dye question [Fwd: Strange Cy5/3 dye effect]

[Rietdorf, Jens]

Dear list,

Any idea what could cause this effect:

..a colleague of mine has a problem with his fluorescent microscopy
probes.
He labels 2 bacteria with different FISH probes. One oligo is labelled
with Cy5 and the other one with Cy3.
Both dyes behave normally when in solution, but when they are bound to
the bacteria they do not show emission fluorescences as expected, but
both become excitable at 488nm and show emission between 500/550nm. The
signals are very strong and autofluorescece of bacteria can be excluded.
According to the provider both probes are labelled with the correct
dyes.
 
Somehow both dyes change their excitation and emission wavelength after
binding to bacteria. Any idea how this could happen and what we could do
to avoid that?
 
Any help is welcome.
 
Thank you, jens

[JOEL B. SHEFFIELD]

Is this effect visible when each dye is used individually on the bacteria?  --and what kind of bacteria are used?
Joel

On Tue, Nov 10, 2009 at 3:10 AM, Rietdorf, Jens <[hidden email]> wrote:

Dear list,

Any idea what could cause this effect:

..a colleague of mine has a problem with his fluorescent microscopy
probes.
He labels 2 bacteria with different FISH probes. One oligo is labelled
with Cy5 and the other one with Cy3.
Both dyes behave normally when in solution, but when they are bound to
the bacteria they do not show emission fluorescences as expected, but
both become excitable at 488nm and show emission between 500/550nm. The
signals are very strong and autofluorescece of bacteria can be excluded.
According to the provider both probes are labelled with the correct
dyes.

Somehow both dyes change their excitation and emission wavelength after
binding to bacteria. Any idea how this could happen and what we could do
to avoid that?

Any help is welcome.

Thank you, jens

--


Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs

[Arne Seitz]

Dear Jens,

unfortunately I have only a somehow vague explanation what could have happened but no strategy how to avoid it.
The color and the fluorescence of the cyanine dyes can be nicely explained with "free" moving electrons in a potential well. Theoretical considerations show that decreasing the length of the well leads to absorption spectra with higher energy.
This seemed to happen when the probe is entering the bacteria. So I guess that the "free" pi electrons of the two molecules have reacted with a bacterial component. As the absorption spectra is the same for both dyes I think it is very likely that one of the "ring"-nitrogens have reacted. But I don't know any clever strategy to avoid it.

Just my two pence.

Cheers Arne

---------------------------------------------------------------
Dr. Arne Seitz
Head of Bioimaging and Optics Platform (BIOP)
Swiss Institute of Technology (EPFL)
Faculty of Life Sciences
Station 15
CH-1015 Lausanne

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

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rietdorf, Jens
Sent: mardi, 10. novembre 2009 09:10
To: [hidden email]
Subject: dye question [Fwd: Strange Cy5/3 dye effect]

Dear list,

Any idea what could cause this effect:

..a colleague of mine has a problem with his fluorescent microscopy
probes.
He labels 2 bacteria with different FISH probes. One oligo is labelled
with Cy5 and the other one with Cy3.
Both dyes behave normally when in solution, but when they are bound to
the bacteria they do not show emission fluorescences as expected, but
both become excitable at 488nm and show emission between 500/550nm. The
signals are very strong and autofluorescece of bacteria can be excluded.
According to the provider both probes are labelled with the correct
dyes.
 
Somehow both dyes change their excitation and emission wavelength after
binding to bacteria. Any idea how this could happen and what we could do
to avoid that?
 
Any help is welcome.
 
Thank you, jens

[Mario-2]

Jens,

Maybe someone else knows the answer to this for Cy3/Cy5 and I would
check it myself if my fluorometer weren't taken apart, but local
electrostatics can strongly affect absorption/emission spectra. One
simple test would be to check abs/em spectra for each probe versus
pH, failing that, vary the ionic strength. Whenever a probe is bound
to a target that is highly charged (nucleic acids, etc) the local pH
can be an order of magnitude or more different than the bulk pH.
Further, one might naively think that altering the bulk pH can
overcome this effect if active. Even a ten fold increase in the bulk
buffer concentration will have little to no effect on the local pH.
For example, with nucleic acids the phosphate moieties provide a
strong negative electrostatic field increasing the local [H+]. That
will be the proton concentration the fluorophore is exposed to, not
the bulk pH. With FISH probes the situation is more complicated
because of the local hydrogen bonding and dielectric constant shifts.
The latter can also effect the spectra.

Increasing the bulk buffer concentration while maintaining the same
ionic strength will have no effect on the local surface charge, the
negative surface potential, and the local pH. Concentrated clusters
of nucleic acids, negatively charged phospholipids and the like act
like overwhelmingly high concentrations of local buffers. The only
thing one can do is to provide counter ions that can bind locally
providing charge compensation. One might try to use divalent cations
such as Ca+2, Mg+2 10 mM or higher mixed with the buffer or lower
concentrations of trivalent lanthanides which bind very well to DNA
at 10 to 100 uM. HEPES works pretty well with lanthanides in that
there is little or no precipitation or binding that's a problem with
some buffers.

Anyway, I suggest doing some probe pH sensitivity and ionic strength
tests in bulk solution. If there is some sensitivity you might be
able to adjust the bulk pH, ionic strength, and/or multivalents that
give separable spectra of the two probes.

Mario

>Dear Jens,
>
>unfortunately I have only a somehow vague explanation what could
>have happened but no strategy how to avoid it.
>The color and the fluorescence of the cyanine dyes can be nicely
>explained with "free" moving electrons in a potential well.
>Theoretical considerations show that decreasing the length of the
>well leads to absorption spectra with higher energy.
>This seemed to happen when the probe is entering the bacteria. So I
>guess that the "free" pi electrons of the two molecules have reacted
>with a bacterial component. As the absorption spectra is the same
>for both dyes I think it is very likely that one of the
>"ring"-nitrogens have reacted. But I don't know any clever strategy
>to avoid it.
>
>Just my two pence.
>
>Cheers Arne
>
>---------------------------------------------------------------
>Dr. Arne Seitz
>Head of Bioimaging and Optics Platform (BIOP)
>Swiss Institute of Technology (EPFL)
>Faculty of Life Sciences
>Station 15
>CH-1015 Lausanne
>
>Phone: [phone]
>Fax:      [phone]
>http://biop.epfl.ch/
>---------------------------------------------------------------
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On Behalf Of Rietdorf, Jens
>Sent: mardi, 10. novembre 2009 09:10
>To: [hidden email]
>Subject: dye question [Fwd: Strange Cy5/3 dye effect]
>
>Dear list,
>
>Any idea what could cause this effect:
>
>..a colleague of mine has a problem with his fluorescent microscopy
>probes.
>He labels 2 bacteria with different FISH probes. One oligo is labelled
>with Cy5 and the other one with Cy3.
>Both dyes behave normally when in solution, but when they are bound to
>the bacteria they do not show emission fluorescences as expected, but
>both become excitable at 488nm and show emission between 500/550nm. The
>signals are very strong and autofluorescece of bacteria can be excluded.
>According to the provider both probes are labelled with the correct
>dyes.
>
>Somehow both dyes change their excitation and emission wavelength after
>binding to bacteria. Any idea how this could happen and what we could do
>to avoid that?
>
>Any help is welcome.
>
>Thank you, jens

--
________________________________________________________________________________
Mario M. Moronne, Ph.D.
[hidden email]

[George McNamara]

Hi jens,

Dye(-oligo) aggregates.

Suggestions:

a) Label each oligo at one 1 dye per oligo (ex. on 5' or 3' only).
Test the result free in solution (had better be the same as free dye)
vs with target present.

b) label (at one dye per oligo) with a non-Cy dye instead.

best wishes and let us know if it works out for you.

George

At 03:10 AM 11/10/2009, you wrote:

>Dear list,
>
>Any idea what could cause this effect:
>
>..a colleague of mine has a problem with his fluorescent microscopy
>probes.
>He labels 2 bacteria with different FISH probes. One oligo is labelled
>with Cy5 and the other one with Cy3.
>Both dyes behave normally when in solution, but when they are bound to
>the bacteria they do not show emission fluorescences as expected, but
>both become excitable at 488nm and show emission between 500/550nm. The
>signals are very strong and autofluorescece of bacteria can be excluded.
>According to the provider both probes are labelled with the correct
>dyes.
>
>Somehow both dyes change their excitation and emission wavelength after
>binding to bacteria. Any idea how this could happen and what we could do
>to avoid that?
>
>Any help is welcome.
>
>Thank you, jens