A warning if you are thinking about Molecular Probes Inspeck intensity
calibration kits for the confocal.

Molecular probes advertises Inspeck intensity calibration kits for
microscopy.
We have tried these however we have not received any beads that have a
uniform intensity throughout (we tried the FITC and Texas Red kits).

When we contacted MProbes technical support they said that they have
trouble with the lower intensity standards as the dye accumulates on the
inner surface of the beads.
They will refund me and said that due to this they admit they are not
suitable for confocal microscopy.

Wayne

Anna Smallcombe wrote:
>
> Dear Heather - yes, you will need some standards to calibrate each time you do
> the measurements. The lasers should be warmed up and the scan head running for
> about 20mins to bring the PMTs up to a stable temperature. You would be best
> selecting a pair of fluorochromes with non-overlapping emission spectra and your
> staining protocol needs to be very repeatable to have any chance of meaningful
> quantitation. You can buy fluorescent plastic from toy shops, but if you give me
> your full postal address, I may be able to send you some. You should check the
> photobleaching properties of the plastic with the laser intensity and zoom you
> intend to use, and try to reduce the intensity so that there is no bleaching. If
> you do want me to send you something, please deop me a line.
>
> Best regards
>
> Anna Smallcombe PhD  Bio-Rad
> ______________________________ Reply Separator _________________________________
> Subject: dye standards for intensity measurements
> Author:  Confocal Microscopy List <[log in to unmask]> at
> Internet
> Date:    16/06/98 17:14
>
> As a new-comer to the wonderful world of confocal microscopy, I am
> amazed at its potential but at the same time I often find
> myself way out to lunch :)
>
> If you haven't already guessed, I'am a novice user, of a Biorad MRC
> 600 confocal microscope to be exact.  As part of my masters research
> I am using the microscope to observe fluorescently stained yeast
> cells.  The dye I am using stains viable cells a diffuse green
> (cytoplasm) with red intravacuolar structures.  I would like to
> measure (quantitate) the fluorescence intensity of both colours
> independently (I realize there will likely be some overlap - if
> anyone has any suggestions for dealing with this situation your
> advice would be greatly appreciated).  I have already been advised
> that I will require some non-fading fluorescent standards (red &
> green) that I can use to set up the microscope before testing my yeast
> samples.  I have been told that some plastics (some kinds used to make
> cheap toys) can work very well.  I don't know how true this is or
> what plastics would be good.  I would really appreciate any
> suggestions anyone might have on appropriate standards that I could
> use.
>
> Thanks in advance for any advice offered
>
> Heather Heggart
> M.E.Sc. candidate
> University of Western Ontario, Canada
> [log in to unmask]