Wow! This is a fantastic response! 

first of all I would like to say thank you to all of you would took the time
to email me and reply to this thread. 

I'm aware of the commercial products available however as I'm just starting
my PhD and we have a small stalk pile of beads I think it would be better if
I get my hands dirty learning some skills! I have one concern some of you
recommend heating the plate so the beads adhere to them. How can you be sure
that the beads are not becoming deformed and possibly broadening?

Another thing is I would quite like to get the PSF through the complete z
axis. obviously a little invalid if they are adhering to the cover slip. I
also think that it would be more representative of a biological sample
suspended in the solution? 

Another tactic I read was to mix the beads with non fluorescent beads (or
beads that don't fluoresce at the lasers wavelength if you feeling rich) I
thought this is  a fantastic idea as it doesn't matter if they stick. I was
wondering if there are rejected beads in the manufacturing proses at could
be bought cheaply for this purpose? 

I had used 5.7 µm (mainly to make it easy to focus the z axis)  mixed with
0.049 µm beads. these where suspended in agar. 

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Hi Stuart,
 
What Z stepsize are you using, and what is the precision of you Z stage?
(i.e., are you using a Z Galvo-type system, or just a regular Z stepping
motor?)  What's the size of the beads that you are using?  In my experience,
you don't necessarily need to average 4 frames, 2-3 is enough if you have a
decent signal to noise ratio to start with. How much do you zoom in (i.e.
what's your XY pixel size)?   I shouldn't think that using the slowest scan
is advantageous either, I'd scan at more laser power and at a higher
frequency.
  You'll always have some bleaching but the final result should be okay even
without bleaching correction. You can buy pre-fabricated slides from
Invitrogen/Molecular probes, they are great but expensive, still worth the
money if you buy the multi-well version that has 4, 2, 1, 0.2, 0.1 um and
mix. You can buy the suspension cheaper and then follow Mariette's protocol
of course but then you'll end up with a  vial full of same-size beads that
you'll hardly use unless you produce a new test slide very often. 
 I hope this helps a bit,
 
Zoltan

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I am using a humble Z step motor when imaging the 5.7 µm bead my z step was
0.5µm I then went to 0.2µm when imaging the 0.049 µm beads. I had the zoom
set to the full 10X so I think my pixel size would represent 0.02 µm. I
realise  that is ridiculously small I was simply trying to gain the highest
image quality possible. 

Once again thank you 

Stuart McIntyre
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