Photoactivation of Dendra and Kaede using 2P

[Stephen Firth-2]

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I have two projects using photoactivatable FPs Dendra and Kaede in living
tissue where I would like to constrain the activation zone in Z using 2P
microscopy, but I'm not having any success in 2P mode.

A literature search does not provide much hope for the Dendra, and I have
not been able to replicate the papers that mention 2P on Kaede.
I am using a Leica SP8 with Mai Tai Deep See, both FPs photoactivate well
in single photon mode.
Can anyone provide some advice for these two fluorophores?
Cheers,
Stephen

--
*Stephen Firth* | Manager | Monash Micro Imaging - Advanced Optical
Microscopy
15 Innovation Walk, STRIP 1 Monash Biotechnology, Monash University,
Clayton, VIC 3800, Australia
Email: [hidden email] | TEL: [phone] | MOB: [phone]
*http://platforms.monash.edu/mmi <http://platforms.monash.edu/mmi>*

[Moulding, Dale]

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Hi Stephen,
I've tried this too, Zeiss 880 with 20x1.0 water objective and the same laser as you. No luck with photo activation with 2p I'm afraid. Great with single photon, and we were able to get good single cell Z targeting at 405nm. Struggled to penetrate far into tissue tho.
I'd love to here if this can be done with 2P.
Cheers
Dale


http://www.ucl.ac.uk/ich/core-scientific-facilities-centres/confocal-microscopy


-------- Original message --------
From: Stephen Firth <[hidden email]>
Date: 23/01/2018 02:01 (GMT+00:00)
To: [hidden email]
Subject: Photoactivation of Dendra and Kaede using 2P

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

I have two projects using photoactivatable FPs Dendra and Kaede in living
tissue where I would like to constrain the activation zone in Z using 2P
microscopy, but I'm not having any success in 2P mode.

A literature search does not provide much hope for the Dendra, and I have
not been able to replicate the papers that mention 2P on Kaede.
I am using a Leica SP8 with Mai Tai Deep See, both FPs photoactivate well
in single photon mode.
Can anyone provide some advice for these two fluorophores?
Cheers,
Stephen

--
*Stephen Firth* | Manager | Monash Micro Imaging - Advanced Optical
Microscopy
15 Innovation Walk, STRIP 1 Monash Biotechnology, Monash University,
Clayton, VIC 3800, Australia
Email: [hidden email] | TEL: [phone] | MOB: [phone]
*http://platforms.monash.edu/mmi <http://platforms.monash.edu/mmi>*

[Raphael J]

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Hi Stephen,

Around 6 years ago I tried the Dendra for months with 2P and never succeed
to make it works. One of my old supervisors told me later that he never
find how either. There are some people on the internet saying that they
succeed "30mW of 815nm for 300s using a parked beam", but it seems to be a
very complicated (if not impossible) task.
But if someone really has some success, I would be pleased to know about it
too!
cheers,
Raf

2018-01-23 8:50 GMT+00:00 Moulding, Dale <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Stephen,
> I've tried this too, Zeiss 880 with 20x1.0 water objective and the same
> laser as you. No luck with photo activation with 2p I'm afraid. Great with
> single photon, and we were able to get good single cell Z targeting at
> 405nm. Struggled to penetrate far into tissue tho.
> I'd love to here if this can be done with 2P.
> Cheers
> Dale
>
>
> http://www.ucl.ac.uk/ich/core-scientific-facilities-centres/
> confocal-microscopy
>
>
> -------- Original message --------
> From: Stephen Firth <[hidden email]>
> Date: 23/01/2018 02:01 (GMT+00:00)
> To: [hidden email]
> Subject: Photoactivation of Dendra and Kaede using 2P
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> I have two projects using photoactivatable FPs Dendra and Kaede in living
> tissue where I would like to constrain the activation zone in Z using 2P
> microscopy, but I'm not having any success in 2P mode.
>
> A literature search does not provide much hope for the Dendra, and I have
> not been able to replicate the papers that mention 2P on Kaede.
> I am using a Leica SP8 with Mai Tai Deep See, both FPs photoactivate well
> in single photon mode.
> Can anyone provide some advice for these two fluorophores?
> Cheers,
> Stephen
>
> --
> *Stephen Firth* | Manager | Monash Micro Imaging - Advanced Optical
> Microscopy
> 15 Innovation Walk, STRIP 1 Monash Biotechnology, Monash University,
> Clayton, VIC 3800, Australia
> Email: [hidden email] | TEL: [phone] | MOB: [phone]
> 425
> *http://platforms.monash.edu/mmi <http://platforms.monash.edu/mmi>*
>