Hi Stuart,
You've learned the first lesson of confocal microscopy!  The problem  
is you are probably following very outdated instructions for the  
confocal.  The manuals and factory training for older confocals  
emphasized noise reduction through slow scan rates and repetitive  
averaging.  These gave dim, noise-free images of bleached specimens.  
Start with the 'Normal' scan rate and Kalman averaging at 3.  This  
will scan with a 5-fold reduction in exposure and gives nearly the  
same noise reduction on our MRC-1024.  Normal scan is 2 us/pixel, Slow  
scan is 8 us with the resulting intensities divided by 4.

If you are deconvolving the PSF and your specimens, try collecting a  
stack at Normal rate, without averaging.

You can test noise reduction as a function of dwell time and averaging  
to see where the noise reduction levels off as a function of Kalman  
repetition to get a sense of how much averaging is really necessary  
for your system.

Apply a coverslip with immersion oil to a fluorescent plastic  
reference slide and focus about 20 microns into the plastic using your  
favorite oil lens, with a zoom of  4 to get a uniform intensity across  
the field.
  Adjust laser and Gain to fill the histogram about 3/4 for a single  
image using Normal scan speed and Direct mode. Use these settings for  
collecting images at Slow Scan and Normal Scan using the Direct mode  
and with a Kalman of 2 to 8 or so..
Move the slide to a new field for each capture.
Graph the mean and Std. dev for each resulting image. Then graph the  
coefficients of variance (var=Std. dev/mean intensity) for each image.

Regards,
Glen

On Nov 24, 2008, at 1:18 AM, stu_the_flat wrote:

> Hi
>
> I'm new to this forum. I have tried searching this forum but I can't  
> find a
> topic. I apologize if I missed it.
>
> I'm a PHD student I'm relatively new to confocal imaging I am trying  
> to
> image sub resolution beads to measure the point spread function.
>
> I am using a bio rad system I use a 60X water immersion lens I set the
> system to 1024 X 1024 at 16 bit resolution. Kalman sampling was set  
> to 4 and
> the scanning head was as slow as possible and the laser power was as  
> low as
> possible to measure the beads.
>
> I have found that this is the perfect recipe for measuring photo  
> bleaching!
>
> I tried looking around for some sort of protocol on imaging sub  
> resolution
> beads but I was unable to find anything. Also as I understand it  
> there will
> always be some element of photo bleaching? Is it easy to compensate  
> for if I
> can calculate how much beaching as occurred.
>
> Similarly if anybody as any tips on creating the slides with the  
> beads I
> would be grateful mine are quite messy.
>
> Many thanks
>
> Stuart McIntyre
>
>
> -- 
> View this message in context: http://n2.nabble.com/Protocol-for-imaging-micro-beads--tp1571444p1571444.html
> Sent from the Confocal Microscopy List mailing list archive at  
> Nabble.com.