weird emission spectral peak of a blank buffer

[yuansheng sun]

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Dear Listers,

Recently, we got two weird peaks in the emission spectrum of our blank
buffer (1-molar Tris at pH 7.3), measured on a spectrofluometer.  The Tris
buffer is what we use to make our dye solutions to be measured on the
spectrofluometer.  It is always freshly prepared.  One small peak of the
blank buffer shows at about 20 nm plus the excitaion wavlength
and the other huge peak appears at about twice of the excitation
wavelength.  We found this with the emission spectrum measurements using 5
different excitation wavelengths - 250 nm, 280 nm, 285 nm 300 nm, and 350
nm.  We also observed the similar things with DI water.  Any explanation
and suggestion would be appreciated.  Thanks a lot in advance.

Yuansheng Sun

[Craig Brideau]

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Maybe the spectrometer needs to be calibrated, or the optical path is
misaligned.  If you are getting signal at twice the excitation then it
could be 'wrap-around' off the grating or whatever your spectrometer uses
to split the light.  Sometimes this can be because the slit in your
spectrometer is misaligned, but it could also be the optical path leading
up to the spectrometer.

Craig


On Wed, Oct 24, 2012 at 11:26 AM, yuansheng sun <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Listers,
>
> Recently, we got two weird peaks in the emission spectrum of our blank
> buffer (1-molar Tris at pH 7.3), measured on a spectrofluometer.  The Tris
> buffer is what we use to make our dye solutions to be measured on the
> spectrofluometer.  It is always freshly prepared.  One small peak of the
> blank buffer shows at about 20 nm plus the excitaion wavlength
> and the other huge peak appears at about twice of the excitation
> wavelength.  We found this with the emission spectrum measurements using 5
> different excitation wavelengths - 250 nm, 280 nm, 285 nm 300 nm, and 350
> nm.  We also observed the similar things with DI water.  Any explanation
> and suggestion would be appreciated.  Thanks a lot in advance.
>
> Yuansheng Sun
>

[Beam, Brooke M - (bbeam)]

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Yuansheng,
The lower wavelength small peak is the water Raman signal.  The water Raman shift is 3382 cm-1 and the wavelength depends on the excitation wavelength.  For your 350 excitation spectra, the Raman peak would occur at 397 nm.  For the others you can calculate the shift using this equation: Raman shift (cm-1) = ((1/excitation wavelength(nm)) -(1/Raman peak (nm))) *10^7.

The peak at twice the excitation wavelength is the second order diffraction peak of your excitation line.  To get rid of this you will need to purchase a filter to remove this light from entering the emission monochromator.  You can purchase order suppressing filters or long pass filters to accomplish this (you will need to talk to your fluorimeter manufacturer to see how this filter can be added to your system).  The other option is to choose dyes with emission peaks that are less than 2*excitation wavelength.
Best,
Brooke Beam

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Brooke Beam, Ph.D.
W.M. Keck Center for Surface and Interface Imaging
University of Arizona Chemistry & Biochemistry
1306 E. University Blvd.
Tucson, AZ 85721
website: www.chem.arizona.edu/rss/keck/keck.html
email: [hidden email]
Ph: [phone]
Fax: [phone]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of yuansheng sun
Sent: Wednesday, October 24, 2012 10:27 AM
To: [hidden email]
Subject: weird emission spectral peak of a blank buffer

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Dear Listers,

Recently, we got two weird peaks in the emission spectrum of our blank buffer (1-molar Tris at pH 7.3), measured on a spectrofluometer.  The Tris buffer is what we use to make our dye solutions to be measured on the spectrofluometer.  It is always freshly prepared.  One small peak of the blank buffer shows at about 20 nm plus the excitaion wavlength and the other huge peak appears at about twice of the excitation wavelength.  We found this with the emission spectrum measurements using 5 different excitation wavelengths - 250 nm, 280 nm, 285 nm 300 nm, and 350 nm.  We also observed the similar things with DI water.  Any explanation and suggestion would be appreciated.  Thanks a lot in advance.

Yuansheng Sun

[Craig Brideau]

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Raman is usually pretty weak.  How long are you acquiring?  If this is over
a (relatively) long acquisition then yes, it could be Raman.  If you look
up the Raman lines for the plastics and liquids in your sample that might
give you a clue.

Craig


On Wed, Oct 24, 2012 at 11:56 AM, Beam, Brooke M - (bbeam) <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Yuansheng,
> The lower wavelength small peak is the water Raman signal.  The water
> Raman shift is 3382 cm-1 and the wavelength depends on the excitation
> wavelength.  For your 350 excitation spectra, the Raman peak would occur at
> 397 nm.  For the others you can calculate the shift using this equation:
> Raman shift (cm-1) = ((1/excitation wavelength(nm)) -(1/Raman peak (nm)))
> *10^7.
>
> The peak at twice the excitation wavelength is the second order
> diffraction peak of your excitation line.  To get rid of this you will need
> to purchase a filter to remove this light from entering the emission
> monochromator.  You can purchase order suppressing filters or long pass
> filters to accomplish this (you will need to talk to your fluorimeter
> manufacturer to see how this filter can be added to your system).  The
> other option is to choose dyes with emission peaks that are less than
> 2*excitation wavelength.
> Best,
> Brooke Beam
>
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> Brooke Beam, Ph.D.
> W.M. Keck Center for Surface and Interface Imaging
> University of Arizona Chemistry & Biochemistry
> 1306 E. University Blvd.
> Tucson, AZ 85721
> website: www.chem.arizona.edu/rss/keck/keck.html
> email: [hidden email]
> Ph: [phone]
> Fax: [phone]
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of yuansheng sun
> Sent: Wednesday, October 24, 2012 10:27 AM
> To: [hidden email]
> Subject: weird emission spectral peak of a blank buffer
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Listers,
>
> Recently, we got two weird peaks in the emission spectrum of our blank
> buffer (1-molar Tris at pH 7.3), measured on a spectrofluometer.  The Tris
> buffer is what we use to make our dye solutions to be measured on the
> spectrofluometer.  It is always freshly prepared.  One small peak of the
> blank buffer shows at about 20 nm plus the excitaion wavlength and the
> other huge peak appears at about twice of the excitation wavelength.  We
> found this with the emission spectrum measurements using 5 different
> excitation wavelengths - 250 nm, 280 nm, 285 nm 300 nm, and 350 nm.  We
> also observed the similar things with DI water.  Any explanation and
> suggestion would be appreciated.  Thanks a lot in advance.
>
> Yuansheng Sun
>

[yuansheng sun]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Dear Craig and Brooke,

Thanks a lot for your explanations, which are really helpful.  The Raman
signal for the small peak makes sense to me, since it is relatively very
small compared to the dye peak.  Now, I need to figure out how to get rid
of the huge peak at the twice of the excitation wavelength.  Thanks again.

Sheng

On Wed, Oct 24, 2012 at 2:11 PM, Craig Brideau <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Raman is usually pretty weak.  How long are you acquiring?  If this is over
> a (relatively) long acquisition then yes, it could be Raman.  If you look
> up the Raman lines for the plastics and liquids in your sample that might
> give you a clue.
>
> Craig
>
>
> On Wed, Oct 24, 2012 at 11:56 AM, Beam, Brooke M - (bbeam) <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Yuansheng,
> > The lower wavelength small peak is the water Raman signal.  The water
> > Raman shift is 3382 cm-1 and the wavelength depends on the excitation
> > wavelength.  For your 350 excitation spectra, the Raman peak would occur
> at
> > 397 nm.  For the others you can calculate the shift using this equation:
> > Raman shift (cm-1) = ((1/excitation wavelength(nm)) -(1/Raman peak (nm)))
> > *10^7.
> >
> > The peak at twice the excitation wavelength is the second order
> > diffraction peak of your excitation line.  To get rid of this you will
> need
> > to purchase a filter to remove this light from entering the emission
> > monochromator.  You can purchase order suppressing filters or long pass
> > filters to accomplish this (you will need to talk to your fluorimeter
> > manufacturer to see how this filter can be added to your system).  The
> > other option is to choose dyes with emission peaks that are less than
> > 2*excitation wavelength.
> > Best,
> > Brooke Beam
> >
> > ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> > Brooke Beam, Ph.D.
> > W.M. Keck Center for Surface and Interface Imaging
> > University of Arizona Chemistry & Biochemistry
> > 1306 E. University Blvd.
> > Tucson, AZ 85721
> > website: www.chem.arizona.edu/rss/keck/keck.html
> > email: [hidden email]
> > Ph: [phone]
> > Fax: [phone]
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> > On Behalf Of yuansheng sun
> > Sent: Wednesday, October 24, 2012 10:27 AM
> > To: [hidden email]
> > Subject: weird emission spectral peak of a blank buffer
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Dear Listers,
> >
> > Recently, we got two weird peaks in the emission spectrum of our blank
> > buffer (1-molar Tris at pH 7.3), measured on a spectrofluometer.  The
> Tris
> > buffer is what we use to make our dye solutions to be measured on the
> > spectrofluometer.  It is always freshly prepared.  One small peak of the
> > blank buffer shows at about 20 nm plus the excitaion wavlength and the
> > other huge peak appears at about twice of the excitation wavelength.  We
> > found this with the emission spectrum measurements using 5 different
> > excitation wavelengths - 250 nm, 280 nm, 285 nm 300 nm, and 350 nm.  We
> > also observed the similar things with DI water.  Any explanation and
> > suggestion would be appreciated.  Thanks a lot in advance.
> >
> > Yuansheng Sun
> >
>

[Iain Johnson]

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As Brooke already mentioned, the way to get rid of the peak at twice the
excitation wavelength is using a blocking filter on the emission side of
the sample.  The point being that if you are exciting at 350 nm, the peak
that is being detected at 700 nm is due to second order diffraction of 350
nm light (i.e. the monochromator reading is "wrong" in relation to the
actual wavelength of light that is being diffracted in this specific
instance).  The filter needed is generally a simple glass transmission
filter that  blocks the excitation light and transmits all wavelengths of
fluorescence that you are interested in.  For UV excitation, a simple piece
of flat glass (which has a transmission cutoff  at about 350 nm) will often
work fine.  For other wavelengths, the required colored glass filters can
be purchased inexpensively from optical supply houses such as Melles-Griot
or Edmunds.  They're often called Corning or Schott glasses after the
manufacturers.  Typically they come in 2 inch squares and can be simply
leaned against the emission face of the sample holders in most commercial
fluorometers.

Another way to get rid of second order diffracted light is by placing
perpendicularly oriented linear polarizers on the excitation and emission
sides of the sample.  This method incurs bigger light losses and is more
expensive to implement than transmission blocking, so generally less
preferred.

For Raman, the easiest way to deal with it is just to numerically subtract
a buffer blank spectrum recorded under the same slit width and
photomultiplier gain conditions used for the fluorescent sample.  The
intensity of Raman scattering is proportional to one over excitation
wavelength to the fourth power, so it is much more prominent with UV
excitation compared to visible.  I've always regarded Raman a useful thing,
in that it is an internal calibrator that tells you that you have a very
weak (and perhaps  practically useless) fluorescence signal.

Iain

Iain Johnson Consulting
Eugene, OR
[phone]

On Wed, Oct 24, 2012 at 1:15 PM, yuansheng sun <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Craig and Brooke,
>
> Thanks a lot for your explanations, which are really helpful.  The Raman
> signal for the small peak makes sense to me, since it is relatively very
> small compared to the dye peak.  Now, I need to figure out how to get rid
> of the huge peak at the twice of the excitation wavelength.  Thanks again.
>
> Sheng
>
> On Wed, Oct 24, 2012 at 2:11 PM, Craig Brideau <[hidden email]
> >wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Raman is usually pretty weak.  How long are you acquiring?  If this is
> over
> > a (relatively) long acquisition then yes, it could be Raman.  If you look
> > up the Raman lines for the plastics and liquids in your sample that might
> > give you a clue.
> >
> > Craig
> >
> >
> > On Wed, Oct 24, 2012 at 11:56 AM, Beam, Brooke M - (bbeam) <
> > [hidden email]> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > *****
> > >
> > > Yuansheng,
> > > The lower wavelength small peak is the water Raman signal.  The water
> > > Raman shift is 3382 cm-1 and the wavelength depends on the excitation
> > > wavelength.  For your 350 excitation spectra, the Raman peak would
> occur
> > at
> > > 397 nm.  For the others you can calculate the shift using this
> equation:
> > > Raman shift (cm-1) = ((1/excitation wavelength(nm)) -(1/Raman peak
> (nm)))
> > > *10^7.
> > >
> > > The peak at twice the excitation wavelength is the second order
> > > diffraction peak of your excitation line.  To get rid of this you will
> > need
> > > to purchase a filter to remove this light from entering the emission
> > > monochromator.  You can purchase order suppressing filters or long pass
> > > filters to accomplish this (you will need to talk to your fluorimeter
> > > manufacturer to see how this filter can be added to your system).  The
> > > other option is to choose dyes with emission peaks that are less than
> > > 2*excitation wavelength.
> > > Best,
> > > Brooke Beam
> > >
> > > ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> > > Brooke Beam, Ph.D.
> > > W.M. Keck Center for Surface and Interface Imaging
> > > University of Arizona Chemistry & Biochemistry
> > > 1306 E. University Blvd.
> > > Tucson, AZ 85721
> > > website: www.chem.arizona.edu/rss/keck/keck.html
> > > email: [hidden email]
> > > Ph: [phone]
> > > Fax: [phone]
> > >
> > >
> > > -----Original Message-----
> > > From: Confocal Microscopy List [mailto:
> [hidden email]]
> > > On Behalf Of yuansheng sun
> > > Sent: Wednesday, October 24, 2012 10:27 AM
> > > To: [hidden email]
> > > Subject: weird emission spectral peak of a blank buffer
> > >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > *****
> > >
> > > Dear Listers,
> > >
> > > Recently, we got two weird peaks in the emission spectrum of our blank
> > > buffer (1-molar Tris at pH 7.3), measured on a spectrofluometer.  The
> > Tris
> > > buffer is what we use to make our dye solutions to be measured on the
> > > spectrofluometer.  It is always freshly prepared.  One small peak of
> the
> > > blank buffer shows at about 20 nm plus the excitaion wavlength and the
> > > other huge peak appears at about twice of the excitation wavelength.
>  We
> > > found this with the emission spectrum measurements using 5 different
> > > excitation wavelengths - 250 nm, 280 nm, 285 nm 300 nm, and 350 nm.  We
> > > also observed the similar things with DI water.  Any explanation and
> > > suggestion would be appreciated.  Thanks a lot in advance.
> > >
> > > Yuansheng Sun
> > >
> >
>