Raman spectroscopy gives us some amazing insight into ancient life. To pick just example, Raman researchers have analysed ancient microbes on Earth to teach us how to recognise life on other planets (Marshall et al 2006). Rather than highlight the advantages of Raman spectroscopy though, I thought I might focus on one of the biggest drawbacks of this technique: Autofluorescence, which is more commonly just called fluorescence.
“The natural enemy of Raman spectroscopy is fluorescence” – Olaf Hollricher, Raman Instrumentation for Confocal Raman Microscopy.
[Check out this post for a basic refresher on Raman spectroscopy]. Fluorescence is what happens when a substance absorbs and then immediately emits light. In essence, the substance glows when a light is shone on it. This is kind-of but-not-quite like a glow-in-the-dark toy: you shine light on the toy, and when you remove the light you can still see the toy. The difference here is that the toy has stored the light and is slowly releasing it, instead of receiving and releasing the light at nearly the same time. Glow-in-the-dark is an example of phosphorescence. We are talking about fluorescence.
Have you ever been to a museum with rocks and minerals that are being lit with a ‘black light’? Did the minerals look all bright red and green and purple like in the picture from Wikipedia below? The bright colours of these minerals are examples of fluorescence.
For a plain-language video explanation of fluorescence (…that uses lasers), check out this Youtube clip:
So the important point here is that a higher energy (shorter wavelength) light source can make substances emit lower energy (longer wavelength) light. A Raman spectrometer that uses a green wavelength (shorter wavelength) might cause a bone to emit near infrared light (longer wavelength). How does this make fluorescence the “…natural enemy…” of Raman spectroscopy? Quite simply, it’s because the business end of a Raman spectrometer is a CCD or CMOS detector, like the one in your camera. The Raman instrument is designed around producing and gathering light, and because of the way Raman scattering works, the detectors need to be very sensitive. Also, detectors don’t discriminate between Raman scattered light and the light produced by fluorescence. Light emitted during fluorescence is much more intense than Raman scattered light: emitted light drowns out the scattered light.
So, why is fluorescence the “…natural enemy…” of Raman spectroscopy? A sample that fluoresces during a Raman analysis is unlikely to give you a meaningful Raman spectrum. So the trick is to stop a sample from fluorescing…
Marshall CP, Carter EA, Leuko S, Javaux EJ. 2006. Vibrational spectroscopy of extant and fossil microbes: Relevance for the astrobiological exploration of Mars. Vibrational Spectroscopy 41: 182-189