Sprecher
Beschreibung
Single-grain pollen analysis holds significant promise for advancing chemical palynology, enabling the potential future classification of morphologically similar taxa based on taxon-specific sporopollenin chemistry. Fourier-Transform Infrared (FT-IR) spectroscopy and microspectroscopy (µFT-IR) are widely used to differentiate morphologically similar grains but face limitations from Mie scattering and coarse spatial resolution. Here, we present the first application of Optical-PhotoThermal Infrared (O-PTIR) spectroscopy to chemically treated single pollen grains, using Molinia caerulea as a model taxon to fully evaluate the technical capabilities and limitations of this technique. O-PTIR achieves sub-micron "super-resolution" infrared spectroscopy through non-contact, non-destructive measurements by exploiting the photothermal effect. O-PTIR spectra exhibited close similarity to FT-IR datasets, with all major sporopollenin bands present and only minor peak shifts (~ 4 cm⁻¹). Reliable application of sub-micron spectra for classification purposes requires a robust understanding of intra- and inter-grain variability. This can now be assessed using O-PTIR to distinguish whether intra-grain surface heterogeneity or true inter-grain biochemical differences drives grain separation. Multivariate analysis of intra- and inter-grain variability indicated that greater intra-grain variability does not drive greater inter-grain variability, confirming that observed grain separation reflects genuine biochemical differences rather than technical or surface-related artefacts. Similarly, intra- and inter-grain variability did not differ significantly between plants. While a single O-PTIR measurement can produce a spectrum closely resembling the species centroid, averaging three to four measurements per grain yields reliable and representative results. This work establishes the methodological foundations necessary for future multi-taxon chemotaxonomic studies using O-PTIR.