yes,
I first put the zoom at 10(because I calculated the size of the pixel
in object space and it matched by putting zoom at 10) and I run the
experience by 10x zoom but I thought maybe I bleached a part of the
sample so I zoomed back when I zoomed back still didn't see any thing.
On 6/21/07, Glen MacDonald <[log in to unmask]> wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Sarah,
> Had you zoomed in to the bead? consider a few mW on the full field
> of the objective at zoom = 1, but after zooming in to adequately
> sample the bead those same few mW are now concentrated onto a
> fraction of the area. Your bead may have moved, but they are very
> easy to bleach. that is a very small volume of fluorophores.
>
> Åt least you have more.
>
> Glen
>
>
> Glen MacDonald
> Core for Communication Research
> Virginia Merrill Bloedel Hearing Research Center
> Box 357923
> University of Washington
> Seattle, WA 98195-7923 USA
> (206) 616-4156
> [log in to unmask]
>
> ************************************************************************
> ******
> The box said "Requires WindowsXP or better", so I bought a Macintosh.
> ************************************************************************
> ******
>
>
> On Jun 20, 2007, at 2:03 PM, Sarah Kefayati wrote:
>
> > 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 μL 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
> >>
> >>
> >>
>
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