FTIR microspectroscopy, in combination with spectral averaging procedure, enables precise analysis of pollen grains for chemical characterization and identification studies of fresh and fossilised pollen in botany, ecology and palaeosciences. Infrared microspectroscopy (µFTIR) of Pinaceae pollen can provide valuable information on plant phenology, ecophysiology and paleoecology, but measurements are challenging, resulting in unreproducible spectra. The comparative analysis of µFTIR spectra belonging to morphologically different Pinaceae pollen, namely bisaccate Pinus and monosaccate Tsuga pollen, was conducted. The study shows that the main cause of spectral variability is non-radial symmetry of bisaccate pollen grains, while additional variation is caused by Mie scattering. Averaging over relatively small number of single pollen grain spectra (approx. 5-10) results with reproducible data on pollen chemical composition. The practical applicability of the µFTIR spectral averaging method has been demonstrated by the partial least-squares regression-based differentiation of the two closely related Pinus species with morphologically indistinguishable pollen: Pinus mugo (mountain pine) and Pinus sylvestris (Scots pine). The study has demonstrated that the µFTIR approach can be used for identification, differentiation and chemical characterization of pollen with complex morphology. The methodology enables analysis of fresh pollen, as well as fossil pollen from sediment core samples, and can be used in botany, ecology and paleoecology for study of biotic and abiotic effects on plants.
Keywords: Fourier transform infrared spectroscopy; Mie scattering; Multivariate analysis; Pinus mugo; Pinus sylvestris; Tsuga canadensis.