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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]

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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
>>
>>
>>