S 4300 e n fesem

Pollens were visualized using a scanning electron microscope (SEM) (Hitachi S-4300 E/N FESEM, NY, USA). For SEM imaging, pollens were first coated with gold and palladium (Technics Hummer V sputter coater, Anatech USA, CA, USA). To observe the internal morphology of pollens, they were broken using a mortar and pestle, and then observed under the SEM. Pollens were also characterized using a CHN analyzer (PerkinElmer 2400 Series II CHNS/O Analyzer, MA, USA) to determine their protein content. A multiplication factor of 6.25 was used to convert percent nitrogen to percent protein in pollens (Atwe et al., 2014 (link); Vanderplanck et al., 2014 (link)).

To examine the interior of the RW shell, samples of RW pollen were manually cracked by grinding with dry ice, mounting on an aluminum stub, and coating with gold-palladium in a sputter coater (Technics Hummer V sputter coater, Anatech USA, CA, USA). The structure of RW was visualized with a scanning electron microscope (Hitachi S-4300 E/N FESEM, NY, USA). Carbon, hydrogen, and nitrogen content in the pollen samples were analyzed using PerkinElmer 2400 Series II CHNS/O Analyzer (PerkinElmer, Inc., Waltham, MA, USA). Protein percentage was calculated from total nitrogen content by multiplying it with a nitrogen-to-protein conversion factor of 6.25 (Atwe et al., 2014 (link)). All measurements were performed in triplicate.

To investigate the interior of the pollen shells, samples of RW pollen were mixed with dry ice to make them brittle and manually cracked in a mortar and pestle. Samples were then coated with gold and palladium (Technics Hummer V sputter coater, Anatech USA, CA, USA). The morphology of the raw and processed RW was examined by a scanning electron microscope (SEM) (Hitachi S-4300 E/N FESEM, NY, USA). Elemental analysis using PerkinElmer 2400 Series II CHNS/O Analyzer (PerkinElmer, Inc., Waltham, MA, USA) was also performed to quantify the amount of nitrogen remaining in RW. Percent nitrogen obtained from sample analysis was then multiplied by a factor of 6.25 to convert it into percent protein [1 (link)]. All measurements were conducted in triplicate.
Chemically processed RW pollens suspended in phosphate buffered saline (PBS) were counted using hemocytometer and an automated cell counter (Countess II FL automated cell counter, Thermo Fisher Scientific, Waltham, MA, USA) to determine the number of pollens present per milligram of the sample.