2-color photoconversion?

[Julian Smith III]

Dear all (with apologies for duplicate posting to those of you who
belong to both the confocal and microscopy listservs)
  We're about to start a series of correlative experiments relating
confocal fluorescence to info from LM  sections. We're using
AlexaFluor488 and AlexaFluor568, and using wholemounts of small (2mm by
200µm) worms.  The current plan is to photoconvert the dye using DAB,
dehydrate, and embed in either paraffin or "Epon"/Araldite for  sectioning.
  I've found plenty of protocols for DAB photoconversion, and we're
starting those experiments this week.  Does anyone know of a protocol
for the photoconversion of another readily-oxidized dye to give a
different color (other than the brown of DAB), so we can see both dyes
at once in our sections?
TIA,
Julian

--
Julian P.S. Smith III
Director, Winthrop Microscopy Facility
Dept. of Biology
Winthrop University
520 Cherry Rd.
Rock Hill, SC  29733

803-323-2111 x6427 (vox)
803-323-3448 (fax)
803-524-2347 (cell)

[Tamara Howard]

Are you dead set on the Alexa pair? You should take a look
at Nanoprobes' Fluoronanogold - fluorescent tag and small
gold together, you could do your FM then silver enhance
the gold, yielding a black signal (also visible in
darkfield). I don't know that the two will be compatible
(silver enhancement and photoconversion in the same
sample).

For 2 fluors with photoconversion....Are there ways to
selectively photovoncert only 1 fluor at a time in a
sample with 2 fluors? I thought it was an all-or-none type
deal. But I've never looked into it, so am probably just
not up on that area of the literature.

Good luck!

Tamara (Oh, no financial interest in Nanoprobes; I'm just
familiar with that product)



On Tue, 2 Jun 2009 09:39:52 -0400
  Julian Smith III <[hidden email]> wrote:

> Dear all (with apologies for duplicate posting to those
>of you who belong to both the confocal and microscopy
>listservs)
>  We're about to start a series of correlative
>experiments relating
> confocal fluorescence to info from LM  sections. We're
>using AlexaFluor488 and AlexaFluor568, and using
>wholemounts of small (2mm by 200µm) worms.  The current
>plan is to photoconvert the dye using DAB, dehydrate, and
>embed in either paraffin or "Epon"/Araldite for
> sectioning.
>  I've found plenty of protocols for DAB photoconversion,
>and we're
> starting those experiments this week.  Does anyone know
>of a protocol for the photoconversion of another
>readily-oxidized dye to give a different color (other
>than the brown of DAB), so we can see both dyes at once
>in our sections?
> TIA,
> Julian
>
> --
> Julian P.S. Smith III
> Director, Winthrop Microscopy Facility
> Dept. of Biology
> Winthrop University
> 520 Cherry Rd.
> Rock Hill, SC  29733
>
> 803-323-2111 x6427 (vox)
> 803-323-3448 (fax)
> 803-524-2347 (cell)

***************************
Tamara Howard
Cell Biology & Physiology
UNM-HSC
Albuquerque, NM
***************************

[Ray Gilbert]

You can also silver enhance DAB to give a black colour, although it is easier just to nickel enhance and get a black product to contrast against the brown. Once again though these need to be done in a stepwise pattern. I can't see how you could selectively photoactivate only one.

Are you taking this through to EM? If so you could try embedding in acrylic resin and using anti Alexa probes and different sized gold particles for en grid staining

Cheers

Ray G


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Tamara A Howard
Sent: Wednesday, 3 June 2009 2:54 a.m.
To: [hidden email]
Subject: Re: 2-color photoconversion?

Are you dead set on the Alexa pair? You should take a look
at Nanoprobes' Fluoronanogold - fluorescent tag and small
gold together, you could do your FM then silver enhance
the gold, yielding a black signal (also visible in
darkfield). I don't know that the two will be compatible
(silver enhancement and photoconversion in the same
sample).

For 2 fluors with photoconversion....Are there ways to
selectively photovoncert only 1 fluor at a time in a
sample with 2 fluors? I thought it was an all-or-none type
deal. But I've never looked into it, so am probably just
not up on that area of the literature.

Good luck!

Tamara (Oh, no financial interest in Nanoprobes; I'm just
familiar with that product)



On Tue, 2 Jun 2009 09:39:52 -0400
  Julian Smith III <[hidden email]> wrote:

> Dear all (with apologies for duplicate posting to those
>of you who belong to both the confocal and microscopy
>listservs)
>  We're about to start a series of correlative
>experiments relating
> confocal fluorescence to info from LM  sections. We're
>using AlexaFluor488 and AlexaFluor568, and using
>wholemounts of small (2mm by 200µm) worms.  The current
>plan is to photoconvert the dye using DAB, dehydrate, and
>embed in either paraffin or "Epon"/Araldite for
> sectioning.
>  I've found plenty of protocols for DAB photoconversion,
>and we're
> starting those experiments this week.  Does anyone know
>of a protocol for the photoconversion of another
>readily-oxidized dye to give a different color (other
>than the brown of DAB), so we can see both dyes at once
>in our sections?
> TIA,
> Julian
>
> --
> Julian P.S. Smith III
> Director, Winthrop Microscopy Facility
> Dept. of Biology
> Winthrop University
> 520 Cherry Rd.
> Rock Hill, SC  29733
>
> 803-323-2111 x6427 (vox)
> 803-323-3448 (fax)
> 803-524-2347 (cell)

***************************
Tamara Howard
Cell Biology & Physiology
UNM-HSC
Albuquerque, NM
***************************