Imaging nanoscale pollen morphology with Superresolution Structured Illumination Microscopy

Applications in the plant sciences that stem from disciplines as diverse as phylogenetics, physiology, and paleoecology all require ever-increasing imaging resolutions for accurate investigations of morphological hypotheses. Of these applications, the visualization of nanoscale plant morphology, such as the taxonomically diagnostic surface texture of individual pollen grains, is a special challenge for researchers. However, a combination of high-resolution imaging and computational analyses promises to unveil such nanoscale plant morphology for a whole spectrum of hypotheses, including those that address the taxonomic resolution of fossil pollen records. Therefore, the choice of imaging method for fossil pollen and hypothesized modern plant affinities is critical to research concerning the ecology and evolution of Earth’s biomes. However, the options for visualizing such nanoscale plant morphologies that are smaller than the diffraction limit of light are often limited to electron microscopy, which presents significant disadvantages in routine palynological work compared to optical microscopy. Superresolution Structured Illumination Microscopy (SR-SIM) is an emerging method that presents a powerful, non-destructive, and optically-sectioned way of imaging pollen that avoids certain disadvantages of electron microscopy. We examined and optimized the performance of SR-SIM in recovering the nanoscale surface morphology of the pollen of nine Poaceae species and compare our results to images obtained using Scanning Electron Microscopy (SEM) and an advanced transmitted light method: Laser-Scanning High-Resolution Differential Interference Contrast (LS-HR-DIC). Through our comparisons of resulting images, we appreciated that SR-SIM, LS-HR-DIC, and SEM represent three very different imaging methods. SR-SIM uses fluorescence, LS-HR- DIC uses transmitted light, and SEM uses reflected electrons. Therefore, the results of our study are expected in that the morphological information gathered by SR-SIM, LS-HR-DIC, and SEM is complementary, not identical: SR-SIM recovers three-dimensional features smaller than the diffraction limit, LS-HR-DIC produces diffraction-limited high contrast 3D representations, and SEM provides two-dimensional high-resolution images of an object’s surface. The morphological detail recovered from the SR-SIM is qualitatively comparable to the SEM. SR-SIM also represents an entirely new source of information on nanoscale plant morphologies, such as fine-scale pollen ornamentation and the interior structure of the pollen exine that could be used in conjunction with other standard approaches in optical and electron microscopy. SR-SIM is not a replacement for existing microscopic approaches, but is a viable alternative for material that is, by necessity or choice, mounted on microscopic slides.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Ecol, Evol, Conservation Biol

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Wesseln, Cassandra J

• superresolution
• Superresolution Structured Illumination Microscopy (SR-SIM)
• pollen
• Poaceae
• microscopy applications to plant biology

Statement dc:rights

• Copyright 2015 Cassandra Wesseln

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/78659

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/78659