Raman Microspectroscopy on Complex Samples

The Raman spectrum of a complex biological sample is brought about by spectral contributions from all Raman molecular groups contained in the sample and hence can serve as a fingerprint-like identifier for a specific biomaterial. Raman spectroscopy has long been known to be useful for characterization and classification of various types of complex biological objects.

Fast identification and biochemical characterization of pollen

We have been working towards the goal of a Raman-based characterization of natural bioaerosols, a matter of increasing interest not only to people suffering from allergies caused by pollen. We have demonstrated the potential of Raman spectroscopy in combination with multivariate statistics to classify pollen species of allergologic relevance based on full spectral fingerprints (380 cm-1-1700 cm-1). The pollen species that were studied were partly closely related, a factor that is of interest in current pollen identification (which is mainly based on visual inspection).

Schulte, F.; Lingott, J.; Panne, U.; Kneipp, J., Chemical Characterization and Classification of Pollen. Analytical Chemistry 2008, 80 (24), 9551–9556 Abstract

Schulte, F.; Mäder, J.; Kroh, L. W.; Panne, U.; Kneipp, J., Characterization of Pollen Carotenoids with in situ and High-Performance Thin-Layer Chromatography Supported Resonant Raman Spectroscopy. Analytical Chemistry 2009, 81, 8426-8433 Abstract

To elucidate the contribution of the different histological structures to the average Raman spectral signatures from intact pollen grains, we conducted spatially resolved measurements on sections of pollen grains. The figure below shows results of four examples of such mapping experiments. The plots of the intensities of selected characteristic bands as a function of lateral position (so-called chemical maps) provide information on the amount of the respective molecules or molecular groups in the different morphological structures.

In the Raman microscope, we can also follow dynamic processes such as the growth of the pollen tube, when pollen grains germinate in nutrient solutions. We find that utilization of metabolite storage is species-specific, and that the pollen outer coat is altered during the process. Furthermore, discharge of molecules into the nutrient medium may depend on the nutrient conditions in such germination experiments.

Schulte, F.; Panne, U.; Kneipp, J., Molecular changes during pollen germination can be monitored by Raman microspectroscopy. Journal of Biophotonics 2010, 3, 542-547 Abstract


To improve the sensitivity and lateral resolution in vibrational spectroscopic experiments for bioanalysis we also apply surface-enhanced Raman spectroscopy (SERS) with different noble metal substrates to the analysis of pollen. Sporopollenin, the biopolymer shell of pollen grains of higher plants, is a very stable and versatile yet so far greatly ill-defined material. Sporopollenin of most pollen species has escaped analysis by Raman spectroscopy because of a high fluorescence background. It is possible to synthesize silver nanoparticles in the presence of purified pollen shells and to thereby embed silver nanoparticles as SERS substrates in the ultrastructure of the sporopollenin biomatrix. Thereby we achieve a vibrational characterization of the local molecular structure of sporopollenin.

Joseph, V.; Schulte, F.; Rooch, H.; Feldmann, I.; Dörfel, I.; Österle, W.; Panne, U.; Kneipp, J., Surface- enhanced Raman scattering with silver nanostructures generated in situ in a sporopollenin biopolymer matrix. Chem. Commun. 2011, 47, 3236-3238 Abstract