2photon conversion of Dendra2

[aget]

Hi, could anyone be kind enough to provide some advice about photonconversion of Dendra2 by two photon laser? We tried the 810nm laser according to a paper (http://cshprotocols.cshlp.org/content/2011/10/pdb.top065904.full), but still have not succeeded yet. Anything tricky about it? Thanks.
Aget

[Manja Schubert]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Aget,

I was very surprised by the findings of the mentioned paper because I
have also tried to switch dendra2 with a Ti:Saph laser a couple years
ago, but I never managed to do so in the whole range of the laser (690 -
1040nm). After contacting the developer of Dendra2
(http://www.nature.com/nprot/journal/v2/n8/full/nprot.2007.291.html) and
discussing this issue we concluded  it is maybe not possible to do so
with an IR laser. They suggested to us a 405 nm laser if available or
exposing it to violet light for 5 min. Because two photon microscopy is
a non-linear process the excitation spectra can show some unexpected
shifts, f.e. Alexa 488 = 750nm 2P or eGFP = 950nm 2P. I found the
following home pages always helpful for a good starting point if you use
a fluorescence dye at 2P.
(http://www.drbio.cornell.edu/cross_sections.html ;
http://www.spectra.arizona.edu/) .
If I right recall I think Kaede can be converted with a Ti:Saph laser at
750nm (http://adsabs.harvard.edu/abs/2008SPIE.6860E..26W). You could try
this wavelength for Dendra2 too.

Good luck.

Manja


On 16/07/2012 18:54, aget wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi, could anyone be kind enough to provide some advice about photonconversion
> of Dendra2 by two photon laser? We tried the 810nm laser according to a
> paper (http://cshprotocols.cshlp.org/content/2011/10/pdb.top065904.full),
> but still have not succeeded yet. Anything tricky about it? Thanks.
> Aget
>
> --
> View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/2photon-conversion-of-Dendra2-tp7578658.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

--
Dr. Manja Schubert
University of Bergen
Department of Clinical Medicine
Paraneoplastic Neurological Syndromes Group
Haukeland University Hospital
5020 Bergen
Norway
Tel:[phone]
Fax: + 47-55 58 63 60

[aget]

Thank you so much Manja, the websites are really helpful! I also wrote to the developer of Dendra2 who politely replied that they dont have experience with 2p conversion. Then i wrote to the authors of the paper describing 2p conversion by 810nm but got no reply so far (more than a month). It seems IR laser is not good at doing this job. Maybe i can try Kaede which can also be used to track cells.

[aget]

Good news, the author of the paper i provided the link, Dr.John Condeelis replied, he said 'We have found the best multiphoton photoconversion to be at 30mW of 815nm for 300s using a parked beam'. Now i can try this, though i wonder how can i park the beam to the whole cell(?).

[Unruh, Jay]

Hi Aget,

In our hands PAGFP and PSCFP2 are by far the best at two photon photoswitching.  We have struggled to get Dendra, KikGR, and mEOS to work well, though given the right conditions (low power, long photoactivation times) perhaps things would work better.  I haven't tried Kaede, but it appears that the chromophore sequence is similar to the others I mentioned.

Jay Unruh
Research Advisor
Stowers Institute for Medical Research

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of aget
Sent: Tuesday, July 17, 2012 4:46 AM
To: [hidden email]
Subject: Re: 2photon conversion of Dendra2

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Thank you so much Manja, the websites are really helpful! I also wrote to the developer of Dendra2 who politely replied that they dont have experience with 2p conversion. Then i wrote to the authors of the paper describing 2p conversion by 810nm but got no reply so far (more than a month). It seems IR laser is not good at doing this job. Maybe i can try Kaede which can also be used to track cells.

-----
Institute of Physiology II,University of Tübingen Keplerstraße 15, 72074, Tübingen, Germany
--
View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/2photon-conversion-of-Dendra2-tp7578658p7578668.html
Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

[Craig Brideau]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

If you really need to, you can frequency-double a Ti:Saph to get 405.  I've
done this for some spectrophotometry to get a highly tunable visible
source.  You fire the Ti:Saph beam through a crystal like BBO, then rotate
the crystal until you see conversion.  The resulting light will be half the
wavelength your laser is tuned to.  For instance to get 405, tune the
Ti:Saph to 810nm.  It's surprisingly simple to do.

Craig


On Tue, Jul 17, 2012 at 10:20 AM, Unruh, Jay <[hidden email]> wrote:

> Hi Aget,
>
> In our hands PAGFP and PSCFP2 are by far the best at two photon
> photoswitching.  We have struggled to get Dendra, KikGR, and mEOS to work
> well, though given the right conditions (low power, long photoactivation
> times) perhaps things would work better.  I haven't tried Kaede, but it
> appears that the chromophore sequence is similar to the others I mentioned.
>
> Jay Unruh
> Research Advisor
> Stowers Institute for Medical Research
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of aget
> Sent: Tuesday, July 17, 2012 4:46 AM
> To: [hidden email]
> Subject: Re: 2photon conversion of Dendra2
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Thank you so much Manja, the websites are really helpful! I also wrote to
> the developer of Dendra2 who politely replied that they dont have
> experience with 2p conversion. Then i wrote to the authors of the paper
> describing 2p conversion by 810nm but got no reply so far (more than a
> month). It seems IR laser is not good at doing this job. Maybe i can try
> Kaede which can also be used to track cells.
>
> -----
> Institute of Physiology II,University of Tübingen Keplerstraße 15, 72074,
> Tübingen, Germany
> --
> View this message in context:
> http://confocal-microscopy-list.588098.n2.nabble.com/2photon-conversion-of-Dendra2-tp7578658p7578668.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>

[aget]

Thanks Jay. i checked Kaede and realized that it might be an issue for its diffusion in host cells. Dendra2 still seems ideal mammalian cell tracker.

[aget]

That sounds a great idea, i will try it. Thanks Craig.

[Jerry (Gerald) Sedgewick]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

July 30, 12:00pm (noon) Central Daylight Time (U.S.).  For Photoshop
Extended versions CS3 (version 10) - CS6 (version 13).  To sign up, go to:

www.Imagingandanalysis.com/web072012cm.html.

You will learn to post-process movie files and things like putting
movies side-by-side, adding arrows and other symbols, adding captions,
and so on.  Here is a list of what will be shown:

- Open Image stacks as image sequences; or as a z- or time-series saved
in the uncompressed AVI format
- Maintain a log of what you did to movie files
- Open DICOM series as movies
- Add text, arrows, captions, etc. to movies
- Filter movies all at once: De-Interlace, Sharpen, etc.
- Place movies side-by-side; overlay movies to merge
- Adjust tonal range for optimizing to output devices
- Export completed movies for frame-by-frame "scientific" replay

Cheers,
Jerry

--
Jerry (Gerald) Sedgewick
Scientific Imaging & Image Analysis Consultant
Technical Writer / Regulatory Consultant
965 Cromwell Avenue
Saint Paul, MN  55114
651-788-2261
http://www.imagingandanalysis.com
http://www.quickphotoshop.com
Author: “Scientific Imaging with Photoshop: Methods, Measurement and Output”

[Craig Brideau]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Glad I could help!  If you don't have a crystal, most laser crystal dealers
can direct you to the best type and cut of crystal for what you want to do.
 I have a piece of BBO that works quite well and is easy to tune.  Other
crystals may be more efficient for 405nm production though, so ask around.

Craig



On Thu, Jul 19, 2012 at 8:11 AM, aget <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> That sounds a great idea, i will try it. Thanks Craig.
>
> -----
> Institute of Physiology II,University of Tübingen
> Keplerstraße 15,
> 72074, Tübingen, Germany
> --
> View this message in context:
> http://confocal-microscopy-list.588098.n2.nabble.com/2photon-conversion-of-Dendra2-tp7578658p7578695.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>