Dear Stuart,
Why are you using .049 um beads? A PSF bead only needs to be smaller  
than the optical resolution of the objective lens. compare the volume  
of that bead to a 120 or 170 um bead to get an idea of the difference  
in possible number of fluorophores available to generate a signal.  
Such small beads and prolonged scanning creates an exercise in  
photobleaching. The RI difference between coverglass and the bead can  
definitely be an issue. However, if you are trying to generate a PSF  
for imaging monolayers of cells adhered to glass, that could be a  
useful PSF. A mounting medium with an RI close to that of the glass  
would reduce the coverglass issue.  A bigger issue when working close  
to the coverslip may be reflection.  The question is why are  
generating the PSF? To characterize the microscope or to describe the  
imaging conditions through a sample? I find that adding a few larger  
beads, ca 6 um or 15 um, is very useful for finding the coverslip.  
they also create a spacer for when I mount the beads. For a thicker  
volume, DAKO Glycergel (RI 1.44-1.47) or Fluormount G (RI 1.39) are  
useful hardening media. I've heard reports of latex beads melting in  
agarose that is too hot.

Regards,
Glen

> 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.
>
> --------------------------------------------------------------------------------------------------
>
> 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
>
> -------------------------------------------------------------------------------------------------
>
> 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
> -- 
> View this message in context: http://n2.nabble.com/Protocol-for-imaging-micro-beads--tp1571444p1575704.html
> Sent from the Confocal Microscopy List mailing list archive at  
> Nabble.com.