Before using the beads stock solution sonicate the tube shortly to 
destroy bead aggregates.

__
This is how I make my bead slides. Some do not use a coating but my 
beads always started swimming without coating._
_Mariette Kemner-van de Corput
Erasmus MC, Rotterdam, NL

_
_

_TetraSpeck beads: http://probes.invitrogen.com/media/pis/mp07279.pdf _

200nm TetraSpeck 4 colour polystyrene beads (Invitrogen)

500nm TetraSpeck 4 colour polystyrene beads (Invitrogen)

ex/em 365/430; 505/515; 560/580; 660/680

 

100nm green fluorescent polystyrene beads (Duke Scientific) for blue, 
green and red beads

 

Use the same chemicals, microscope slides and cover slips as you use in 
the experiments with your cells.

 

   1. pipet a big drop of 0.1% poly-L lysine (PLL) in dH_2 O on your #
      1.5 cover slip
   2. allow the glass to coat for 30-60min at RT
   3. remove PLL as much as possible
   4. air dry till all liquid has evaporated
   5. dry for another hour at RT
   6. pipet drop of bead solution straight from the tube on the coated
      cover slip
   7. allow to adhere for 5 min
   8. pipet off as much as possible
   9. allow to dry for 30-60min in the dark
  10. put embedding medium on the microscope slide
  11. put cover slip with attached beads on the microscope slide
  12. allow curing embedding medium to cure
  13. seal cover slip to the slide with non-fluorescent nail-polish
  14. store at 4°C in the dark
  15. beads slides should remain re-usable for at least 6 months.



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 
>
>
>