conversion of mEOS2 by 2-photon

[Sarah Richert]

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Dear all,
we are working with mEOS2 and would like to photo-convert it by 2-photon.
With 405 nm laser it works but not in 2-photon mode.
Has anybody successfully done that?
Many thanks in advance!
Sarah

[Johannes Helm]

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Hi, Sarah,

I am a complete ignorant in any matters of life science but assume that,
what you are tyring to do, is a non-coherent process.

You do not describe at which wavelength(s) you tried to do it in your
multi photon approach. I have sometimes met life science researchers, who
took the term "two photon" a little bit too literate.
Did you possibly just go for 810nm (by multiplying 405 * 2)?
Since, as I assume, you are having a non coherent process, you most
probably have to try at all the wavelengths shorter than 810nm available
on your laser.

Which laser model do you use?

Also, are there possibly any photo chemical reactions in your preparation
going on - competing with the one you want to accomplish - that would "eat
all the laser light" - or, more correctly speaking, would have a much
larger cross section than the reaction you want to induce - from you IR
laser? Can you try it "in solution", outside a specimen, in order to just
have the chemistry and not a lot of biology around, which could cause
problems?

Best wishes,

Johannes

> *****
> To join, leave or search the confocal microscopy listserv, go to:
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>
> Dear all,
> we are working with mEOS2 and would like to photo-convert it by 2-photon.
> With 405 nm laser it works but not in 2-photon mode.
> Has anybody successfully done that?
> Many thanks in advance!
> Sarah
>

--
P. Johannes Helm, M.Sc. PhD
Seniorengineer
CMBN
University of Oslo
Institute of Basic Medical Science
Department of Anatomy
Postboks 1105 - Blindern
NO-0317 Oslo

Voice: [phone]
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[Andrew York]

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We tried mEOS2 activation with 2-photon in COS7 cells. It didn't work very
well. I thought this was described in one of our papers, but I guess we
snipped it out to make length limits. Here's the relevant paragraph:

"""
Purified mEos2 proteins deposited nonspecifically on a glass coverslip
displayed partial conversion from dark to bright states under 2PA at 800
nm, but after all apparent molecules were activated we found a much larger
remaining population that could be activated only under 1P activation at
405 nm (data not shown). Similar results were obtained when attempting to
2PA mEos2 fusion proteins in fixed cells. However, we observed complete
photoconversion of purified PA-mCherry1 proteins deposited  on a surface,
and improved axial activation confinement under scanning temporal focus
compared to conventional temporal focus or 1PA
"""

The paper this paragraph was removed from:
http://www.ncbi.nlm.nih.gov/pubmed/21317909

PA-mCherry1 worked great in COS-7 cells, but I've heard of some people who
couldn't get it to 2PA in zebrafish.

On Wed, Oct 31, 2012 at 10:38 AM, Sarah Richert <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
> we are working with mEOS2 and would like to photo-convert it by 2-photon.
> With 405 nm laser it works but not in 2-photon mode.
> Has anybody successfully done that?
> Many thanks in advance!
> Sarah
>

[John Oreopoulos]

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Sarah,

It's been done before using photoactivatable mCherry (PM-mCherry) by the Shroff group:

York, A.G., et al., Confined activation and subdiffractive localization enables whole-cell PALM with genetically expressed probes. Nat Meth, 2011.

Andrew York was the lead author on that paper, and he is a subscriber to this listserver. During those studies, they may have tried 2-photon photoactivation with mEos2 as well. There is one mention of the mEos2 probe in the acknowledgements section of that paper. Try sending him an email.

Cheers,

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


On 2012-10-31, at 10:38 AM, Sarah Richert wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
> we are working with mEOS2 and would like to photo-convert it by 2-photon.
> With 405 nm laser it works but not in 2-photon mode.
> Has anybody successfully done that?
> Many thanks in advance!
> Sarah

[Chen, De (NIH/NCI) [C]]

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We tested this mechanism before. mEOS-FP is very efficient to be converted to red by 810nm two photon illumination. Wish this info is useful to you.

________________________________________
From: Sarah Richert [[hidden email]]
Sent: Wednesday, October 31, 2012 10:38 AM
To: [hidden email]
Subject: conversion of mEOS2 by 2-photon

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Dear all,
we are working with mEOS2 and would like to photo-convert it by 2-photon.
With 405 nm laser it works but not in 2-photon mode.
Has anybody successfully done that?
Many thanks in advance!
Sarah

[Ekaterina PAPUSHEVA-2]

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Hi Sarah,

This protocol worked for us

You have to photoconvert at 800nm with the laser power not more than 0.5 mW at the objective plane.
You need a long time: 3-5min for one z plane  of a square with a size of half a cell. To detect the red form you have to do imaging at 970nm.
If the laser power is higher, you bleach immediately the red form.

Play around these lines!

Best regards,
Ekaterina

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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sarah Richert
Sent: Wednesday, October 31, 2012 3:39 PM
To: [hidden email]
Subject: conversion of mEOS2 by 2-photon

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Dear all,
we are working with mEOS2 and would like to photo-convert it by 2-photon.
With 405 nm laser it works but not in 2-photon mode.
Has anybody successfully done that?
Many thanks in advance!
Sarah

[Sarah Richert]

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Many thanks for your answers and suggestions!
Good to hear that photoconversion of mEOS by 2P seems to work with mEOS-FP
at 810 nm. It would be great to know, how many scans it takes and ideally
which laser power should be used. Would you be so kind to share these
details, Chen?

Unfortunately, AG York has stated it did not work (efficiently) at 800 nm
with mEOS2.
I am wondering if this could be due to differences between mEOS-FP and
mEOS2. Or if it is a difference between 800 versus 810 nm excitation?

Thanks a lot,
Sarah

[Andrew York]

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We never tried the long exposures Ekaterina Papusheva describes. Worth a
try for sure.

One caveat: We used temporal focusing (lower peak power, higher average
power), not point-scanning. I suspect EP's description is for a
point-scanning system. For a nonlinear process like this, it's possible
that's an important difference.



On Wed, Nov 7, 2012 at 8:12 AM, Sarah Richert <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Many thanks for your answers and suggestions!
> Good to hear that photoconversion of mEOS by 2P seems to work with mEOS-FP
> at 810 nm. It would be great to know, how many scans it takes and ideally
> which laser power should be used. Would you be so kind to share these
> details, Chen?
>
> Unfortunately, AG York has stated it did not work (efficiently) at 800 nm
> with mEOS2.
> I am wondering if this could be due to differences between mEOS-FP and
> mEOS2. Or if it is a difference between 800 versus 810 nm excitation?
>
> Thanks a lot,
> Sarah
>