Second color fluorophore for dSTORM experiments?

[lechristophe]

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Dear all,

I am currently using super-resolution images of fixed/immunolabeled cells
using a Nikon N-STORM microscope. I want to do 2-color dSTORM (to compare
with 2-color STORM with 2 activators/1 reporter).

Of course the first channel is Alexa Fluor 647 which is by far the best
fluorophore for dSTORM. But which fluorophore to use for the second
channel? On the Leica GSD scope we use Alexa Fluor 532 excited by the 532
nm laser, which is not an option on the N-STORM scope that lacks this laser
(it has solid state 405, 488, 561 and 647 lasers). I have tried Alexa Fluor
488, but I don't get good results. People have suggested to switch to Atto
488, so I'll try that (for those who use it, should I get Atto 488-labeled
secondary abntibodies from Sigma or from Rockland?). I have also read that
people use orange dyes like Cy3B or Alexa555 as a second channel.

I'd be very happy to hear about your experience on this subject, and to try
the fluorophore that woks best in your hands.

Thanks for the help,

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France

[John Oreopoulos]

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Cy3B and Atto488 would be my probes of choice in that order for a secondary label. I too find that the bluer probes are a little more difficult to get switching. The Heilemann group suggests that the switching buffer chemistry might need to be tuned a bit to help in this regard. See these papers:

Heilemann, M., et al., Super-resolution imaging with small organic fluorophores. Angewandte Chemie-International Edition, 2009. 48(37): p. 6903-6908.
van de Linde, S., et al., Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nature Protocols, 2011. 6(7): p. 991-1009.

John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca



On 2013-10-09, at 4:18 AM, Christophe Leterrier wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> I am currently using super-resolution images of fixed/immunolabeled cells
> using a Nikon N-STORM microscope. I want to do 2-color dSTORM (to compare
> with 2-color STORM with 2 activators/1 reporter).
>
> Of course the first channel is Alexa Fluor 647 which is by far the best
> fluorophore for dSTORM. But which fluorophore to use for the second
> channel? On the Leica GSD scope we use Alexa Fluor 532 excited by the 532
> nm laser, which is not an option on the N-STORM scope that lacks this laser
> (it has solid state 405, 488, 561 and 647 lasers). I have tried Alexa Fluor
> 488, but I don't get good results. People have suggested to switch to Atto
> 488, so I'll try that (for those who use it, should I get Atto 488-labeled
> secondary abntibodies from Sigma or from Rockland?). I have also read that
> people use orange dyes like Cy3B or Alexa555 as a second channel.
>
> I'd be very happy to hear about your experience on this subject, and to try
> the fluorophore that woks best in your hands.
>
> Thanks for the help,
>
> --
> Christophe Leterrier
> Researcher
> Axonal Domains Architecture Team
> CRN2M CNRS UMR 7286
> Aix Marseille University, France

[Kurt Thorn]

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We have had good luck with Atto488 / Alexa647, using 10 mM MEA in the
buffer.  Getting Atto488 into the dark state requires fairly high laser
power - on our N-STORM scope we use ~140 mW 488 nm light out of the
fiber to drive it into the dark state, and then image at about 70 mW power.

Kurt

On 10/9/2013 4:28 AM, John Oreopoulos wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Cy3B and Atto488 would be my probes of choice in that order for a secondary label. I too find that the bluer probes are a little more difficult to get switching. The Heilemann group suggests that the switching buffer chemistry might need to be tuned a bit to help in this regard. See these papers:
>
> Heilemann, M., et al., Super-resolution imaging with small organic fluorophores. Angewandte Chemie-International Edition, 2009. 48(37): p. 6903-6908.
> van de Linde, S., et al., Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nature Protocols, 2011. 6(7): p. 991-1009.
>
> John Oreopoulos
> Staff Scientist
> Spectral Applied Research
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
>
>
> On 2013-10-09, at 4:18 AM, Christophe Leterrier wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear all,
>>
>> I am currently using super-resolution images of fixed/immunolabeled cells
>> using a Nikon N-STORM microscope. I want to do 2-color dSTORM (to compare
>> with 2-color STORM with 2 activators/1 reporter).
>>
>> Of course the first channel is Alexa Fluor 647 which is by far the best
>> fluorophore for dSTORM. But which fluorophore to use for the second
>> channel? On the Leica GSD scope we use Alexa Fluor 532 excited by the 532
>> nm laser, which is not an option on the N-STORM scope that lacks this laser
>> (it has solid state 405, 488, 561 and 647 lasers). I have tried Alexa Fluor
>> 488, but I don't get good results. People have suggested to switch to Atto
>> 488, so I'll try that (for those who use it, should I get Atto 488-labeled
>> secondary abntibodies from Sigma or from Rockland?). I have also read that
>> people use orange dyes like Cy3B or Alexa555 as a second channel.
>>
>> I'd be very happy to hear about your experience on this subject, and to try
>> the fluorophore that woks best in your hands.
>>
>> Thanks for the help,
>>
>> --
>> Christophe Leterrier
>> Researcher
>> Axonal Domains Architecture Team
>> CRN2M CNRS UMR 7286
>> Aix Marseille University, France
>

[Matt Kofron]

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We've had good luck with Cy3B or Alexa 568 paired with Alexa 647. We
generally directly conjugate our primary antibodies for STORM experiments.
Invitrogen makes both Alexa 647 and Alexa 568 version of their Apex
conjugation kits. If you want to to the secondary AB route, Abcam is selling
some Alexa 568 conjugated secondaries. As far as I know, Cy3B is only
available as an NHS ester, so you have to work out the conjugation
conditions on your own.