Re: mEos4 photoconversion

Posted by mcammer on
URL: http://confocal-microscopy-list.275.s1.nabble.com/mEos4-photoconversion-tp7583802p7583821.html

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Early last week somebody brought us mEos to image by TIRF.  Even with the 488 nm laser low with ND filters in the path and the EMCCD gain all the way up, we could not image more than 2 or 3 seconds.  (It was TIRF, so I guess we effectively showed that there isn't transport of new protein from above down to the substratum, but this wasn't the experiment...)

However, when we set the 405 laser low and the 561 laser low and used both or a second of 405 alone followed by continuous 561, we were able to image continuously in the red for 1-2 minutes.  

So imaging green nearly impossible,  Imaging the photoconverted in red, easy.

_________________________________________
Michael Cammer, Optical Microscopy Specialist
http://ocs.med.nyu.edu/microscopy
http://microscopynotes.com/
Cell: [phone]

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From: Confocal Microscopy List [[hidden email]] on behalf of Stuckey, Jeff [[hidden email]]
Sent: Saturday, May 30, 2015 11:40 AM
To: [hidden email]
Subject: Re: mEos4 photoconversion

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Hi Jordan

We weren't using the transmitted LED the other day.  For visualizing with transmitted light we could put a filter in the path that would block the absorption wavelengths for eos if that was a concern.

Best

Jeff

Sent from my iPhone

> On May 30, 2015, at 10:15 AM, "Jordan Becker" <[hidden email]> wrote:
>
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> 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.
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>
> Jeff,
>
> It's great to find you on here! I wondered if the LED was sending some
> shorter wavelength light that was prematurely switching some to red. From
> what I can find in previous literature, low power on the 488 laser and high
> on the 561 is a good starting place for visualizing. I think if I can
> decrease the premature red signal, the photoconversion ratio should improve
> accordingly.
>
> Thanks for the help Sarah and I'll hope to see you around next week Jeff.
>
> Best,
> Jordan