Is DMSO photobleaching autofluorescent proteins?

[Josef Gotzmann]

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Dear List,

I have a technical questions:
When we live image cells (in buffered saline, pH7.4) expressing GFP- and
mCherry-labeled proteins, respectively, on our confocal and we add a drug
(dissolved in DMSO) we rapidly (within seconds) lose fluorescence of both
dyes. After a very sharp drop down to 50% of intensity within the first 2
minutes, we reach a plateau (2-5minutes), followed by another less sharp,
though steady, decrease in fluorescence for another 2 minutes.
This is the effect we see with an end concentration of 1% DMSO, which is
completely absent when are scaling down the DMSO concentration to 0,1% !
We have already ruled out that it's simple bleaching due to lightning
conditions.
My understanding was that the OH-radical scavenging properties of DMSO
should more stabilize fluorescence!?
Anyone an idea how DMSO can act like that?

thx
Josef

[Kelly Lundsten-2]

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Hi Josef,

It's much more likely that you are denaturing the fluorescent protein.  Simple synthetic organic fluors like Alexa Fluors or Cy dyes, are often reconsistituted entirely in DMSO in their reactive form, for that purpose to protect them from oxygen.  DMSO does not hurt the actual fluorescent molecule itself.  I would absolutely imagine it can hurt protein, though.  With fluorescent proteins, the chromophore at the center of the beta barrel of a GFP requires the protein component to remain rigid.  Should the protein component relax or denature, the chromophore at the center of the molecule is no longer rigid itself and cannot resonate the energy it absorbs efficiently (quantum efficiency).  The same effect is seen with methanol or other denaturing solvents and fluorescent proteins.  The effect is not photobleaching, just a loss of quantum efficiency.  

Your cells also can't be so happy with the presence of the DMSO.  What most people would do in this instance is ask themselves 1. Can the drug be dissolved in something besides DMSO and 2. If not, can you concentrate the drug more so as to deliver the least amount of DMSO as is possible.

Good luck,
Kelly

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Josef Gotzmann
Sent: Thursday, March 14, 2013 9:42 AM
To: [hidden email]
Subject: Is DMSO photobleaching autofluorescent proteins?

*****
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Dear List,

I have a technical questions:
When we live image cells (in buffered saline, pH7.4) expressing GFP- and
mCherry-labeled proteins, respectively, on our confocal and we add a drug
(dissolved in DMSO) we rapidly (within seconds) lose fluorescence of both
dyes. After a very sharp drop down to 50% of intensity within the first 2
minutes, we reach a plateau (2-5minutes), followed by another less sharp,
though steady, decrease in fluorescence for another 2 minutes.
This is the effect we see with an end concentration of 1% DMSO, which is
completely absent when are scaling down the DMSO concentration to 0,1% !
We have already ruled out that it's simple bleaching due to lightning
conditions.
My understanding was that the OH-radical scavenging properties of DMSO
should more stabilize fluorescence!?
Anyone an idea how DMSO can act like that?

thx
Josef

[Jurek Dobrucki]

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Dear Josef,

indeed, it is possible that DMSO quenches the fluorescence FPs:

http://pubs.acs.org/doi/abs/10.1021/j100372a091
http://www.ncbi.nlm.nih.gov/pubmed/2457301

If it is the case, the effect ought to be reversible (immediately); if
the protein moiety is dentured, I guess it should be irreversible.

Hope this helps,
Regards,
Jurek Dobrucki
--
Jerzy Dobrucki, Ph.D., D.Sc.
Professor of Biophysics
Head, Division of Cell Biophysics
Faculty of Biochemistry, Biophysics and
    Biotechnology
Jagiellonian University
ul. Gronostajowa 7
30-387 Krakow, Poland
tel. [phone]; fax. [phone]
[hidden email]
http://www.wbbib.uj.edu.pl/helios




On Thu, Mar 14, 2013 at 8:01 PM, Kelly Lundsten <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Josef,
>
> It's much more likely that you are denaturing the fluorescent protein.  Simple synthetic organic fluors like Alexa Fluors or Cy dyes, are often reconsistituted entirely in DMSO in their reactive form, for that purpose to protect them from oxygen.  DMSO does not hurt the actual fluorescent molecule itself.  I would absolutely imagine it can hurt protein, though.  With fluorescent proteins, the chromophore at the center of the beta barrel of a GFP requires the protein component to remain rigid.  Should the protein component relax or denature, the chromophore at the center of the molecule is no longer rigid itself and cannot resonate the energy it absorbs efficiently (quantum efficiency).  The same effect is seen with methanol or other denaturing solvents and fluorescent proteins.  The effect is not photobleaching, just a loss of quantum efficiency.
>
> Your cells also can't be so happy with the presence of the DMSO.  What most people would do in this instance is ask themselves 1. Can the drug be dissolved in something besides DMSO and 2. If not, can you concentrate the drug more so as to deliver the least amount of DMSO as is possible.
>
> Good luck,
> Kelly
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Josef Gotzmann
> Sent: Thursday, March 14, 2013 9:42 AM
> To: [hidden email]
> Subject: Is DMSO photobleaching autofluorescent proteins?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear List,
>
> I have a technical questions:
> When we live image cells (in buffered saline, pH7.4) expressing GFP- and
> mCherry-labeled proteins, respectively, on our confocal and we add a drug
> (dissolved in DMSO) we rapidly (within seconds) lose fluorescence of both
> dyes. After a very sharp drop down to 50% of intensity within the first 2
> minutes, we reach a plateau (2-5minutes), followed by another less sharp,
> though steady, decrease in fluorescence for another 2 minutes.
> This is the effect we see with an end concentration of 1% DMSO, which is
> completely absent when are scaling down the DMSO concentration to 0,1% !
> We have already ruled out that it's simple bleaching due to lightning
> conditions.
> My understanding was that the OH-radical scavenging properties of DMSO
> should more stabilize fluorescence!?
> Anyone an idea how DMSO can act like that?
>
> thx
> Josef

[Tobias Baskin]

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Greetings,
        Some years ago, we tested our biological material (which
happens to be plant cells but principle is general) against solvents.
We found biological effects of 1% DMSO (and of 0.1% ethanol). For
that reason we never allow our cells to see concentrations as high as
1% DMSO (or 0.1% ethanol). I would not be surprised if animal/fungal
cells also were affected by 1% DMSO. Over the years we have used a
large variety of inhibitors and they almost always are able to be
disolved in 1000x DMSO, allowing us to add 1 miroL inhibitor stock
per mL of growth medium.

        Hope this helps,
                        Tobias

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear Josef,
>
>indeed, it is possible that DMSO quenches the fluorescence FPs:
>
>http://pubs.acs.org/doi/abs/10.1021/j100372a091
>http://www.ncbi.nlm.nih.gov/pubmed/2457301
>
>If it is the case, the effect ought to be reversible (immediately); if
>the protein moiety is dentured, I guess it should be irreversible.
>
>Hope this helps,
>Regards,
>Jurek Dobrucki
>--
>Jerzy Dobrucki, Ph.D., D.Sc.
>Professor of Biophysics
>Head, Division of Cell Biophysics
>Faculty of Biochemistry, Biophysics and
>     Biotechnology
>Jagiellonian University
>ul. Gronostajowa 7
>30-387 Krakow, Poland
>tel. [phone]; fax. [phone]
>[hidden email]
>http://www.wbbib.uj.edu.pl/helios
>
>
>
>
>On Thu, Mar 14, 2013 at 8:01 PM, Kelly Lundsten
><[hidden email]> wrote:
>>  *****
>>  To join, leave or search the confocal microscopy listserv, go to:
>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  *****
>>
>>  Hi Josef,
>>
>>  It's much more likely that you are denaturing the fluorescent
>>protein.  Simple synthetic organic fluors like Alexa Fluors or Cy
>>dyes, are often reconsistituted entirely in DMSO in their reactive
>>form, for that purpose to protect them from oxygen.  DMSO does not
>>hurt the actual fluorescent molecule itself.  I would absolutely
>>imagine it can hurt protein, though.  With fluorescent proteins,
>>the chromophore at the center of the beta barrel of a GFP requires
>>the protein component to remain rigid.  Should the protein
>>component relax or denature, the chromophore at the center of the
>>molecule is no longer rigid itself and cannot resonate the energy
>>it absorbs efficiently (quantum efficiency).  The same effect is
>>seen with methanol or other denaturing solvents and fluorescent
>>proteins.  The effect is not photobleaching, just a loss of quantum
>>efficiency.
>>
>>  Your cells also can't be so happy with the presence of the DMSO.
>>What most people would do in this instance is ask themselves 1. Can
>>the drug be dissolved in something besides DMSO and 2. If not, can
>>you concentrate the drug more so as to deliver the least amount of
>>DMSO as is possible.
>>
>>  Good luck,
>>  Kelly
>>
>>  -----Original Message-----
>>  From: Confocal Microscopy List
>>[mailto:[hidden email]] On Behalf Of Josef
>>Gotzmann
>>  Sent: Thursday, March 14, 2013 9:42 AM
>>  To: [hidden email]
>>  Subject: Is DMSO photobleaching autofluorescent proteins?
>>
>>  *****
>>  To join, leave or search the confocal microscopy listserv, go to:
>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  *****
>>
>>  Dear List,
>>
>>  I have a technical questions:
>>  When we live image cells (in buffered saline, pH7.4) expressing GFP- and
>>  mCherry-labeled proteins, respectively, on our confocal and we add a drug
>>  (dissolved in DMSO) we rapidly (within seconds) lose fluorescence of both
>>  dyes. After a very sharp drop down to 50% of intensity within the first 2
>>  minutes, we reach a plateau (2-5minutes), followed by another less sharp,
>>  though steady, decrease in fluorescence for another 2 minutes.
>>  This is the effect we see with an end concentration of 1% DMSO, which is
>>  completely absent when are scaling down the DMSO concentration to 0,1% !
>>  We have already ruled out that it's simple bleaching due to lightning
>  > conditions.
>>  My understanding was that the OH-radical scavenging properties of DMSO
>>  should more stabilize fluorescence!?
>>  Anyone an idea how DMSO can act like that?
>>
>>  thx
>>  Josef

--
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