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mEos2 live-cell imaging 12h

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Sarah Richert

mEos2 live-cell imaging 12h

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Hi everyone,
i´m using mEos2 to follow organelle turnover. We successfully image live
cells with photoactivated mEos2 on our confocal setup for more than 20
hours. For different reason though i as well would like to use our wide
field microscope, but here is the problem. Cells only survive
photoconversion for a couple of hours (3-5) on the wide field microscope. If
somebody has experience with this i would be extremely grateful as i have
been trying for month.

Details:
cells: Fibroblasts/ Oli-neu cell line/ primary oligodendrocytes

medium: tried simple (DMEM+HS) or complex media (according to cell type)
always without phenol red, +/- vitamin C as a radical scavenger....

photoconversion: we have a halogen lamp which can be set to different
intensities (1-4) (im sorry i dont know the amount of light it emits but
will find out if necessary). For photoconverting i use
a) the highest setting (4) and irradiate for about 1000-1500 ms
b) the sec. highest setting (3) and irradiate for about 2000-3000 ms
filter: i have a filter which 420-470 nm light, but i as well have used one
with a smaller spectrum (390-420).

Thanks for any kind of advice in advance!
Sarah

Julio Vazquez

Re: mEos2 live-cell imaging 12h

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Hi Sarah,

Did you check how long the cells survive if you just image them on the widefield scope (without the conversion)? This would allow to narrow down the problem and determine if the short viability is related to the conversion step, or to the imaging itself...

--
Julio Vazquez,
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024

http://www.fhcrc.org

On Apr 25, 2013, at 2:59 AM, Sarah Richert wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone,
> i´m using mEos2 to follow organelle turnover. We successfully image live
> cells with photoactivated mEos2 on our confocal setup for more than 20
> hours. For different reason though i as well would like to use our wide
> field microscope, but here is the problem. Cells only survive
> photoconversion for a couple of hours (3-5) on the wide field microscope. If
> somebody has experience with this i would be extremely grateful as i have
> been trying for month.
>
> Details:
> cells: Fibroblasts/ Oli-neu cell line/ primary oligodendrocytes
>
> medium: tried simple (DMEM+HS) or complex media (according to cell type)
> always without phenol red, +/- vitamin C as a radical scavenger....
>
> photoconversion: we have a halogen lamp which can be set to different
> intensities (1-4) (im sorry i dont know the amount of light it emits but
> will find out if necessary). For photoconverting i use
> a) the highest setting (4) and irradiate for about 1000-1500 ms
> b) the sec. highest setting (3) and irradiate for about 2000-3000 ms
> filter: i have a filter which 420-470 nm light, but i as well have used one
> with a smaller spectrum (390-420).
>
> Thanks for any kind of advice in advance!
> Sarah

Julio Vazquez

Re: mEos2 live-cell imaging 12h

In reply to this post by Sarah Richert

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Either way, one possible culprit may be the filter sets leaking out UV light.... If your widefield system is old and/or has old filter sets (or low grade filter sets), you may want to consider purchasing a high qualiy filterset for your experiment, and/or add a UV-blocking filter. Check with your scope rep. Otherwise, or in addition, you may need to sacrifice a bit of image quality and cut down illumination intensity and/or exposure times. This may also depend on teh grade of camera you have A better camera will typically allow you to use shorter exposure times.
--
Julio Vazquez,
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024

http://www.fhcrc.org

On Apr 25, 2013, at 2:59 AM, Sarah Richert wrote:

Mike Ignatius-2

Re: mEos2 live-cell imaging 12h

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That are a few recent papers documenting the negative effects that vitamins
in complete media can have on the photostability of fluorescent proteins -
up to a nine fold improvement for some FP forms.

"Cell culture medium affects GFP photostability: a solution." Bogdanov AM,
Bogdanova EA, Chudakov DM, Gorodnicheva TV, Lukyanov S, Lukyanov KA. Nat
Methods. 2009 Dec;6(12):859-60.

If FPs are bleaching, it has to be considered that other native protein
function is being altered.

Culture media is optimized for cell growth, not cell imaging. A long list
of problems includes flavins that are autofluorescent, vitamins that are
photolabile and become toxic with imaging, pH changes outside of C02 changes
and phenol red quenching green emission. For those working with scopes with
even minor UV leak, all these negative influences can be further compounded.

A brief commercial moment - Marker Gene Tech sells a live cell imaging
buffer (Opti-Klear) that avoids most of the pitfalls of media formulations
for extended live cell imaging sessions outside of CO2.

Mike Ignatius

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Julio Vazquez
Sent: Thursday, April 25, 2013 10:57 AM
To: [hidden email]
Subject: Re: mEos2 live-cell imaging 12h

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Either way, one possible culprit may be the filter sets leaking out UV
light.... If your widefield system is old and/or has old filter sets (or low
grade filter sets), you may want to consider purchasing a high qualiy
filterset for your experiment, and/or add a UV-blocking filter. Check with
your scope rep. Otherwise, or in addition, you may need to sacrifice a bit
of image quality and cut down illumination intensity and/or exposure times.
This may also depend on teh grade of camera you have A better camera will
typically allow you to use shorter exposure times.
--
Julio Vazquez,
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024

http://www.fhcrc.org

On Apr 25, 2013, at 2:59 AM, Sarah Richert wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone,
> i´m using mEos2 to follow organelle turnover. We successfully image
> live cells with photoactivated mEos2 on our confocal setup for more
> than 20 hours. For different reason though i as well would like to use
> our wide field microscope, but here is the problem. Cells only survive
> photoconversion for a couple of hours (3-5) on the wide field
> microscope. If somebody has experience with this i would be extremely
> grateful as i have been trying for month.
>
> Details:
> cells: Fibroblasts/ Oli-neu cell line/ primary oligodendrocytes
>
> medium: tried simple (DMEM+HS) or complex media (according to cell
> type) always without phenol red, +/- vitamin C as a radical scavenger....
>
> photoconversion: we have a halogen lamp which can be set to different
> intensities (1-4) (im sorry i dont know the amount of light it emits
> but will find out if necessary). For photoconverting i use
> a) the highest setting (4) and irradiate for about 1000-1500 ms
> b) the sec. highest setting (3) and irradiate for about 2000-3000 ms
> filter: i have a filter which 420-470 nm light, but i as well have
> used one with a smaller spectrum (390-420).
>
> Thanks for any kind of advice in advance!
> Sarah