Hi everyone! I'm trying to work on the acquisition of the Raman Spectra of a leaf section using Confocal Raman Spectroscopy. The samples to be used are pure, dried, and powdered leaf samples. I am going to use a 785 nm laser source.
However, the only thing I get was a spectra with no peaks or it is strongly masked by fluorescence. Do you have any tricks/sample preparations to avoid the fluorescence because I'm afraid that it covers the raman signal or enhance the Raman Signal because the compounds might have a relatively weak Raman Signal compare to the background signal and the fluorescence? Are there any sample preparations that can be done without the use of water or an immersion objective like the use of solid matrices which can be mixed with the sample? Thank you.
Dear John,
As you have noted, fluorescense is often a serious problem in biomedical Raman spectroscopy. Strategies to overcome the fluorescence problem can be subdivided into three main categories:
a) reducing the fluorescence signal and/or at enhancing the Raman signal - photobleaching,fluorescence quenching, removal of the fluorophores e.g. by sample washing, or filtration, etc.
b) utilizing different physical properties of the Raman scattered light and the fluorescence: using a NIR laser with longer wavelengths (1064 nm), anti-Stokes Raman spectroscopy (see link attached), etc.
c) application of computational methods like baseline subtraction, etc. pp
In your case I would try first carefully pre-bleaching the samples. You could try also anti-Stokes Raman spectroscopy - the fluorescence signal is usually red-shifted, however the anti-Stokes bands are less intense than the Stokes bands. I am afraid, however, that without washing the samples in water or non-polar liquids you will not be able to remove the fluorophores.
http://doi.org/10.1016/0030-3992(86)90026-5
Dr, Lasch covers it. But you may also find that using a shorter wavelength works. The offset from the (variable) laser wavelength is constant for Raman, or nearly so, while the fluorescence wavelength is fixed. OTOH the higher-energy laser may excite additional fluorescence modes, so no guarantees are offered.
I refer to my previous answer to a similar question:
Fluorescence from samples can dominate their Raman spectra: Shifted excitation Raman methods can reduce this background, an example being Sequentially Shifted Excitation Raman [1 - 2].
Another shifted excitation Raman method is shown in [3],
[1] Sequentially Shifted Excitation Raman Spectroscopy: Novel Algorithm and Instrumentation for Fluorescence-Free Raman Spectroscopy in Spectral Space, Applied Spectroscopy Vol. 67, Issue 8, pp. 973-984 (2013)
[2] https://www.bruker.com/fileadmin/user_upload/8-PDF-Docs/OpticalSpectrospcopy/Raman/BRAVO/Flyers/BRAVO_Flyer_EN.pdf
[3] A shifted-excitation Raman difference spectroscopy (SERDS) evaluation strategy for the efficient isolation of Raman spectra from extreme fluorescence interference, Journal of Raman Spectroscopy, 47(2), 198–209, 2016: http://onlinelibrary.wiley.com/doi/10.1002/jrs.4775/epdf
Alternatively, the use of time gated methods may also give Raman spectra from difficult samples: http://www.timegate.fi/timegated-raman/
Could you help with a Raman Spectrum technique?. Available from: https://www.researchgate.net/post/Could_you_help_with_a_Raman_Spectrum_technique [accessed Apr 21, 2017].
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