Myelin / DiD labeling

[Vladimir Ghukasyan-2]

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Dear Colleagues,

We're trying to image myelin and face a strange problem.
a. Over the time course the DiD lipophilic dye is getting brighter.
When observing the sample after LSM with mercury light an overly
bright square can be visualized.
b. Signal in green channel from myelin particles is increasing with
time as well. Since DiD is supposed to fluoresce in 650 - 750, this is
strange. Non-labeled myelin didn't seem to have the same signal. This
can't be a cross-talk - we are using spectral separation, sequential
scanning (FV1000). Same effect observed on Zeiss 710.

Any suggestions?

Thank you in advance,
Vladimir

==================
Vladimir Ghukasyan, Ph.D.
Confocal and Multiphoton Imaging Facility
Neuroscience Center
University of North Carolina

115 Mason Farm Rd., Bld. 245, Rm. 7109F
Chapel Hill 27599-7250
NC

Tel.: [phone]
Fax: [phone]

[Julio Vazquez]

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We observed something similar with a mitochondria label (mitotracker), that was getting brighter over time. It turned out the person doing the experiment was using way too much dye. After they diluted the dye one hundred fold or more, the dye started behaving as expected. My interpretation is that the dye was self-quenching, and as dye was being photobleached, it was losing its ability to quench itself faster than it was being bleached, if that makes any sense. In any event, diluting it solved the problem. Don't know if this is what you are observing, but its easy to try...

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

http://www.fhcrc.org/




On Nov 1, 2011, at 2:43 PM, Vladimir Ghukasyan wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Colleagues,
>
> We're trying to image myelin and face a strange problem.
> a. Over the time course the DiD lipophilic dye is getting brighter.
> When observing the sample after LSM with mercury light an overly
> bright square can be visualized.
> b. Signal in green channel from myelin particles is increasing with
> time as well. Since DiD is supposed to fluoresce in 650 - 750, this is
> strange. Non-labeled myelin didn't seem to have the same signal. This
> can't be a cross-talk - we are using spectral separation, sequential
> scanning (FV1000). Same effect observed on Zeiss 710.
>
> Any suggestions?
>
> Thank you in advance,
> Vladimir
>
> ==================
> Vladimir Ghukasyan, Ph.D.
> Confocal and Multiphoton Imaging Facility
> Neuroscience Center
> University of North Carolina
>
> 115 Mason Farm Rd., Bld. 245, Rm. 7109F
> Chapel Hill 27599-7250
> NC
>
> Tel.: [phone]
> Fax: [phone]

[John Oreopoulos]

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Before reading Julio's response, I was going to suggest the same thing. It sounds like the dye concentration is too high and is de-quenching. As you image, some of the DiD begins to photobleach, and the self-quenching effect isn't as strong. Hence the sample appears to get brighter with time.

I have observed the same thing when using similar lipophilic probes like DiI and DiO at too high a concentration.

Try diluting your probe.

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


On 2011-11-01, at 5:57 PM, Julio Vazquez wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> We observed something similar with a mitochondria label (mitotracker), that was getting brighter over time. It turned out the person doing the experiment was using way too much dye. After they diluted the dye one hundred fold or more, the dye started behaving as expected. My interpretation is that the dye was self-quenching, and as dye was being photobleached, it was losing its ability to quench itself faster than it was being bleached, if that makes any sense. In any event, diluting it solved the problem. Don't know if this is what you are observing, but its easy to try...
>
> --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> Seattle, WA
>
> http://www.fhcrc.org/
>
>
>
>
> On Nov 1, 2011, at 2:43 PM, Vladimir Ghukasyan wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Colleagues,
>>
>> We're trying to image myelin and face a strange problem.
>> a. Over the time course the DiD lipophilic dye is getting brighter.
>> When observing the sample after LSM with mercury light an overly
>> bright square can be visualized.
>> b. Signal in green channel from myelin particles is increasing with
>> time as well. Since DiD is supposed to fluoresce in 650 - 750, this is
>> strange. Non-labeled myelin didn't seem to have the same signal. This
>> can't be a cross-talk - we are using spectral separation, sequential
>> scanning (FV1000). Same effect observed on Zeiss 710.
>>
>> Any suggestions?
>>
>> Thank you in advance,
>> Vladimir
>>
>> ==================
>> Vladimir Ghukasyan, Ph.D.
>> Confocal and Multiphoton Imaging Facility
>> Neuroscience Center
>> University of North Carolina
>>
>> 115 Mason Farm Rd., Bld. 245, Rm. 7109F
>> Chapel Hill 27599-7250
>> NC
>>
>> Tel.: [phone]
>> Fax: [phone]

[Iain Johnson]

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In addition to the concentration of DiD already mentioned, the method of
delivering it to the cells or tissue is important.  If you introduce DiD
(or any of the other lipophilic carbocyanines like DiI) from a DMSO or
ethanol stock solution into medium you inevitably produce a population of
stable dye aggregates that don't directly label cells.  If you leave the
preparation over time, there will be slow equilibration of aggregate and
monomer and some of the monomer will eventually label the cells.  The
monomer-aggregate equilibrium is extremely dependent on solution conditions
(salt concentration, osmolarity etc).   Overall, this is not a good
prescription for reproducible labeling.  In my experience, premixing the
dye with a carrier such as BSA before dispersing it in aqueous medium
produces more controlled labeling of cultures with lipophilic carbocyanines.

Iain

Iain Johnson Consulting
Eugene, OR
[phone]

On Tue, Nov 1, 2011 at 3:08 PM, John Oreopoulos <[hidden email]
> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Before reading Julio's response, I was going to suggest the same thing. It
> sounds like the dye concentration is too high and is de-quenching. As you
> image, some of the DiD begins to photobleach, and the self-quenching effect
> isn't as strong. Hence the sample appears to get brighter with time.
>
> I have observed the same thing when using similar lipophilic probes like
> DiI and DiO at too high a concentration.
>
> Try diluting your probe.
>
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
>
> On 2011-11-01, at 5:57 PM, Julio Vazquez wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > We observed something similar with a mitochondria label (mitotracker),
> that was getting brighter over time. It turned out the person doing the
> experiment was using way too much dye. After they diluted the dye one
> hundred fold or more, the dye started behaving as expected. My
> interpretation is that the dye was self-quenching, and as dye was being
> photobleached, it was losing its ability to quench itself faster than it
> was being bleached, if that makes any sense. In any event, diluting it
> solved the problem. Don't know if this is what you are observing, but its
> easy to try...
> >
> > --
> > Julio Vazquez
> > Fred Hutchinson Cancer Research Center
> > Seattle, WA
> >
> > http://www.fhcrc.org/
> >
> >
> >
> >
> > On Nov 1, 2011, at 2:43 PM, Vladimir Ghukasyan wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Dear Colleagues,
> >>
> >> We're trying to image myelin and face a strange problem.
> >> a. Over the time course the DiD lipophilic dye is getting brighter.
> >> When observing the sample after LSM with mercury light an overly
> >> bright square can be visualized.
> >> b. Signal in green channel from myelin particles is increasing with
> >> time as well. Since DiD is supposed to fluoresce in 650 - 750, this is
> >> strange. Non-labeled myelin didn't seem to have the same signal. This
> >> can't be a cross-talk - we are using spectral separation, sequential
> >> scanning (FV1000). Same effect observed on Zeiss 710.
> >>
> >> Any suggestions?
> >>
> >> Thank you in advance,
> >> Vladimir
> >>
> >> ==================
> >> Vladimir Ghukasyan, Ph.D.
> >> Confocal and Multiphoton Imaging Facility
> >> Neuroscience Center
> >> University of North Carolina
> >>
> >> 115 Mason Farm Rd., Bld. 245, Rm. 7109F
> >> Chapel Hill 27599-7250
> >> NC
> >>
> >> Tel.: [phone]
> >> Fax: [phone]
>