Bleaching of background?

[xavier Sanjuan]

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Dear listers,

One of our users is making experiments to measure GFP signal in a cell
culture. Background fluorescence is quite strong and GFP signal is just
slightly a bit stronger than that. The thing we do not have an explanation
for is we are detecting an exponential decrease in background signal
intensity. Briefly the details of the experiment are:

Cells are cultured in Lab-Tek chambers in a collagen matrix (prepared with
phenol red from MEM 10x medium) and (in their initial experiments) kept in
medium with FBS + phenol red. Plates were imaged in a Zeiss Cell Observer
using a 20x dry objective and, as GFP signal is really low, an EMCCD
camera. Images were obtained every 30 min during 24h and exposure time was
between 750 ms and 2.5 s depending on the experiment.

As mentioned, under these conditions background signal is quite high but
over time we detect an exponential decrease in its intensity. As phenol red
is a known source of autofluorescence, we advised him to use medium without
it, but in these new experiments we observed the same phenomena. Wells with
only collagen + medium also showed that issue. He will try to prepare the
collagen also without phenol red just to totally discard this component as
a background source, but we still have the issue of "bleaching" of the
background. I don't expect bleaching to be an issue with that modest amount
of exposure, at least for GFP; and we all know autofluorescent components
tend to be more resistant to photobleaching than the labels we introduce in
our samples...

Anyone has an explanation for this exponential decrease in background
intensity then?

Thank you very much for your help,

Xavi.
___________________________________

*Xavier Sanjuan**
*Advanced Light Microscopy Unit

Parc de Recerca Biomèdica de Barcelona
Doctor Aiguader, 88
08003 Barcelona - Spain
Tel: + 34 93 316 0206 (ext 1206 dins el PRBB)
Fax: + 34 93 316 09 01
E-mail: [hidden email]
Web:
http://pasteur.crg.es/portal/page/portal/Internet/03_CORES/Core_Facilities/Advanced%20Light%20Microscopy%20Unit

[Arne Seitz]

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Dear Xavi,

unfortunately I do not have an explanation for your background. Nevertheless I just wanted to make some remarks regarding your conclusions:

> exposure time was
> between 750 ms and 2.5 s depending on the experiment.

>I don't expect bleaching to be an issue with that modest amount
> of exposure, at least for GFP; and we all know autofluorescent components
> tend to be more resistant to photobleaching than the labels we introduce in
> our samples...

I personally would consider 750 ms-2.5 s as a long exposure times. Therefore I don't think that bleaching can be ruled out.
But you can easily test it experimentally. Just image with a higher temporal resolution (e.g. image every 5 minutes) and  check whether the drop in intensity is faster or as fast as before. In the latter case you can be sure that bleaching is not an issue.

In the link below they describe that collagen itself is auto-fluorescent. So maybe the background is coming from the matrix. And as the matrix protein is probably higher concentrated (at least locally) as the GFP is could make an considerable contribution. This auto fluorescence can be increased due to an leakage of your filter-cube in the UV. It is known that a small amount of UV light is always present (therefore cells tend to be longer happy when the are illuminated with LED's).  

http://www.uhnresearch.ca/facilities/wcif/PDF/Autofluorescence.pdf

Just my 2c

Cheers Arne


---------------------------------------------------------------
Arne Seitz
Head of Bioimaging and Optics Platform (PT-BIOP)
Ecole Polytechnique Fédérale de Lausanne (EPFL)
Faculty of Life Sciences
Station 15, AI 0241
CH-1015 Lausanne

Phone: [phone]
Fax:      [phone]
http://biop.epfl.ch/
---------------------------------------------------------------

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of SANJUAN
> SAMARRA, XAVIER
> Sent: lundi 16 juillet 2012 12:26
> To: [hidden email]
> Subject: Bleaching of background?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear listers,
>
> One of our users is making experiments to measure GFP signal in a cell
> culture. Background fluorescence is quite strong and GFP signal is just slightly
> a bit stronger than that. The thing we do not have an explanation for is we
> are detecting an exponential decrease in background signal intensity. Briefly
> the details of the experiment are:
>
> Cells are cultured in Lab-Tek chambers in a collagen matrix (prepared with
> phenol red from MEM 10x medium) and (in their initial experiments) kept in
> medium with FBS + phenol red. Plates were imaged in a Zeiss Cell Observer
> using a 20x dry objective and, as GFP signal is really low, an EMCCD camera.
> Images were obtained every 30 min during 24h and exposure time was
> between 750 ms and 2.5 s depending on the experiment.
>
> As mentioned, under these conditions background signal is quite high but
> over time we detect an exponential decrease in its intensity. As phenol red is
> a known source of autofluorescence, we advised him to use medium without
> it, but in these new experiments we observed the same phenomena. Wells
> with only collagen + medium also showed that issue. He will try to prepare
> the collagen also without phenol red just to totally discard this component as
> a background source, but we still have the issue of "bleaching" of the
> background. I don't expect bleaching to be an issue with that modest amount
> of exposure, at least for GFP; and we all know autofluorescent components
> tend to be more resistant to photobleaching than the labels we introduce in
> our samples...
>
> Anyone has an explanation for this exponential decrease in background
> intensity then?
>
> Thank you very much for your help,
>
> Xavi.
> ___________________________________
>
> *Xavier Sanjuan**
> *Advanced Light Microscopy Unit
>
> Parc de Recerca Biomèdica de Barcelona
> Doctor Aiguader, 88
> 08003 Barcelona - Spain
> Tel: + 34 93 316 0206 (ext 1206 dins el PRBB)
> Fax: + 34 93 316 09 01
> E-mail: [hidden email]
> Web:
> http://pasteur.crg.es/portal/page/portal/Internet/03_CORES/Core_Facilities
> /Advanced%20Light%20Microscopy%20Unit

[Alessandro Esposito]

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Dear Sanjuan,
      I can confirm that you may see autofluorescence in many media
formulations. We observe the same with Leibovitz media. I never
investigated the issue as it is not impacting our experiments, but there is
clearly some component of Leibovitz (I've seen identical results with
DMEM) that is autofluorescent. I would also add that I assume the source
of autofluorescence is a relativelty large molecule as it diffuse very slowly
to the photobleached field of view. I mention this because it has possible
impact on imaging. Very often, by the time you select a cell and find the
proper imaging parameters, autofluorescence is significantly bleached and
(with the appropriate filters) not visible on the acquired images. However,
for time lapse imaging, autofluorescence is recoverying because of the
long time between subsequent exposures and contributing to background.

I am not sure if there is anything better than Leibovitz in terms of
autofluorescence, perhaps other users have more experience.
Cheers,

Alessandro

[B. Prabhakar Pandian]

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Hello,
            Can someone suggest a stable fluorescent stain for live cell
imaging of leukocytes upto 4 hours.  We are using Rhodamine but the
signal goes away rapidly.
We keep the lamp off during the non-acquisition time, hence
photo-bleaching is not the main issue.

Thanks,

-Prabhakar

[David Baddeley]

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My money would be on the collagen. I've seen very similar background and bleaching behaviour in collagen containing mountant (glycerol jelly).

Cheers,
David

------------------------------
On Mon, Jul 16, 2012 11:02 PM NZST Seitz Arne wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear Xavi,
>
>unfortunately I do not have an explanation for your background. Nevertheless I just wanted to make some remarks regarding your conclusions:
>
>> exposure time was
>> between 750 ms and 2.5 s depending on the experiment.
>
>>I don't expect bleaching to be an issue with that modest amount
>> of exposure, at least for GFP; and we all know autofluorescent components
>> tend to be more resistant to photobleaching than the labels we introduce in
>> our samples...
>
>I personally would consider 750 ms-2.5 s as a long exposure times. Therefore I don't think that bleaching can be ruled out.
>But you can easily test it experimentally. Just image with a higher temporal resolution (e.g. image every 5 minutes) and  check whether the drop in intensity is faster or as fast as before. In the latter case you can be sure that bleaching is not an issue.
>
>In the link below they describe that collagen itself is auto-fluorescent. So maybe the background is coming from the matrix. And as the matrix protein is probably higher concentrated (at least locally) as the GFP is could make an considerable contribution. This auto fluorescence can be increased due to an leakage of your filter-cube in the UV. It is known that a small amount of UV light is always present (therefore cells tend to be longer happy when the are illuminated with LED's).  
>
>http://www.uhnresearch.ca/facilities/wcif/PDF/Autofluorescence.pdf
>
>Just my 2c
>
>Cheers Arne
>
>
>---------------------------------------------------------------
>Arne Seitz
>Head of Bioimaging and Optics Platform (PT-BIOP)
>Ecole Polytechnique Fédérale de Lausanne (EPFL)
>Faculty of Life Sciences
>Station 15, AI 0241
>CH-1015 Lausanne
>
>Phone: [phone]
>Fax:      [phone]
>http://biop.epfl.ch/
>---------------------------------------------------------------
>
>
>
>
>
>
>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of SANJUAN
>> SAMARRA, XAVIER
>> Sent: lundi 16 juillet 2012 12:26
>> To: [hidden email]
>> Subject: Bleaching of background?
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear listers,
>>
>> One of our users is making experiments to measure GFP signal in a cell
>> culture. Background fluorescence is quite strong and GFP signal is just slightly
>> a bit stronger than that. The thing we do not have an explanation for is we
>> are detecting an exponential decrease in background signal intensity. Briefly
>> the details of the experiment are:
>>
>> Cells are cultured in Lab-Tek chambers in a collagen matrix (prepared with
>> phenol red from MEM 10x medium) and (in their initial experiments) kept in
>> medium with FBS + phenol red. Plates were imaged in a Zeiss Cell Observer
>> using a 20x dry objective and, as GFP signal is really low, an EMCCD camera.
>> Images were obtained every 30 min during 24h and exposure time was
>> between 750 ms and 2.5 s depending on the experiment.
>>
>> As mentioned, under these conditions background signal is quite high but
>> over time we detect an exponential decrease in its intensity. As phenol red is
>> a known source of autofluorescence, we advised him to use medium without
>> it, but in these new experiments we observed the same phenomena. Wells
>> with only collagen + medium also showed that issue. He will try to prepare
>> the collagen also without phenol red just to totally discard this component as
>> a background source, but we still have the issue of "bleaching" of the
>> background. I don't expect bleaching to be an issue with that modest amount
>> of exposure, at least for GFP; and we all know autofluorescent components
>> tend to be more resistant to photobleaching than the labels we introduce in
>> our samples...
>>
>> Anyone has an explanation for this exponential decrease in background
>> intensity then?
>>
>> Thank you very much for your help,
>>
>> Xavi.
>> ___________________________________
>>
>> *Xavier Sanjuan**
>> *Advanced Light Microscopy Unit
>>
>> Parc de Recerca Biomèdica de Barcelona
>> Doctor Aiguader, 88
>> 08003 Barcelona - Spain
>> Tel: + 34 93 316 0206 (ext 1206 dins el PRBB)
>> Fax: + 34 93 316 09 01
>> E-mail: [hidden email]
>> Web:
>> http://pasteur.crg.es/portal/page/portal/Internet/03_CORES/Core_Facilities
>> /Advanced%20Light%20Microscopy%20Unit

[simon walker (BI)-2]

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We routinely use the lipophilic dye Cell Mask Orange (Invitrogen) to label neutrophils in chemotaxis assays.  Our exps are not usually longer than half an hour, but we see no loss of signal during this time.
Simon

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of B. Prabhakar Pandian
Sent: 16 July 2012 21:18
To: [hidden email]
Subject: Fluorescent stain for leukocytes

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Hello,
            Can someone suggest a stable fluorescent stain for live cell imaging of leukocytes upto 4 hours.  We are using Rhodamine but the signal goes away rapidly.
We keep the lamp off during the non-acquisition time, hence photo-bleaching is not the main issue.

Thanks,

-Prabhakar
The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT Registered Charity No. 1053902.
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[Chris Guerin]

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Hi Prabhakar:

        We have successfully used DiI and DiO for staining blood derived macrophages (see:Blood 2009 114: 4664-4674). They are very photostable and used at the proper concentration relatively non-toxic.

Best wishes,

Chris

Christopher Guérin, Ph.D.
Leader, DMBR Microscopy Core
Manager, VIB Center for Life Science Imaging
V.I.B., D.M.B.R., Univ. Gent
Fiers-Schell-Van Montagu' building
Technologiepark 927, B-9052 Ghent (Zwijnaarde) - Belgium
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