Re: weird emission spectral peak of a blank buffer

Posted by yuansheng sun on
URL: http://confocal-microscopy-list.275.s1.nabble.com/weird-emission-spectral-peak-of-a-blank-buffer-tp7579220p7579226.html

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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:

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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:
>
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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
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
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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
> >
>