I supplemented bacteria with 13C labelled substrate, and measured the incorporation of labelled growth substrate at regular time interval with Raman Microscope. Incorporation of labelled substrate was proved by shift in wavenumbers but the intensity was also different for different cell at different time point. Can somebody please explain what does the intensity represents for bacterial sample?
Dear Priyanka,
I guess you are using a confocal Raman microscope. If that is the case, the absolute intensity scales with the excitation laser power and the number of molecules. Therefore it depends on experimental parameters (laser power), geometric parameters of the setup (focal plane vs plane where the bacteria are) and also the state of the sample (humidity). As these parameters cannot be controlled, researches use a statistical normalization and only use a relative intensity. Hope that is helping ... You might want to look to the paper: ‘How to pre-process Raman spectra for reliable and stable models?’ or ‘Demonstration of Carbon Catabolite Repression in Naphthalene Degrading Soil Bacteria via Raman Spectroscopy Based Stable Isotope Probing’.
Best,
Thomas
Dear Priyanka,
One should always keep in mind that microbial cells of the same genotype may express different phenotypes. When characterizing individual microbial cells by (confocal?) Raman microspectroscopy one will always observe some level of biological variance and perhaps also phenotypic heterogeneity. I would say that biological variance / phenotypic heterogeneity are the main sources of the observed intensity variations of the isotope (13C) shifted Raman bands (see also the paper below).
Having said that, in the theory of Raman scattering at least four variables define Raman intensities: (i) the intensity of the excitation laser, (ii) the laser wavelength (lambda^(-4) dependence) and (iii) the Raman cross section. Furthermore, Raman intensities are (linearly) dependent on the concentration (iv). Last but not least, in case of confocal Raman microspectroscopy the signal comes from a confocal volume which means that the optical configuration of your system (N.A., etc.) also affects the intensities of Raman bands.
Best regards,
Peter Lasch
Your question is somewhat vague. As pointed out by Thomas and Peter, absolute intensities depend on instrumental parameters (and therefore variations/fluctuations) and also sample heterogeneity. If, however, your (relative) intensity variations as a function of time are systematic, then they may tell you about metabolic processes. If you think they are, plot normalised or relative peak heights as a function of time and look for systematic variations. If they are evident, a multivariate regression of the full spectrum against time may tell you more.
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