Comparison of 3-D subcellular imaging techniques based on scanned electron probes: application to blood platelet ultrastructure

Authors

• MA Aronova
• Q He
• MD Guay
• RP Desai
• MP Tobin
• EL McBride
• A Rao
• G Zhang
• ID Pokrovskaya
• B Storrie
• RD Leapman

Abstract

Microscopies based on focused electron probes allow the cell biologist to image the 3-D ultrastructure of eukaryotic cells and tissues extending over large volumes, thus providing new insight on the relationship between cellular architecture and function of organelles. These approaches can involve very different types of instrumentation, including scanning transmission electron microscopes (STEMs) operating at a primary beam energy of 300 keV, and scanning electron microscopes (SEMs) operating at a primary beam energy of only 1 keV. Here we compare two such techniques: axial bright-field STEM tomography, and serial block face (SBF)-SEM, by considering the 3-D ultrastructure of human blood platelets. We find that many features of the complex membranes composing the platelet organelles can be determined from both approaches. However, STEM tomography provides higher spatial resolution (~3 nm isotropic resolution) relative to SBF-SEM (~ 5 nm in the plane of the block face and 25 nm perpendicular to the block face). On the other hand, SBF-SEM enables visualization of large numbers of entire platelets, each of which extends ~2 µm in minimum dimension, whereas STEM tomography can only visualize ~75% of the platelet volume due to a rapid non-linear loss of signal in specimens of thickness greater than ~1.5 µm. The time required to segment ultrastructural components manually is currently a limiting factor for both techniques, but we are developing deep learning approaches that promise to greatly accelerate image segmentation.

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