Abstract Detail

Anatomy and Morphology

Gladish, Daniel K. [1], Saito, Susumu [2], Miki, Yasushi [2], Niki, Teruo [2].

Special thin-section preparation makes possible high-resolution "reverse" tomography by light microscopy and laptop computer.

A novel slide preparation technique emerged from the systematic use of histochemistry and diagnostic staining to identify the composition of an unusual ultrastructural feature in rapidly-growing maize root tips.  We discovered that higher contrast among major cell features, such as walls and nuclei, in a cytoplasmically-dense meristematic tissue could be obtained by one of those procedures: treatment with RNase followed by toluidine blue staining.  This works because toluidine blue stains carbohydrates and nucleic acids strongly, and RNase eliminates most of the RNA that is abundant in meristematic cytosol associated with polysomes and on ER.   DNA and carbohydrate structures remain sharply defined.   Later we learned that enzyme treatment could follow staining with very similar results.  By using high magnification Plan Fluor ∞/0 lenses, which do not require a cover slip, we were able to make a detailed record of cells' initial staining patterns, then "clear" them to increase contrast and photograph the same sections again to examine tissue patterns.  Precision serial sectioning of resin-embedded tissue at 1 or 2 µm provides excellent resolution of detail.  It was apparent this technique could streamline the ability to align digital micrographs of serial sections for studying cell lineages and tissue ontogeny and for measuring distances.  We succeeded in reassessing and comparing details of promeristem organization in a wild subspecies (a teosinte) and a domesticated cultivar of Zea mays L.  The high resolution and contrast of the serial digital micrographs we obtained suggested an opportunity for a new, relatively inexpensive type of computerized tomography.  We recently refined a procedure that uses a standard laptop computer equipped with commercial software to align section images, measure and correct possible sectioning-induced distortion, color-label target cell types, then use these to generate high-resolution 3D virtual reconstructions of transversely sectioned root tips that can ultimately be virtually sectioned longitudinally.  We are currently using this technique to analyze in detail the ontogeny of late-maturing metaxylem vessels in Zea mays procambium.

Related Links:
Recent publication about the methods

1 - Miami University-Hamilton, Biology, 1601 University Blvd., Hamilton, OH, 45011, United States
2 - MikiOn LLC, Image Processing Section, 103 Ishikawa Heights, 1737 Hazama-machi, Hachioji, Tokyo 193-0941, 193-0941, Japan

high-resolution tomography
light microscopy
root meristem structure
3D virtual reconstruction
Metaxylem ontogeny.

Presentation Type: Oral Paper
Session: AM1, Anatomy and Morphology
Location: Virtual/Virtual
Date: Wednesday, July 29th, 2020
Time: 10:00 AM
Number: AM1001
Abstract ID:599
Candidate for Awards:None

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