Evaluation of Signal Reabsorption and Sample Heating in NIR-Raman Measurements

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Abstract
In order to assess the magnitude of the experimental problem of NIR absorption in NIR-Raman measurements, 1064 nm-Raman spectra of representative scattering lipid suspensions were measured, the spectral artifacts were compared, and the relative magnitude of sample heating was determined by using principal component analysis to measure the shift in a thermotropic phase transition. As in a previous evaluation of the problem, solvent O-H absorption was found to be the main difficulty because it significantly attenuates the Raman signal from C-H stretching. This is true even though the effective sample thickness was only 175 μm. Small Raman intensity artifacts were created by the changes in NIR absorption or optical scattering that occur with changing lipid concentration or state of liposome aggregation. Though use of D2O as the suspending solvent greatly improved the intensity of the C-H stretching region, in comparison to H2O suspensions, observed laser heating was reduced by only a factor of two. C-H stretching absorption can contribute to the heating when D2O is the solvent. In D2O the lipid sample heating was reduced to an acceptable level (1°C) when the laser illumination was 740 mW over a 2.5-mm circular spot. Thus power density needs to be kept at less than 1/10 that typically used in similar visible Raman experiments. O-H containing samples without strong optical scattering show pronounced spectral attenuation in the 180° geometry, if the spectrometer optics collect from deep within the specimen. This consideration places limitations on the use of long pathlengths to improve signal intensity. Extinction pathlengths available from the literature provide a convenient way to extrapolate these results to other NIR excitation wavelengths. Shifting excitation from 1064 nm to 910 nm would avoid most of the Raman spectral attenuation by O-H and reduce the H2O lipid suspension sample heating by a factor of two. Unfortunately, heating of the D2O suspensions will not be significantly reduced even if the excitation is moved to 830 nm.