Re: mEos4 photoconversion

Posted by Michelle Peckham on
URL: http://confocal-microscopy-list.275.s1.nabble.com/mEos4-photoconversion-tp7583802p7583833.html

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I have imaged mEos2 in live cells, using a Zeiss 700 confocal with no
problems at all.
It is important to keep laser intensities low of course as Eos does bleach
quite quickly in the green. Meanwhile - no fluorescence in the red
channel.  
I have also photo activated mEos2 using the 405nm laser, and measured
changes to red-fluorescence (an inverted FRAP experiment) - (using quite
low levels of blue light, and not many interations, - set up as a FRAP
experiment)

A bit of fiddling to get it to work, but it did work well in the end.

Can send images to anyone who is interested.

All best

Michelle



On 31/05/2015 23:28, "Cammer, Michael" <[hidden email]> wrote:

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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]
>
>________________________________________
>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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>>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