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Bleaching the beads will be difficult - we haven't managed to bleach single labelled fluorospheres down to a level where they'd be suitable as fiduciaries even in our very bright laser spot, although, as far as I recall, tetraspecks bleach significantly faster. At any rate you're going to have a hard time doing it in an aliquot. Ultimately you're after something which is only marginallly brighter than a single molecule, so assuming you do manage to bleach the beads to this level you're either going to loose them completely or have problems with intermittency. As to labelling your own - you're pretty much limited to surface attachment, which will be in the same chemical environment as the dyes you're imaging and will thus bleach and blink similarly (beads with surface attached dye do make got test samples though). An out of band bead, however, sounds like a really good idea.
Colloidal gold also works, although the results can be a bit variable (at least with the relatively cheap gold nanoparticles we've tried). We've also seen intermittency (blinking) of the gold nanoparticles and are as yet at a loss to explain it. One promising option that we've yet to try is nanodiamonds (they are very stable and typically have a small number of nitrogen vacancy centres and a long lived excited state so are sufficiently weak so as not to interfere).
cheers,
David
________________________________
From: Christophe Leterrier <[log in to unmask]>
To: [log in to unmask]
Sent: Tuesday, 13 March 2012 1:58 AM
Subject: Re: Protocols for fluorescent beads (fiducial markers)
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Dear Daniel,
If you have the 200 nm Tetraspeck beads they are indeed very bright.
Here are three other options :
- Use PS-speck beads (170 nm) that are fluorescent in only one channel
rather than Tetraspeck beads that fluoresce in all standard 4
channels. This way you can use bleed-through form beads that fluoresce
in another channel to minimize fluorescence. Example, for
high-intensity illumination with a 488 nm laser (dSTORM), I use red or
far-red PS-Speck beads that have a very weak fluorescence if excited
at 488 nm and observed around 515 nm.
- Try smaller Tetraspeck beads (100 nm do exist), their fluorescence
is still strong but a lot less than the 200 nm ones (4 or 8 times ?
Would be a way to know if the dye is a coating or a fill).
- Some people use colloidal gold (50-100 nm) that exist in a variety
of sizes and are visible in all channels. I have yet to source and try
some to see if it's better than fluorescent beads.
Cheers,
Christophe
--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France
On Mon, Mar 12, 2012 at 13:47, Dan Metcalf <[log in to unmask]> wrote:
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> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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>
> I've been using Invitrogen's 'fluospheres' but they are too bright for my
> application (STORM super-resolution microscopy). I'm thinking about making
> my own so that I can choose which dyes to use and can titrate down the
> amount of dye. Alternatively I could deliberately bleach the Invitrogen
> fluospheres I guess.
>
> Can anyone point me in the direction of protocols for:
>
> (1) Staining ~200 nm beads with dyes
> (2) Bleaching a large aliquot of beads in a relatively uniform way
>
> Thanks,
>
> Dan Metcalf,
>
> National Physical Laboratory
> Teddington
> UK
>
>
> --
> View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/Protocols-for-fluorescent-beads-fiducial-markers-tp7365190p7365190.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
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