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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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>
> 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
>
>
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