct depth, and hence find the RI of the biofilm - which you can reuse as a
correction, assuming the RI of the biofilm is unchanging.  Cutting will be
easier if the biofilm if grown on a plastic coverslip

Jeremy Adler
CIAL
NIMR
Mill Hill
London


"Wijnholds, Anita" wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear all,
>
> For Theresa, Stephen, Mario and all the others: Thank you for the good
> thougths about this topic. I hope you get even more ideas when I explain the
> measurements a little bit further.
> We are measuring on a glass slide on several places and collect Z-stacks of
> the forming biofilm at these places. The point is that we want to measure
> influences of light, season and temperature on the development of the
> biofilms. We are not interested, at the moment in getting very nice
> pictures, but just want to quantify the effects of light and so on. We have
> software to analyse the stacks 2D (areas) of three channels and combine that
> with the z-steps to 3D (volume) numbers. The main parameter we measure in a
> slice is (the percentage of) the area which is covered by the biofilm. To
> see what the influence of for instance light is, it is not necessary to do
> z-sampling according to Nyquist I think.
> But what is important to quantify the effects of several treatments? Is it
> just a matter of statistics? Can we say for instance, we are going to
> measure each time at 5 points in z: if the biofilm is 50 micron thick than
> we measure at 0, 10, 20, 30 and 40 micron? Or are there other criteria which
> are important? I am interested in your ideas about this.
>
> A merry Christmas and a Happy New Year to all of you!
>
> With kind regards,
>
> Anita Wijnholds