Application of High-Throughput Screening Raman Spectroscopy (HTS-RS) for Label-Free Identification and Molecular Characterization of Pollen

Abstract

Pollen studies play a critical role in various fields of science. In the last couple of decades, replacement of manual identification of pollen by image-based methods using pollen morphological features was a great leap forward, but challenges for pollen with similar morphology remain, and additional approaches are required. Spectroscopy approaches for identification of pollen, such as Raman spectroscopy has potential benefits over traditional methods, due to the investigation of the intrinsic molecular composition of a sample. However, current Raman-based characterization of pollen is complex and time-consuming, resulting in low throughput and limiting the statistical significance of the acquired data. Previously demonstrated high-throughput screening Raman spectroscopy (HTS-RS) eliminates the complexity as well as human interaction by incorporation full automation of the data acquisition process. Here, we present a customization of HTS-RS for pollen identification, enabling sampling of a large number of pollen in comparison to other state-of-the-art Raman pollen investigations. We show that using Raman spectra we are able to provide a preliminary estimation of pollen types based on growth habits using hierarchical cluster analysis (HCA) as well as good taxonomy of 37 different Pollen using principal component analysis-support vector machine (PCA-SVM) with good accuracy even for the pollen specimens sharing similar morphological features. Our results suggest that HTS-RS platform meets the demands for automated pollen detection making it an alternative method for research concerning pollen.

Chemical variations in Quercus pollen as a tool for taxonomic identification: Implications for long‐term ecological and biogeographical research

“…Indeed, our results indicate the potential for FTIR to surpass traditional LM methods used in palynology, and work at a comparable level to SEM (Denk & Grimm, 2009;Denk & Tekleva, 2014;Grímsson, Grimm, Meller, Bouchal, & Zetter, 2016;Grímsson et al, 2015). However, the extensive automatedclassification possibilities offered by future IR analysis (Mondol et al, 2019), and in the ease of sample preparation and data collection, may mean it will be easier to expand these technologies compared with the more time-consuming SEM methods in the long term.…”

Chemical Analysis of Pollen by FT-Raman and FTIR Spectroscopies

“…Vibrational studies of pollen, by diverse infrared and Raman techniques, have shown that vibrational spectroscopy achieves economical and rapid identification and classification of pollen according to taxonomy and phylogenetic relationship (Pappas et al, 2003;Ivleva et al, 2005;Gottardini et al, 2007;Schulte et al, 2008;Dell'anna et al, 2009;Zimmermann, 2010Zimmermann, , 2018Guedes et al, 2014;Zimmermann and Kohler, 2014;Zimmermann et al, 2015b;Julier et al, 2016;Seifert et al, 2016;Woutersen et al, 2018;Jardine et al, 2019;Mondol et al, 2019). For example, the recent FTIR microspectroscopy study on individual pollen grains has achieved more accurate classification than optical microscopy, which is the benchmark method in pollen identification (Zimmermann et al, 2016).…”

Discrimination of grass pollen of different species by FTIR spectroscopy of individual pollen grains

“…The fact that identification is based here on spectra from individual pollen grains rather than averages from one plant adds another source of variation here, as was recently also discussed when we compared different spectroscopic methods that probe either bulk samples or individual pollen grains and their potential for pollen identification [ 24 ]. Nevertheless, the possibility to study pollen spectra in mixtures could in the future open possibilities for FTIR imaging-based identification of mixed grass pollen samples, similar to existing high-throughput and mapping approaches [ 9 , 10 ].…”

“…In most cases, identification to species level is not possible, and some pollen types can be identified only to higher taxonomic level, such as family level for grasses (Poaceae) [8]. For that reason, spectroscopic and spectrometric methods, including mass spectrometry, Raman scattering, and Fourier-transform infrared (FTIR) spectroscopy, are currently being harnessed by several groups in order to obtain not only more precise identification but also highthroughput pollen chemical analysis [9][10][11][12][13].…”