Tom,
the 0.1 um and 0.2 um Tetra Speck beads seem not to have enough dye for a
good signal considering the bad matching with the 488nm line. That's why
we switched to the 0.5 um Tetra Speck for measuring chromatic shift. For
measuring the PSF we use the PS-Speck from molecular probes. They have a
diameter of 175nm and are labelled with a single dye. Their signal is ok.
I used the green, the red and the dep red ones on confocals.
Good luck,
Joachim
On Tue, 27 Mar 2001, Tom Phillips wrote:
> We have been trying to use the Molecular Probes fluorescent bead size
> kit which includes 0.1 um through 4 um beads that are each stained
> with four fluorescent dyes (blue, green, orange/red, and dark red).
> In the complete kit, there are three slides, five areas that are
> supposed to contain the five separate sizes of beads, and then one
> area of mixed beads.
>
> The set we have has unusable 0.2 um and 0.1 um beads for the blue and
> green channels for what we are doing. The 0.2 um beads have high
> background fluorescence in the blue and green channels, so that the
> bead is not easily distinguishable. When the psf stack of the bead
> is gathered, the background is around 20 - 30 on a 255 greyscale. The
> 0.1 um bead area has so much other stuff - dust? - and variation in
> size that I can't tell which objects are beads and which are junk.
> And the blue and green channels have high background fluorescence (as
> with the 0.2 um beads).
>
>
>
>
> >On Mon, 26 Mar 2001, Tom Phillips wrote:
> >
> >> I would appreciate hearing how the experts out there prepare their
> >> microscope slides for measuring a PSF. We got a couple of prepared
> >> slides from Molecular Probes and were quite disappointed with the
> >> quality of the Blue and Green beads (the red ones were fine). This
> >> was somewhat surprising since they usually have great quality. We
> >> bought the beads and are making our own but I would be interested in
> >> any protocols or alternative sources for prepared slides. Thanks in
> >> advance, Tom
> >
> > Which type of beads did you use? What light source? We use beads
> >from Molecular Probes (we have tried both ready slides and slides
> >prepared from their bead suspensions with different embedding media)
> >and they work VERY well in all colors and they do not bleach at all.
> >We use 100W mercury lamp + filters. Probably you could get worse
> >performance with lasers in blue and green because their dyes do
> >not exactly match DAPI and FITC. The excitation/emission maxima
> >for their blue and green are 365/430 nm and 505/515 nm, respectively.
> >We do use DAPI and FITC filters but with a mercury lamp we get
> >a broader spectrum, not just a single wavelength.
> >
> > Just a thought.
> > With best regards,
> > Michal Kozubek
> >
> >P.S. For the preparation of slides from the bead suspension we use
> > protocol from Molecular Probes handbook - so we basically drop
> > the suspension on the slide (or coverglass), spread with a
> > pipette tip, let it dry, apply embedding medium, cover with
> > a coverglass (or put the coverglass on the slide) and seal.
> >
> >~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> >Michal Kozubek, Ph.D.
> >Laboratory of Optical Microscopy, Faculty of Informatics,
> >Masaryk University, Botanicka 68a, CZ-60200, Brno, Czech Republic
> >Tel/Fax/Ans: +420-5-41512467 E-mail: [log in to unmask]
> >Internet home page: http://www.fi.muni.cz/~kozubek
>
> --
> Thomas E. Phillips, Ph.D.
> Associate Professor of Biological Sciences
> Director, Molecular Cytology Core Facility
>
> 3 Tucker Hall
> Division of Biological Sciences
> University of Missouri
> Columbia, MO 65211-7400
> (573)-882-4712 (voice)
> (573)-882-0123 (fax)
>
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Joachim Walter, Dipl. Phys.
Institut für Anthropologie und Humangenetik der LMU München
AG Cremer
Richard-Wagner-Straße 10/I
D-80333 München Tel. +49 - 89-2180-6713
Germany Fax -6719
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