X-ray Microscopic Examination of Lily Pollen Grains

Figure 1: Representative image of lilium tigrinum ‘Splendens’ flower from which pollen grains were extracted for imaging1

Pollen is studied by archaeologists to learn about ancient communities, by paleobotanists to learn about former environments, and by scientists as part of forensic research, to study climate change, and to explore possibilities for plant breeding. Size, shape, number of apertures, and the surface texture are key characteristics used to identify pollen types.

For our imaging series this month, we utilized the SkyScan 2214 nano-CT to examine lily pollen at two different scales.

X-Ray Microscopic Imaging of Pollen and Plant Materials

Pollen grains were manually extracted from the lily flower’s anther and deposited in a working solution of 0.3% phosphotungstic acid (PTA) in ethanol. After sufficient exposure, we removed the pollen grains using a pipette and deposited the grain solution onto either a polystyrene bead or into a porous polymeric foam prior to imaging. The ethanol was allowed to evaporate from the supporting matrix, leaving behind a dry layer of pollen grains on the underlying support material used for imaging. By incorporating the PTA staining step, we aimed to increase our local attenuation of X-ray energy within the pollen granules, which can often be a challenge for very small, low-density organic samples like pollen grains. We examined the grains using our ultra high-resolution SkyScan 2214 nano-CT at an isotropic voxel size of 900 nm, and then imaged a smaller sample size at an isotropic voxel size of 200 nm. The SkyScan 2214 is a good match for this project due to its high resolution arising from the nano-focused X-ray source that allowed us to produce detailed images similar to those that could be observed from scanning electron microscopy (SEM).

Figure 2: Planar 2D views of the 900 nm overview scan

As shown in Figure 2, after reconstruction with NRecon utilizing single distance phase retrieval to boost our contrast between the pollen grains and the polystyrene and plastic tube used to contain the sample, DataViewer was used to straighten and align datasets. While individual grains are present and visible all around the surface of the polystyrene bead used as a mount, structural details of the pollen grains are limited at this imaging resolution due to their small size.

Figure 3: Rendered cut 3D view through the 900 nm dataset showing the location of pollen granules within the sample

Using a combination of transfer functions and clipping planes within CTVox, we digitally dissected a live view of the pollen dataset, as shown in Figure 3. In this view, we removed the signal from the polystyrene and focused primarily on the pollen granules with some remaining signal from the polymeric tube used to contain the sample during imaging.

Figure 4: 2D view through the 200 nm dataset of pollen grains dispersed within polymeric foam

To better elucidate structural details on the individual grains, we prepared a new sample with a smaller diameter and increased our magnification on the SkyScan 2214 down to a 200 nm voxel size. Imaging this sample under these conditions improved our resolution of each pollen grain, as shown in Figure 4. SkyScan CTAnalyzer was then used to create a volume of interest from the 200 nm dataset where we isolated a single grain of pollen from the remaining grains and foam signal.

Figure 5: Comparison of SEM micrograph of lilium auratum pollen grains2 (top) and our isolated lilium tigrinium pollen grain rendered in SkyScan CTVox (bottom)

As shown in Figure 5, the 200 nm dataset acquired on the SkyScan 2214 nano-CT produced detailed structural information of similar quality to what is often observed using SEM imaging. While the SEM images are sharper and more detailed due to the superior resolution of the technique, no 3D structural data is present from SEM imaging since the technique is a surface imaging technique only. Thus, our X-ray microscopy data, while not as resolved, does provide useful structural data for the pollen grains as a 3D dataset instead of a static 2D image.

Figure 6: Maverick 3D rendering from isolated pollen grain VOI extracted from the 200 nm dataset

After extracting the single pollen grain from the 200 nm dataset and producing a volumetric mesh within Synopsys’ Simpleware ScanIP software with the CAD add-on module (Synopsys, Inc., Mountain View, USA), we imported the mesh into Maverick Render Indie to create high resolution photo realistic renderings and videos of our pollen grain shown in Figure 6.

Conclusion

Among the SkyScan product line, the SkyScan 2214 represents our highest resolution instrument and is traditionally thought of as a great fit for imaging organic and ceramic samples at resolutions that approach the capabilities of scanning electron microscopy but with the added benefit of full 3D structural information.

We hope you found this Image of the Month informative and encourage you to subscribe to our newsletter and social media channels in preparation for the continuation of our Image of the Month series next month.

Scan Specifications

Sample Pollen Overview Pollen VOI
Voltage (kV) 35 40
Current (µA) 150 150
Filter None None
Voxel Size (nm) 900 200
Rotation Step 0.2 0.2
Exposure Time (ms) 7000 8000
Rotation Extent (deg.) 180 360
Scan Time (HH:MM:SS) 14:46:55 16:01:29

These scans were completed on our SkyScan 2214 nano-CT system at the Micro Photonics Imaging Laboratory in Allentown, PA. Reconstructions were completed using NRecon 2.0 while visualization and volumetric inspection of the 2D and 3D results were completed using DataViewer and CTVox. The isolated pollen grains were converted to STL volumetric models using Synopsys’ Simpleware ScanIP software with the CAD add-on module (Synopsys, Inc., Mountain View, USA) before 3D rendering using Maverick Render Indie (Random Control, Madrid, Spain).

Would you like your work to be featured in our monthly newsletter? If so, please contact us by calling Seth Hogg at 610-366-7103 or emailing seth.hogg@microphotonics.com.

References

1 https://www.wilsonbrosgardens.com/lilium-tigrinum-splendens-orange-tiger-lily-1-gallon.html

2 https://en.m.wikipedia.org/wiki/File:Lilium_auratum_-_pollen.jpg

*Simpleware software (Synopsys, Inc., Mountain View, USA) enables you to comprehensively process 3D image data (MRI, CT, micro-CT, FIB-SEM…) and export models suitable for CAD, CAE and 3D printing. Use Simpleware software’s capabilities to visualize, analyze, and quantify your data, and to export models for design and simulation workflows. Simpleware™ is a trademark of Synopsys, Inc. in the U.S. and/or other countries.

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