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mEos4 photoconversion

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Jordan Becker

mEos4 photoconversion

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Hey I hope somebody can help me out. I've started working with a fusion
protein of mEos4a and the photoconversion has been working poorly. I'm using
a swept field confocal and Prairie View software.

I am using a 488 laser at 20% power and 561 at 35% power for visualizing the
protein in live cells. I notice that the green form of mEos bleaches quite
quickly even as I'm identifying cells of interest to image. In addition,
there tends to be quite a bit of red signal already detectable in these
cells without photoconversion by a 405 laser.

I am able to get ~500% increase in red signal and maybe a 50% decrease in
green signal upon 405 conversion (but still far from complete and it was
difficult to find cells with minimal red signal). I converted using 20 fast
repetitions of a relatively low powered 405 (~10%) at a single point
(0.500micron diameter).

Does anyone have any advice for best visualization laser and photoconversion
laser settings? I'm looking to use mEos4 for FRAP/FLAP. Is mEos sensitive to
ambient light

Best,
Jordan

Sarah Richert

Re: mEos4 photoconversion

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Dear Jordan,

i am working with mEos2 but dont have experience with mEos4, but still i
would like to suggest two things:

1) i have observed photoconversion to some extent by illumination with a
UV-Lamp and a green filter when the lamp is very strong. Is it possible to
identify the cells in brightfield and image in the red channel first to see
whether the 488nm illumination already photoconverts?

2) regarding the photoconversion for me it works better to have a high power
output and less repetitions, but i dont know if thats genereally the case.
In the beginning i tried to photoconvert the total green signal to red
aswell, but realised that i dont yield a strong signal at the end anymore
most likely due to bleaching caused by high energy input. Additionally, at
least my cells, would not survive this procedure. Now i´m working with
approximately 50/50 green to red signal.

Best,

Sarah

Stuckey, Jeff

Re: mEos4 photoconversion

In reply to this post by Jordan Becker

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Hi Jordan,

I'll be around Bill's lab next week a few times collecting data. In line with Sarah's suggestion, I can show you how to use the transmitted light along with DIC in order to find your cells. Jimmy and I talked about this last night after you left. It's a new capability where the system can do DIC imaging through the confocal scanner.

Best

Jeff Stuckey

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jordan Becker
Sent: Friday, May 29, 2015 8:35 AM
To: [hidden email]
Subject: mEos4 photoconversion

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hey I hope somebody can help me out. I've started working with a fusion protein of mEos4a and the photoconversion has been working poorly. I'm using a swept field confocal and Prairie View software.

I am using a 488 laser at 20% power and 561 at 35% power for visualizing the protein in live cells. I notice that the green form of mEos bleaches quite quickly even as I'm identifying cells of interest to image. In addition, there tends to be quite a bit of red signal already detectable in these cells without photoconversion by a 405 laser.

I am able to get ~500% increase in red signal and maybe a 50% decrease in green signal upon 405 conversion (but still far from complete and it was difficult to find cells with minimal red signal). I converted using 20 fast repetitions of a relatively low powered 405 (~10%) at a single point (0.500micron diameter).

Does anyone have any advice for best visualization laser and photoconversion laser settings? I'm looking to use mEos4 for FRAP/FLAP. Is mEos sensitive to ambient light

Best,
Jordan

Jordan Becker

Re: mEos4 photoconversion

In reply to this post by Jordan Becker

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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

Stuckey, Jeff

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:
>
> *****
> 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.
> *****
>
> 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

mcammer

Re: mEos4 photoconversion

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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: (914) 309-3270

________________________________________
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

*****
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 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

mcammer

Re: mEos4 photoconversion

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

Early last week somebody brought us mEos to image by TIRF (first time for us, disclaimer: n=1). 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: (914) 309-3270

________________________________________
From: Cammer, Michael
Sent: Sunday, May 31, 2015 6:28 PM
To: Confocal Microscopy List
Subject: RE: mEos4 photoconversion

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: (914) 309-3270

________________________________________
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

*****
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 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

Arvydas Matiukas

Advice on CCD camera

In reply to this post by Stuckey, Jeff

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Hello list,

Please share your experience and advice on CCD camera
for imaging fixed slides on widefield fluorescent microscope.
We are looking for a reasonably priced, reliable and sensitive
camera. High speed is not very important. Also how newest sCMOS
compare to CCD?

Our 5 year old 1.4MP cooled CCD camera died, and as it
is discontinued, we prefer to replace it instead of repairing.
It has been mostly used for imaging fixed brain slices
labeled by immunofluorescence or fluorescent proteins,
some labels being quite dim.

Thanks,
Arvydas

Arvydas Matiukas, Ph.D.
Director of Confocal&Two-Photon Core
SUNY Upstate Medical University
Neuroscience & Physiology Dept

George McNamara

Re: Advice on CCD camera

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Hi Arvydas,

The sCMOS cameras from (alphabetical order) Andor, Hamamatsu and PCO
have similar pixel size but 5x or 5x as many pixels as your old CCD, so
a lot more area covered. The sCMOS focusing is a lot faster: 100 fps
full frame ... the lower price models are 30 fps (ex. USB3), still
faster than an old CCD.

If you have limited budget, go with an A,H, or P sCMOS that is USB3
(lower price, still 16-bit ... play with contrast) and invest in LED
illuminator, maybe for price consider ThorLabs 4LED
https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=3836
new filter(s), -- ideally a matched Quad filter set --- and find more
money for a Sutter + Semrock fast emission wheel with threaded filters -
similar cost to slow wheels, but 'time is money".
http://www.semrock.com/how-to-get-the-most-out-of-your-sutter-filter-wheel.aspx
the right LEDs, right Quad set means no moving parts on the excitation
side. Emission filter wheel (especially assuming wide emission bands on
the Quad set) would enable you to choose what fluorophore(s).
If you can find additional money (neighboring labs? friendly dean?)
maybe time to get at least one new objective lens or lenses.
Finally, "instant gratification" deconvolution is just about here --
http://www.microvolution.com/ and
http://www.microvolution.com/technology.php
(yes, my image is on their home page - my colleague went "Wow" when he
saw the deconvolved data),
along with NVidia TITAN X card or cards (hey, at least you wouldn't need
to buy a new PC) ... new GPU cards would also let you put HD 4K monitors
on your system. Finally, an data center quality solid state drive for
local file saving (and then transfer to the network to a server for long
term storage and other desktop PC's access).

Do me a favor - for any experiment, do not use overlapping emission
filters like this paper did
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3653935/figure/pone-0063286-g004/
the emission filters are about $125 each (a bit more for threaded) -- a
tiny fraction of PLoS One's page charges, to say nothing of the money
spent on salaries for that paper (they also should have multimerized the
mPlum and other dim FPs). If you do like the above paper, see 26010570
for new (one week) paper that cites some of the recent developments in
many color cells ('brainbow 2015'). you may also find of use,
http://works.bepress.com/gmcnamara/65/ and my other writing on
Tattletales and T-Bow.

enjoy,

George

On 6/2/2015 10:12 AM, Arvydas Matiukas wrote:

> *****
> 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.
> *****
>
> Hello list,
>
> Please share your experience and advice on CCD camera
> for imaging fixed slides on widefield fluorescent microscope.
> We are looking for a reasonably priced, reliable and sensitive
> camera. High speed is not very important. Also how newest sCMOS
> compare to CCD?
>
> Our 5 year old 1.4MP cooled CCD camera died, and as it
> is discontinued, we prefer to replace it instead of repairing.
> It has been mostly used for imaging fixed brain slices
> labeled by immunofluorescence or fluorescent proteins,
> some labels being quite dim.
>
> Thanks,
> Arvydas
>
> Arvydas Matiukas, Ph.D.
> Director of Confocal&Two-Photon Core
> SUNY Upstate Medical University
> Neuroscience& Physiology Dept
>
>

--

George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42

Michelle Peckham

Re: mEos4 photoconversion

In reply to this post by mcammer

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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:

>*****
>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.
>*****
>
>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: (914) 309-3270
>
>________________________________________
>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
>
>*****
>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 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:
>>
>> *****
>> 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.
>> *****
>>
>> 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