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Beam, Brooke M - (bbeam) on
URL: http://confocal-microscopy-list.275.s1.nabble.com/weird-emission-spectral-peak-of-a-blank-buffer-tp7579220p7579222.html
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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:
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Ph: (520)621-3395
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From: Confocal Microscopy List [mailto:
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Sent: Wednesday, October 24, 2012 10:27 AM
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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