Effects of sample heating in FT-Raman spectra of biological materials

The effects of laser-induced sample heating and fluorescence of biopolymers in Fourier transform-Raman spectroscopy were investigated. It was shown that these two phenomena are essentially independent of each other. Methods of quantifying the laser-induced healing were developed and it was shown that measurement of the thermal emission generated by the sample is not sufficient to determine its temperature. The use of a marker compound mixed with the test sample to estimate the sample temperature was explored. Monitoring the temperature-dependent phase transitions of NH4NO3 provided a crude indication of a minimum sample temperature. However, using sulphur as a marker and exploiting the relationship between the intensities of its anti-Stokes and Stokes bands, it was possible to estimate the sample temperature with greater confidence. Using these techniques it was shown that sample temperatures could easily reach approximately 400 K under normal laser power inputs. Such temperatures are likely to be damaging to biological samples and it is concluded that great care must be exercised when using FT-Raman.