I'm just trying to figure out why I saw them first and then by trying
refocusing I lost them!
On 6/20/07, Julio Vazquez <[log in to unmask]> wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> -
> Sarah,
>
> "a few mW" of laser power focused onto a single spot can be actually quite a
> lot of light...
>
> I recommend you follow the Molecular Probes procedure, as described below:
>
> "MultiSpeck multispectral and RGB Mix microspheres can be
> mounted on a microscope slide and used as an external fluores-
> cent standard. Add a small drop (<10 ěL for a standard cover-
> slip-size sample) of one of the suspensions to a microscope slide and air
> dry
> protect from dust during drying. We recommend that
> you use microscope slides etched with rings to make it easy to
> identify the position of the microspheres once the drop dries.
> Alternatively, you can make a circle on the bottom of a standard
> microscope slide with a marker and place the sample drop on the
> top of the slide within the circle. When the sample is completely
> dry, add a small drop of mounting medium to cover the spot,
> place a coverslip on the slide and seal. The mounting medium
> will remain liquid; thus, the sample distribution may not be per-
> manent. Visualize the mounted standards with a fluorescence
> microscope equipped with the appropriate filter set. "
>
>
> Once you have your sample, you may try to find the focal plane (find the
> beads) with transmitted light (using DIC/Nomarski), which you probably have
> on your confocal, to minimize exposure to bright light.
>
> Set the appropriate configuration for the detection channel, and set your
> 405 laser power to a reasonable value with the AOTF... maybe 20% should be
> enough for a 1 mW laser, and less if your laser has more power. We use a 10
> mW laser at about 3% power, on average.
>
> Once you have found your beads, you can start checking wether they fade at
> the laser powers you are using, whether they move, etc...
>
> It is better to store those types of slides in the dark, but I don't think
> they should just fade like that in front of your eyes. As a matter of fact,
> even if you bleached the beads in a specific field of view, there should be
> more beads nearby that would not have bleached, which is why I am somewhat
> puzzled by your observation...
>
> I am assuming that you know how to use the microscope... but if you follow
> the recommendations above, and still have the same problem, perhaps you
> ought to find someone who is experienced with the instrument so that they
> can watch you and figure out the cause of the problem... for instance, if
> the droplet of water used as immersion medium for your objective dried out,
> or you lost contact between objective and coverslip, your image would just
> vanish...
>
>
>
> --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> Seattle, WA 98109-1024
>
>
> [log in to unmask]
> http://www.fhcrc.org/
>
>
> On Jun 20, 2007, at 12:36 PM, Sarah Kefayati wrote:
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> But my laser power is just about very few mW(cause I'm using diode
> laser),do you think this much power causes burning?
> also I left the sample to be dried and then I put water on it,maybe
> not dried enough?!
> or do you think being exposed to light may cause this because after
> preparing my sample I didn't keep that in dark.
>
> thanks
> sarah
>
> On 6/20/07, James Beals <[log in to unmask]> wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Sara,
> You may need to let the beads dry onto the cover slip, then use
> mounting media.
> My tetraspeck beads came with mounting media, and instructions.
> The beads may be fading, they may be moving too fast to image.
> James Beals
> [log in to unmask]
> 734.936-2051
>
> 205 Zina Pitcher Place
> 2038 MBNI
> Molecular and Behavioral Neuroscience Institute
> University of Michigan
> Ann Arbor, Mi
> 48109
>
>
>
>
>
> On Jun 20, 2007, at 1:49 PM, Sarah Kefayati wrote:
>
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Hello everyone!
> >
> > I have just started imaging 200 nm Tetraspeck beads with the diode
> > laser(407 nm) and objective lens 60x water immersion and also we are
> > using Fluovew 300 and its soft ware.
> > I also mounted my sample in water.at the beginning I saw an image of
> > my beads,trying refocusing the objective my image faded away and I
> > couldn't get anything at all any more.I think that my sample is
> > photobleached and I thought maybe is good to use another
> > solution(probably prolong gold).
> > Do you have any experience or comment about this regard to share with
> > me,may be some thing else is wrong!
> >
> > cheers
> > sarah
>
>
>
|
